High-resolution high-speed dynamic mechanical spectroscopy of cells and other soft materials with the help of atomic force microscopy

PubMed Central

Dokukin, M.; Sokolov, I.

2015-01-01

Dynamic mechanical spectroscopy (DMS), which allows measuring frequency-dependent viscoelastic properties, is important to study soft materials, tissues, biomaterials, polymers. However, the existing DMS techniques (nanoindentation) have limited resolution when used on soft materials, preventing them from being used to study mechanics at the nanoscale. The nanoindenters are not capable of measuring cells, nanointerfaces of composite materials. Here we present a highly accurate DMS modality, which is a combination of three different methods: quantitative nanoindentation (nanoDMA), gentle force and fast response of atomic force microscopy (AFM), and Fourier transform (FT) spectroscopy. This new spectroscopy (which we suggest to call FT-nanoDMA) is fast and sensitive enough to allow DMS imaging of nanointerfaces, single cells, while attaining about 100x improvements on polymers in both spatial (to 10–70 nm) and temporal resolution (to 0.7s/pixel) compared to the current art. Multiple frequencies are measured simultaneously. The use of 10 frequencies are demonstrated here (up to 300 Hz which is a rather relevant range for biological materials and polymers, in both ambient conditions and liquid). The method is quantitatively verified on known polymers and demonstrated on cells and polymers blends. Analysis shows that FT-nanoDMA is highly quantitative. The FT-nanoDMA spectroscopy can easily be implemented in the existing AFMs. PMID:26218346

Molecular Force Spectroscopy on Cells

NASA Astrophysics Data System (ADS)

Liu, Baoyu; Chen, Wei; Zhu, Cheng

2015-04-01

Molecular force spectroscopy has become a powerful tool to study how mechanics regulates biology, especially the mechanical regulation of molecular interactions and its impact on cellular functions. This force-driven methodology has uncovered a wealth of new information of the physical chemistry of molecular bonds for various biological systems. The new concepts, qualitative and quantitative measures describing bond behavior under force, and structural bases underlying these phenomena have substantially advanced our fundamental understanding of the inner workings of biological systems from the nanoscale (molecule) to the microscale (cell), elucidated basic molecular mechanisms of a wide range of important biological processes, and provided opportunities for engineering applications. Here, we review major force spectroscopic assays, conceptual developments of mechanically regulated kinetics of molecular interactions, and their biological relevance. We also present current challenges and highlight future directions.

Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

PubMed Central

Taubenberger, Anna; Cisneros, David A.; Friedrichs, Jens; Puech, Pierre-Henri; Muller, Daniel J.

2007-01-01

We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility. PMID:17314408

Single-cell force spectroscopy of the medically important Staphylococcus epidermidis-Candida albicans interaction

NASA Astrophysics Data System (ADS)

Beaussart, Audrey; Herman, Philippe; El-Kirat-Chatel, Sofiane; Lipke, Peter N.; Kucharíková, Soňa; van Dijck, Patrick; Dufrêne, Yves F.

2013-10-01

Despite the clinical importance of bacterial-fungal interactions, their molecular details are poorly understood. A hallmark of such medically important interspecies associations is the interaction between the two nosocomial pathogens Staphylococcus aureus and Candida albicans, which can lead to mixed biofilm-associated infections with enhanced antibiotic resistance. Here, we use single-cell force spectroscopy (SCFS) to quantify the forces engaged in bacterial-fungal co-adhesion, focusing on the poorly investigated S. epidermidis-C. albicans interaction. Force curves recorded between single bacterial and fungal germ tubes showed large adhesion forces (~5 nN) with extended rupture lengths (up to 500 nm). By contrast, bacteria poorly adhered to yeast cells, emphasizing the important role of the yeast-to-hyphae transition in mediating adhesion to bacterial cells. Analysis of mutant strains altered in cell wall composition allowed us to distinguish the main fungal components involved in adhesion, i.e. Als proteins and O-mannosylations. We suggest that the measured co-adhesion forces are involved in the formation of mixed biofilms, thus possibly as well in promoting polymicrobial infections. In the future, we anticipate that this SCFS platform will be used in nanomedicine to decipher the molecular mechanisms of a wide variety of pathogen-pathogen interactions and may help in designing novel anti-adhesion agents.

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

PubMed

Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

2014-12-18

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Spectrin-ankyrin interaction mechanics: A key force balance factor in the red blood cell membrane skeleton.

PubMed

Saito, Masakazu; Watanabe-Nakayama, Takahiro; Machida, Shinichi; Osada, Toshiya; Afrin, Rehana; Ikai, Atsushi

2015-01-01

As major components of red blood cell (RBC) cytoskeleton, spectrin and F-actin form a network that covers the entire cytoplasmic surface of the plasma membrane. The cross-linked two layered structure, called the membrane skeleton, keeps the structural integrity of RBC under drastically changing mechanical environment during circulation. We performed force spectroscopy experiments on the atomic force microscope (AFM) as a means to clarify the mechanical characteristics of spectrin-ankyrin interaction, a key factor in the force balance of the RBC cytoskeletal structure. An AFM tip was functionalized with ANK1-62k and used to probe spectrin crosslinked to mica surface. A force spectroscopy study gave a mean unbinding force of ~30 pN under our experimental conditions. Two energy barriers were identified in the unbinding process. The result was related to the well-known flexibility of spectrin tetramer and participation of ankyrin 1-spectrin interaction in the overall balance of membrane skeleton dynamics. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of the adsorption geometry of PTCDA on Ag(111) on the tip-molecule forces in non-contact atomic force microscopy.

PubMed

Langewisch, Gernot; Falter, Jens; Schirmeisen, André; Fuchs, Harald

2014-01-01

Perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) adsorbed on a metal surface is a prototypical organic-anorganic interface. In the past, scanning tunneling microscopy and scanning tunneling spectroscopy studies of PTCDA adsorbed on Ag(111) have revealed differences in the electronic structure of the molecules depending on their adsorption geometry. In the work presented here, high-resolution 3D force spectroscopy measurements at cryogenic temperatures were performed on a surface area that contained a complete PTCDA unit cell with the two possible geometries. At small tip-molecule separations, deviations in the tip-sample forces were found between the two molecule orientations. These deviations can be explained by a different electron density in both cases. This result demonstrates the capability of 3D force spectroscopy to detect even small effects in the electronic properties of organic adsorbates.

Microfluidic chip for non-invasive analysis of tumor cells interaction with anti-cancer drug doxorubicin by AFM and Raman spectroscopy.

PubMed

Zhang, Han; Xiao, Lifu; Li, Qifei; Qi, Xiaojun; Zhou, Anhong

2018-03-01

Raman spectroscopy has been playing an increasingly significant role for cell classification. Here, we introduce a novel microfluidic chip for non-invasive Raman cell natural fingerprint collection. Traditional Raman spectroscopy measurement of the cells grown in a Polydimethylsiloxane (PDMS) based microfluidic device suffers from the background noise from the substrate materials of PDMS when intended to apply as an in vitro cell assay. To overcome this disadvantage, the current device is designed with a middle layer of PDMS layer sandwiched by two MgF 2 slides which minimize the PDMS background signal in Raman measurement. Three cancer cell lines, including a human lung cancer cell A549, and human breast cancer cell lines MDA-MB-231 and MDA-MB-231/BRMS1, were cultured in this microdevice separately for a period of three days to evaluate the biocompatibility of the microfluidic system. In addition, atomic force microscopy (AFM) was used to measure the Young's modulus and adhesion force of cancer cells at single cell level. The AFM results indicated that our microchannel environment did not seem to alter the cell biomechanical properties. The biochemical responses of cancer cells exposed to anti-cancer drug doxorubicin (DOX) up to 24 h were assessed by Raman spectroscopy. Principal component analysis over the Raman spectra indicated that cancer cells untreated and treated with DOX can be distinguished. This PDMS microfluidic device offers a non-invasive and reusable tool for in vitro Raman measurement of living cells, and can be potentially applied for anti-cancer drug screening.

Bacterial adhesion force quantification by fluidic force microscopy

NASA Astrophysics Data System (ADS)

Potthoff, Eva; Ossola, Dario; Zambelli, Tomaso; Vorholt, Julia A.

2015-02-01

Quantification of detachment forces between bacteria and substrates facilitates the understanding of the bacterial adhesion process that affects cell physiology and survival. Here, we present a method that allows for serial, single bacterial cell force spectroscopy by combining the force control of atomic force microscopy with microfluidics. Reversible bacterial cell immobilization under physiological conditions on the pyramidal tip of a microchanneled cantilever is achieved by underpressure. Using the fluidic force microscopy technology (FluidFM), we achieve immobilization forces greater than those of state-of-the-art cell-cantilever binding as demonstrated by the detachment of Escherichia coli from polydopamine with recorded forces between 4 and 8 nN for many cells. The contact time and setpoint dependence of the adhesion forces of E. coli and Streptococcus pyogenes, as well as the sequential detachment of bacteria out of a chain, are shown, revealing distinct force patterns in the detachment curves. This study demonstrates the potential of the FluidFM technology for quantitative bacterial adhesion measurements of cell-substrate and cell-cell interactions that are relevant in biofilms and infection biology.Quantification of detachment forces between bacteria and substrates facilitates the understanding of the bacterial adhesion process that affects cell physiology and survival. Here, we present a method that allows for serial, single bacterial cell force spectroscopy by combining the force control of atomic force microscopy with microfluidics. Reversible bacterial cell immobilization under physiological conditions on the pyramidal tip of a microchanneled cantilever is achieved by underpressure. Using the fluidic force microscopy technology (FluidFM), we achieve immobilization forces greater than those of state-of-the-art cell-cantilever binding as demonstrated by the detachment of Escherichia coli from polydopamine with recorded forces between 4 and 8 nN for many cells. The contact time and setpoint dependence of the adhesion forces of E. coli and Streptococcus pyogenes, as well as the sequential detachment of bacteria out of a chain, are shown, revealing distinct force patterns in the detachment curves. This study demonstrates the potential of the FluidFM technology for quantitative bacterial adhesion measurements of cell-substrate and cell-cell interactions that are relevant in biofilms and infection biology. Electronic supplementary information (ESI) available: Video S1. Detachment of a S. pyogenes cell chain from glass substrate. The cantilever is approached on the outermost adherent cell of a chain and four bacteria were then sequentially detached. The sequential cell detachment suddenly stopped after four bacteria. This possibly occurred because bacteria-glass interactions became too strong or the maximal probe retraction was reached. The cells spontaneously detached from the cantilever flipping back on the surface. Fig. S1. (A) Adhesion force-distance and (B) adhesion force-detaching work correlation of E.coli on PLL for setpoints of 1 and 10 nN. Circle: 1 nN setpoint, square: 10 nN. See DOI: 10.1039/c4nr06495j

Medical applications of atomic force microscopy and Raman spectroscopy.

PubMed

Choi, Samjin; Jung, Gyeong Bok; Kim, Kyung Sook; Lee, Gi-Ja; Park, Hun-Kuk

2014-01-01

This paper reviews the recent research and application of atomic force microscopy (AFM) and Raman spectroscopy techniques, which are considered the multi-functional and powerful toolkits for probing the nanostructural, biomechanical and physicochemical properties of biomedical samples in medical science. We introduce briefly the basic principles of AFM and Raman spectroscopy, followed by diagnostic assessments of some selected diseases in biomedical applications using them, including mitochondria isolated from normal and ischemic hearts, hair fibers, individual cells, and human cortical bone. Finally, AFM and Raman spectroscopy applications to investigate the effects of pharmacotherapy, surgery, and medical device therapy in various medicines from cells to soft and hard tissues are discussed, including pharmacotherapy--paclitaxel on Ishikawa and HeLa cells, telmisartan on angiotensin II, mitomycin C on strabismus surgery and eye whitening surgery, and fluoride on primary teeth--and medical device therapy--collagen cross-linking treatment for the management of progressive keratoconus, radiofrequency treatment for skin rejuvenation, physical extracorporeal shockwave therapy for healing of Achilles tendinitis, orthodontic treatment, and toothbrushing time to minimize the loss of teeth after exposure to acidic drinks.

Nanoscale visualization and characterization of Myxococcus xanthus cells with atomic force microscopy

PubMed Central

Pelling, Andrew E.; Li, Yinuo; Shi, Wenyuan; Gimzewski, James K.

2005-01-01

Multicellular microbial communities are the predominant form of existence for microorganisms in nature. As one of the most primitive social organisms, Myxococcus xanthus has been an ideal model bacterium for studying intercellular interaction and multicellular organization. Through previous genetic and EM studies, various extracellular appendages and matrix components have been found to be involved in the social behavior of M. xanthus, but none of them was directly visualized and analyzed under native conditions. Here, we used atomic force microscopy (AFM) imaging and in vivo force spectroscopy to characterize these cellular structures under native conditions. AFM imaging revealed morphological details on the extracellular ultrastructures at an unprecedented resolution, and in vivo force spectroscopy of live cells in fluid allowed us to nanomechanically characterize extracellular polymeric substances. The findings provide the basis for AFM as a useful tool for investigating microbial-surface ultrastructures and nanomechanical properties under native conditions. PMID:15840722

Probing the nanoscale interaction forces and elastic properties of organic and inorganic materials using force-distance (F-D) spectroscopy

NASA Astrophysics Data System (ADS)

Vincent, Abhilash

Due to their therapeutic applications such as radical scavenging, MRI contrast imaging, Photoluminescence imaging, drug delivery, etc., nanoparticles (NPs) have a significant importance in bio-nanotechnology. The reason that prevents the utilizing NPs for drug delivery in medical field is mostly due to their biocompatibility issues (incompatibility can lead to toxicity and cell death). Changes in the surface conditions of NPs often lead to NP cytotoxicity. Investigating the role of NP surface properties (surface charges and surface chemistry) on their interactions with biomolecules (Cells, protein and DNA) could enhance the current understanding of NP cytotoxicity. Hence, it is highly beneficial to the nanotechnology community to bring more attention towards the enhancement of surface properties of NPs to make them more biocompatible and less toxic to biological systems. Surface functionalization of NPs using specific ligand biomolecules have shown to enhance the protein adsorption and cellular uptake through more favorable interaction pathways. Cerium oxide NPs (CNPs also known as nanoceria) are potential antioxidants in cell culture models and understanding the nature of interaction between cerium oxide NPs and biological proteins and cells are important due to their therapeutic application (especially in site specific drug delivery systems). The surface charges and surface chemistry of CNPs play a major role in protein adsorption and cellular uptake. Hence, by tuning the surface charges and by selecting proper functional molecules on the surface, CNPs exhibiting strong adhesion to biological materials can be prepared. By probing the nanoscale interaction forces acting between CNPs and protein molecules using Atomic Force Microscopy (AFM) based force-distance (F-D) spectroscopy, the mechanism of CNP-protein adsorption and CNP cellular uptake can be understood more quantitatively. The work presented in this dissertation is based on the application of AFM in studying the interaction forces as well as the mechanical properties of nanobiomaterials. The research protocol employed in the earlier part of the dissertation is specifically aimed to understand the operation of F-D spectroscopy technique. The elastic properties of thin films of silicon dioxide NPs were investigated using F-D spectroscopy in the high force regime of few 100 nN to 1 microN. Here, sol-gel derived porous nanosilica thin films of varying surface morphology, particle size and porosity were prepared through acid and base catalyzed process. AFM nanoindentation experiments were conducted on these films using the F-D spectroscopy mode and the nanoscale elastic properties of these films were evaluated. The major contribution of this dissertation is a study exploring the interaction forces acting between CNPs and transferrin proteins in picoNewton scale regime using the force-distance spectroscopy technique. This study projects the importance of obtaining appropriate surface charges and surface chemistry so that the NP can exhibit enhanced protein adsorption and NP cellular uptake.

Combined use of atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry for cell surface analysis.

PubMed

Dague, Etienne; Delcorte, Arnaud; Latgé, Jean-Paul; Dufrêne, Yves F

2008-04-01

Understanding the surface properties of microbial cells is a major challenge of current microbiological research and a key to efficiently exploit them in biotechnology. Here, we used three advanced surface analysis techniques with different sensitivity, probing depth, and lateral resolution, that is, in situ atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry, to gain insight into the surface properties of the conidia of the human fungal pathogen Aspergillus fumigatus. We show that the native ultrastructure, surface protein and polysaccharide concentrations, and amino acid composition of three mutants affected in hydrophobin production are markedly different from those of the wild-type, thereby providing novel insight into the cell wall architecture of A. fumigatus. The results demonstrate the power of using multiple complementary techniques for probing microbial cell surfaces.

Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

PubMed

Andolfi, Laura; Bourkoula, Eugenia; Migliorini, Elisa; Palma, Anita; Pucer, Anja; Skrap, Miran; Scoles, Giacinto; Beltrami, Antonio Paolo; Cesselli, Daniela; Lazzarino, Marco

2014-01-01

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

Quantitative modeling of forces in electromagnetic tweezers

NASA Astrophysics Data System (ADS)

Bijamov, Alex; Shubitidze, Fridon; Oliver, Piercen M.; Vezenov, Dmitri V.

2010-11-01

This paper discusses numerical simulations of the magnetic field produced by an electromagnet for generation of forces on superparamagnetic microspheres used in manipulation of single molecules or cells. Single molecule force spectroscopy based on magnetic tweezers can be used in applications that require parallel readout of biopolymer stretching or biomolecular binding. The magnetic tweezers exert forces on the surface-immobilized macromolecule by pulling a magnetic bead attached to the free end of the molecule in the direction of the field gradient. In a typical force spectroscopy experiment, the pulling forces can range between subpiconewton to tens of piconewtons. In order to effectively provide such forces, an understanding of the source of the magnetic field is required as the first step in the design of force spectroscopy systems. In this study, we use a numerical technique, the method of auxiliary sources, to investigate the influence of electromagnet geometry and material parameters of the magnetic core on the magnetic forces pulling the target beads in the area of interest. The close proximity of the area of interest to the magnet body results in deviations from intuitive relations between magnet size and pulling force, as well as in the force decay with distance. We discuss the benefits and drawbacks of various geometric modifications affecting the magnitude and spatial distribution of forces achievable with an electromagnet.

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy.

PubMed

Hu, Mingqian; Wang, Jiongkun; Cai, Jiye; Wu, Yangzhe; Wang, Xiaoping

2008-09-12

To date, nanoscale imaging of the morphological changes and adhesion force of CD4(+) T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4(+) T cells. The AFM images revealed that the volume of activated CD4(+) T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4(+) T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.

Toward investigating changes in cell mechanoelastic properties in response to nanosecond pulsed electric fields

NASA Astrophysics Data System (ADS)

Coker, Zachary; Troyanova-Wood, Maria; Traverso, Andrew; Meng, Zhaokai; Ballmann, Charles; Petrov, Georgi; Ibey, Bennett L.; Yakovlev, Vladislav

2017-02-01

Nanosecond electric pulses (nsEPs) are known to cause a variety of effects on mammalian cells, ranging from destabilization of cell membranes to changes in cytoskeleton and elastic moduli. Measurement of a cells mechanoelastic properties have previously been limited to only invasive and destructive techniques such as atomic force microscopy or application of optical tweezers. However, due to recent advances, Brillouin spectroscopy has now become viable as a non-contact, non-invasive method for measuring these properties in cells and other materials. Here, we present progress toward applying Brillouin spectroscopy using a unique microscopy system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 50kV/cm. Successful measurement of mechanoelastic changes in these cells will demonstrate Brillouin spectroscopy as a viable method for measuring changes in elastic properties of other cells and living organisms.

Single-molecule dynamic force spectroscopy of the fibronectin-heparin interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Gabriel; Lamontagne, Charles-Antoine; Lebel, Rejean

2007-12-21

The integrity of cohesive tissues strongly depends on the presence of the extracellular matrix, which provides support and anchorage for cells. The fibronectin protein and the heparin-like glycosaminoglycans are key components of this dynamic structural network. In this report, atomic force spectroscopy was used to gain insight into the compliance and the resistance of the fibronectin-heparin interaction. We found that this interaction can be described by an energetic barrier width of 3.1 {+-} 0.2 A and an off-rate of 0.2 {+-} 0.1 s{sup -1}. These dissociation parameters are similar to those of other carbohydrate-protein interactions and to off-rate values reportedmore » for more complex interactions between cells and extracellular matrix components. Our results indicate that the function of the fibronectin-heparin interaction is supported by its capacity to sustain significant deformations and considerable external mechanical forces.« less

Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

NASA Astrophysics Data System (ADS)

Christenson, Wayne B.

Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights.

Probing cellular mechanics with Acoustic Force Spectroscopy.

PubMed

Sorkin, Raya; Bergamaschi, Giulia; Kamsma, Douwe; Brand, Guy; Dekel, Elya; Ofir-Birin, Yifat; Rudik, Ariel; Gironella, Marta; Ritort, Felix; Regev-Rudzki, Neta; Roos, Wouter; Wuite, Gijs J L

2018-06-21

A large number of studies demonstrate that cell mechanics and pathology are intimately linked. In particular, Red Blood Cell (RBC) deformability is key to their function, and is dramatically altered in the time course of diseases such as anemia and malaria. Due to the physiological importance of cell mechanics, many methods for cell mechanical probing have been developed. While single cell methods provide very valuable information, they are often technically challenging and lack high data throughput needed to distinguish differences in heterogeneous populations, while fluid flow high throughput methods miss the accuracy to detect subtle differences. Here, we present a new method for multiplexed single-cell mechanical probing using Acoustic Force Spectroscopy (AFS). We demonstrate that mechanical differences induced by chemical treatments of known effect can be measured and quantified. Furthermore, we explore the effect of extracellular-vesicles (EVs) uptake on RBC mechanics and demonstrate that EVs uptake increases RBC deformability. Our findings demonstrate the ability of AFS to manipulate cells with high stability and precision, and pave the way to further new insights into cellular mechanics and mechanobiology in health and disease, as well as potential biomedical applications.

The molten globule state is unusually deformable under mechanical force

PubMed Central

Elms, Phillip J.; Chodera, John D.; Bustamante, Carlos; Marqusee, Susan

2012-01-01

Recently, the role of force in cellular processes has become more evident, and now with advances in force spectroscopy, the response of proteins to force can be directly studied. Such studies have found that native proteins are brittle, and thus not very deformable. Here, we examine the mechanical properties of a class of intermediates referred to as the molten globule state. Using optical trap force spectroscopy, we investigated the response to force of the native and molten globule states of apomyoglobin along different pulling axes. Unlike natively folded proteins, the molten globule state of apomyoglobin is compliant (large distance to the transition state); this large compliance means that the molten globule is more deformable and the unfolding rate is more sensitive to force (the application of force or tension will have a more dramatic effect on the unfolding rate). Our studies suggest that these are general properties of molten globules and could have important implications for mechanical processes in the cell. PMID:22355138

Investigation of specific interactions between T7 promoter and T7 RNA polymerase by force spectroscopy using atomic force microscope.

PubMed

Zhang, Xiaojuan; Yao, Zhixuan; Duan, Yanting; Zhang, Xiaomei; Shi, Jinsong; Xu, Zhenghong

2018-01-11

The specific recognition and binding of promoter and RNA polymerase is the first step of transcription initiation in bacteria and largely determines transcription activity. Therefore, direct analysis of the interaction between promoter and RNA polymerase in vitro may be a new strategy for promoter characterization, to avoid interference due to the cell's biophysical condition and other regulatory elements. In the present study, the specific interaction between T7 promoter and T7 RNA polymerase was studied as a model system using force spectroscopy based on atomic force microscope (AFM). The specific interaction between T7 promoter and T7 RNA polymerase was verified by control experiments, and the rupture force in this system was measured as 307.2 ± 6.7 pN. The binding between T7 promoter mutants with various promoter activities and T7 RNA polymerase was analyzed. Interaction information including rupture force, rupture distance and binding percentage were obtained in vitro , and reporter gene expression regulated by these promoters was also measured according to a traditional promoter activity characterization method in vivo Using correlation analysis, it was found that the promoter strength characterized by reporter gene expression was closely correlated with rupture force and the binding percentage by force spectroscopy. These results indicated that the analysis of the interaction between promoter and RNA polymerase using AFM-based force spectroscopy was an effective and valid approach for the quantitative characterization of promoters. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Nanostructure and force spectroscopy analysis of human peripheral blood CD4{sup +} T cells using atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu Mingqian; Wang Jiongkun; Cai Jiye

2008-09-12

To date, nanoscale imaging of the morphological changes and adhesion force of CD4{sup +} T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4{sup +} T cells. The AFM images revealed that the volume of activated CD4{sup +} T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times thatmore » of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4{sup +} T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.« less

Measuring the Adhesion Forces for the Multivalent Binding of Vancomycin-Conjugated Dendrimer to Bacterial Cell-Wall Peptide.

PubMed

Peterson, Elizabeth; Joseph, Christine; Peterson, Hannah; Bouwman, Rachael; Tang, Shengzhuang; Cannon, Jayme; Sinniah, Kumar; Choi, Seok Ki

2018-06-19

Multivalent ligand-receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5(Van) n ( n = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5(Van) 4 bound most tightly with a K D of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5(Van) 1 or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5(Van) n and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at n = 4 greater than 96 pN at n = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates.

Quantifying effects of cyclic stretch on cell-collagen substrate adhesiveness of vascular endothelial cells.

PubMed

Omidvar, Ramin; Tafazzoli-Shadpour, Mohammad; Mahmoodi-Nobar, Farbod; Azadi, Shohreh; Khani, Mohammad-Mehdi

2018-05-01

Vascular endothelium is continuously subjected to mechanical stimulation in the form of shear forces due to blood flow as well as tensile forces as a consequence of blood pressure. Such stimuli influence endothelial behavior and regulate cell-tissue interaction for an optimized functionality. This study aimed to quantify influence of cyclic stretch on the adhesive property and stiffness of endothelial cells. The 10% cyclic stretch with frequency of 1 Hz was applied to a layer of endothelial cells cultured on a polydimethylsiloxane substrate. Cell-substrate adhesion of endothelial cells was examined by the novel approach of atomic force microscope-based single-cell force spectroscopy and cell stiffness was measured by atomic force microscopy. Furthermore, the adhesive molecular bonds were evaluated using modified Hertz contact theory. Our results show that overall adhesion of endothelial cells with substrate decreased after cyclic stretch while they became stiffer. Based on the experimental results and theoretical modeling, the decrease in the number of molecular bonds after cyclic stretch was quantified. In conclusion, in vitro cyclic stretch caused alterations in both adhesive capacity and elastic modulus of endothelial cells through mechanotransductive pathways as two major determinants of the function of these cells within the cardiovascular system.

Towards force spectroscopy of single tip-link bonds

NASA Astrophysics Data System (ADS)

Koussa, Mounir A.; Sotomayor, Marcos; Wong, Wesley P.; Corey, David P.

2015-12-01

Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. Molecular dynamics simulations predict that the tip-link bond is mechanically strong enough to withstand forces in hair cells, but its experimentally determined strength is unknown. We have developed molecular tools to facilitate single-molecule force spectroscopy on the tip link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip link nanoswitches are designed to provide a signature force-extension profile. This molecular signature should allow us to identify single-molecule rupture events in pulling experiments.

Atomic Force Microscopy Measurements of the Mechanical Properties of Cell Walls on Living Bacterial Cells

NASA Astrophysics Data System (ADS)

Bailey, Richard; Mullin, Nic; Turner, Robert; Foster, Simon; Hobbs, Jamie

2014-03-01

Staphylococcus aureus is a major cause of infection in humans, including the Methicillin resistant strain, MRSA. However, very little is known about the mechanical properties of these cells. Our investigations use AFM to examine live S. aureus cells to quantify mechanical properties. These were explored using force spectroscopy with different trigger forces, allowing the properties to be extracted at different indentation depths. A value for the cell wall stiffness has been extracted, along with a second, higher value which is found upon indenting at higher forces. This higher value drops as the cells are exposed to high salt, sugar and detergent concentrations, implying that this measurement contains a contribution from the internal turgor pressure. We have monitored these properties as the cells progress through the cell cycle. Force maps were taken over the cells at different stages of the growth process to identify changes in the mechanics throughout the progression of growth and division. The effect of Oxacillin has also been studied, to better understand its mechanism of action. Finally mutant strains of S. aureus and a second species Bacillus subtilis have been used to link the mechanical properties of the cell walls with the chain lengths and substructures involved.

Electrorotation and levitation of cells and colloidal particles

PubMed Central

Foster, Kenneth R.; Sauer, Friedrich A.; Schwan, Herman P.

1992-01-01

We review dielectrophoretic forces on cells and colloidal particles, emphasizing their use for manipulating and characterizing the electrical properties of suspended particles. Compared with dielectric spectroscopy, these methods offer a measure of independence from electrode artifacts and mixture theory. On the assumption that the particles can be modeled as uniform dielectric objects with effective dielectric properties, a simple theory can be developed for the frequency variation in the field-induced forces. For particles exhibiting counterion polarization, dielectrophoretic forces differ considerably from predictions of this theory at low frequencies, apparently because of double layer phenomena. PMID:19431839

Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Valle-Delgado, Juan José; Urbán, Patricia; Fernàndez-Busquets, Xavier

2013-04-01

Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level.Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32821f

Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells.

PubMed

Pi, Jiang; Jin, Hua; Jiang, Jinhuan; Yang, Fen; Cai, Huaihong; Yang, Peihui; Cai, Jiye; Chen, Zheng W

2017-05-01

As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM-SMFS) was used for real-time and in-situ detection of EGF-EGFR interactions in living esophageal cancer KYSE-150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE-150 cells. AFM-SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE-150 cells by decreasing the unbinding force and binding probability for EGF-EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM-SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF-EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF-EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF-EGFR interactions in ROS dependent mechanism, but also highlighted AFM-SMFS as a powerful technique for pharmacodynamic studies by detecting ligand-receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single-molecule force spectroscopy study of interactions between angiotensin II type 1 receptor and different biased ligands in living cells.

PubMed

Li, Wenhui; Xu, Jiachao; Kou, Xiaolong; Zhao, Rong; Zhou, Wei; Fang, Xiaohong

2018-05-01

Angiotensin II type 1 receptor (AT1R), a typical G protein-coupled receptor, plays a key role in regulating many cardiovascular functions. Different ligands can bind with AT1R to selectively activate either G protein (Gq) or β-arrestin (β-arr) pathway, or both pathways, but the molecular mechanism is not clear yet. In this work, we used, for the first time, atomic force microscopy-based single molecule force spectroscopy (SMFS) to study the interactions of AT1R with three types of ligands, balanced ligand, Gq-biased ligand, and β-arr-biased ligand, in living cells. The results revealed their difference in binding force and binding stability. The complex of the Gq-biased ligand-AT1R overcame two energy barriers with an intermediate state during dissociation, whereas that of β-arr-biased ligand-AT1R complex overcame one energy barrier. This indicated that AT1R had different ligand-binding conformational substates and underwent different structural changes to activate downstream signaling pathways with variable agonist efficacies. Quantitative analysis of AT1R-ligand binding in living cells at the single-molecule level offers a new tool to study the molecular mechanism of AT1R biased activation. Graphical Abstract Single-molecule force measurement on the living cell expressing AT1R-eGFP with a ligand modified AFM tip (left), the dynamic force spectra of β-arrestin biased ligands-AT1R (middle), and Gq-biased ligands-AT1R (right). The complexes of β-arr-biased ligand-AT1R overcame one energy barrier, with one linear region in the spectra, whereas the Gq-biased ligand-AT1R complexes overcame two energy barriers with two linear regions.

Mammalian complex I pumps 4 protons per 2 electrons at high and physiological proton motive force in living cells.

PubMed

Ripple, Maureen O; Kim, Namjoon; Springett, Roger

2013-02-22

Mitochondrial complex I couples electron transfer between matrix NADH and inner-membrane ubiquinone to the pumping of protons against a proton motive force. The accepted proton pumping stoichiometry was 4 protons per 2 electrons transferred (4H(+)/2e(-)) but it has been suggested that stoichiometry may be 3H(+)/2e(-) based on the identification of only 3 proton pumping units in the crystal structure and a revision of the previous experimental data. Measurement of proton pumping stoichiometry is challenging because, even in isolated mitochondria, it is difficult to measure the proton motive force while simultaneously measuring the redox potentials of the NADH/NAD(+) and ubiquinol/ubiquinone pools. Here we employ a new method to quantify the proton motive force in living cells from the redox poise of the bc(1) complex measured using multiwavelength cell spectroscopy and show that the correct stoichiometry for complex I is 4H(+)/2e(-) in mouse and human cells at high and physiological proton motive force.

Taking nanomedicine teaching into practice with atomic force microscopy and force spectroscopy.

PubMed

Carvalho, Filomena A; Freitas, Teresa; Santos, Nuno C

2015-12-01

Atomic force microscopy (AFM) is a useful and powerful tool to study molecular interactions applied to nanomedicine. The aim of the present study was to implement a hands-on atomic AFM course for graduated biosciences and medical students. The course comprises two distinct practical sessions, where students get in touch with the use of an atomic force microscope by performing AFM scanning images of human blood cells and force spectroscopy measurements of the fibrinogen-platelet interaction. Since the beginning of this course, in 2008, the overall rating by the students was 4.7 (out of 5), meaning a good to excellent evaluation. Students were very enthusiastic and produced high-quality AFM images and force spectroscopy data. The implementation of the hands-on AFM course was a success, giving to the students the opportunity of contact with a technique that has a wide variety of applications on the nanomedicine field. In the near future, nanomedicine will have remarkable implications in medicine regarding the definition, diagnosis, and treatment of different diseases. AFM enables students to observe single molecule interactions, enabling the understanding of molecular mechanisms of different physiological and pathological processes at the nanoscale level. Therefore, the introduction of nanomedicine courses in bioscience and medical school curricula is essential. Copyright © 2015 The American Physiological Society.

Digital force-feedback for protein unfolding experiments using atomic force microscopy

NASA Astrophysics Data System (ADS)

Bippes, Christian A.; Janovjak, Harald; Kedrov, Alexej; Muller, Daniel J.

2007-01-01

Since its invention in the 1990s single-molecule force spectroscopy has been increasingly applied to study protein (un-)folding, cell adhesion, and ligand-receptor interactions. In most force spectroscopy studies, the cantilever of an atomic force microscope (AFM) is separated from a surface at a constant velocity, thus applying an increasing force to folded bio-molecules or bio-molecular bonds. Recently, Fernandez and co-workers introduced the so-called force-clamp technique. Single proteins were subjected to a defined constant force allowing their life times and life time distributions to be directly measured. Up to now, the force-clamping was performed by analogue PID controllers, which require complex additional hardware and might make it difficult to combine the force-feedback with other modes such as constant velocity. These points may be limiting the applicability and versatility of this technique. Here we present a simple, fast, and all-digital (software-based) PID controller that yields response times of a few milliseconds in combination with a commercial AFM. We demonstrate the performance of our feedback loop by force-clamp unfolding of single Ig27 domains of titin and the membrane proteins bacteriorhodopsin (BR) and the sodium/proton antiporter NhaA.

Ultrafast single molecule technique for the study of force dependent kinetics and conformational changes of actin-protein interaction involved in mechanotransduction

NASA Astrophysics Data System (ADS)

Sergides, M.; Arbore, C.; Pavone, F. S.; Capitanio, M.

2018-02-01

Mechanical signals occurring at the interface between cell membrane and extracellular matrix and at intercellular junctions trigger biochemical signals that are fundamental for cell growth, development and regulation. Adaptor proteins, which link the cell membrane to the actin cytoskeleton, seem to partake in this process of mechanotransduction. In particular, catenins play a key role in intercellular junctions, where they act as a bridge between the cell membrane and actin. Studies suggest that α-catenin contains a domain that normally masks vinculin binding sites, which can become accessible after a conformational change induced by an external force. Here we demonstrate a single-molecule technique for investigating actin-protein interactions at different forces (up to 17 pN) with adequate temporal resolution (sub-ms). This system is based on the ultrafast force-clamp spectroscopy technique that has been recently developed by our group and is adapted to study and measure force-dependent kinetics of the catenin-actin interaction, as well as the amplitude of the expected conformational changes such as force-induced protein unfolding.

Early Adhesion of Candida albicans onto Dental Acrylic Surfaces.

PubMed

Aguayo, S; Marshall, H; Pratten, J; Bradshaw, D; Brown, J S; Porter, S R; Spratt, D; Bozec, L

2017-07-01

Denture-associated stomatitis is a common candidal infection that may give rise to painful oral symptoms, as well as be a reservoir for infection at other sites of the body. As poly (methyl methacrylate) (PMMA) remains the main material employed in the fabrication of dentures, the aim of this research was to evaluate the adhesion of Candida albicans cells onto PMMA surfaces by employing an atomic force microscopy (AFM) single-cell force spectroscopy (SCFS) technique. For experiments, tipless AFM cantilevers were functionalized with PMMA microspheres and probed against C. albicans cells immobilized onto biopolymer-coated substrates. Both a laboratory strain and a clinical isolate of C. albicans were used for SCFS experiments. Scanning electron microscopy (SEM) and AFM imaging of C. albicans confirmed the polymorphic behavior of both strains, which was dependent on growth culture conditions. AFM force-spectroscopy results showed that the adhesion of C. albicans to PMMA is morphology dependent, as hyphal tubes had increased adhesion compared with yeast cells ( P < 0.05). C. albicans budding mother cells were found to be nonadherent, which contrasts with the increased adhesion observed in the tube region. Comparison between strains demonstrated increased adhesion forces for a clinical isolate compared with the lab strain. The clinical isolate also had increased survival in blood and reduced sensitivity to complement opsonization, providing additional evidence of strain-dependent differences in Candida-host interactions that may affect virulence. In conclusion, PMMA-modified AFM probes have shown to be a reliable technique to characterize the adhesion of C. albicans to acrylic surfaces.

Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods

PubMed Central

Cartagena, Alexander; Raman, Arvind

2014-01-01

The measurement of viscoelasticity of cells in physiological environments with high spatio-temporal resolution is a key goal in cell mechanobiology. Traditionally only the elastic properties have been measured from quasi-static force-distance curves using the atomic force microscope (AFM). Recently, dynamic AFM-based methods have been proposed to map the local in vitro viscoelastic properties of living cells with nanoscale resolution. However, the differences in viscoelastic properties estimated from such dynamic and traditional quasi-static techniques are poorly understood. In this work we quantitatively reconstruct the local force and dissipation gradients (viscoelasticity) on live fibroblast cells in buffer solutions using Lorentz force excited cantilevers and present a careful comparison between mechanical properties (local stiffness and damping) extracted using dynamic and quasi-static force spectroscopy methods. The results highlight the dependence of measured viscoelastic properties on both the frequency at which the chosen technique operates as well as the interactions with subcellular components beyond certain indentation depth, both of which are responsible for differences between the viscoelasticity property maps acquired using the dynamic AFM method against the quasi-static measurements. PMID:24606928

Spectroscopy and atomic force microscopy of biomass.

PubMed

Tetard, L; Passian, A; Farahi, R H; Kalluri, U C; Davison, B H; Thundat, T

2010-05-01

Scanning probe microscopy has emerged as a powerful approach to a broader understanding of the molecular architecture of cell walls, which may shed light on the challenge of efficient cellulosic ethanol production. We have obtained preliminary images of both Populus and switchgrass samples using atomic force microscopy (AFM). The results show distinctive features that are shared by switchgrass and Populus. These features may be attributable to the lignocellulosic cell wall composition, as the collected images exhibit the characteristic macromolecular globule structures attributable to the lignocellulosic systems. Using both AFM and a single case of mode synthesizing atomic force microscopy (MSAFM) to characterize Populus, we obtained images that clearly show the cell wall structure. The results are of importance in providing a better understanding of the characteristic features of both mature cells as well as developing plant cells. In addition, we present spectroscopic investigation of the same samples.

Dynamics of diamond nanoparticles in solution and cells.

PubMed

Neugart, Felix; Zappe, Andrea; Jelezko, Fedor; Tietz, C; Boudou, Jean Paul; Krueger, Anke; Wrachtrup, Jörg

2007-12-01

The fluorescence and motional dynamics of single diamond nanocrystals in buffer solution and in living cells is investigated. Stable hydrosols of nanodiamonds in buffer solutions are investigated by fluorescence correlation spectroscopy. Measurement of the effective hydrodynamic radius yields particles of 48 nm diameter, which is in excellent agreement with atomic force microscopy measurements made on the same particles. Fluorescence correlation spectroscopy measurements indicate that nanocrystals easily form aggregates when the buffer pH is changed. This tendency is reduced when the surface of the diamonds is covered with surfactants. Upon incubation, cells spontaneously take up nanocrystals that uniformly distribute in cells. Most of the particles get immobilized within a few minutes. The binding of streptavidin to biotinylated aggregates of 4 nm diameter nanodiamonds is demonstrated.

Cytocompatibility of polyethylene grafted with triethylenetetramine functionalized carbon nanoparticles

NASA Astrophysics Data System (ADS)

Žáková, Pavlína; Slepičková Kasálková, Nikola; Slepička, Petr; Kolská, Zdeňka; Karpíšková, Jana; Stibor, Ivan; Švorčík, Václav

2017-11-01

Various carbon nanostructures are widely researched as scaffolds for tissue engineering. We evaluated the surface properties and cell-substrate interactions of carbon nanoparticles functionalized with triethylenetetramine (CNPs) grafted polymer film. Two forms of polyethylene (HDPE, LDPE) were treated in an inert argon plasma discharge and, subsequently, grafted with CNPs. The surface properties were studied using multiple methods, including Raman spectroscopy, goniometry, atomic force microscopy, X-ray photoelectron spectroscopy and electrokinetic analysis. Cell-substrate interactions were determined in vitro by studying adhesion, proliferation and viability of vascular smooth muscle cells (VSMCs) from the aorta of a rat. Cell-substrate interactions on pristine and modified substrates were compared to standard tissue culture polystyrene. Our results show that CNPs affect surface morphology and wettability and therefore adhesion, proliferation and viability of cultured muscle cells.

Mammalian Complex I Pumps 4 Protons per 2 Electrons at High and Physiological Proton Motive Force in Living Cells*

PubMed Central

Ripple, Maureen O.; Kim, Namjoon; Springett, Roger

2013-01-01

Mitochondrial complex I couples electron transfer between matrix NADH and inner-membrane ubiquinone to the pumping of protons against a proton motive force. The accepted proton pumping stoichiometry was 4 protons per 2 electrons transferred (4H+/2e−) but it has been suggested that stoichiometry may be 3H+/2e− based on the identification of only 3 proton pumping units in the crystal structure and a revision of the previous experimental data. Measurement of proton pumping stoichiometry is challenging because, even in isolated mitochondria, it is difficult to measure the proton motive force while simultaneously measuring the redox potentials of the NADH/NAD+ and ubiquinol/ubiquinone pools. Here we employ a new method to quantify the proton motive force in living cells from the redox poise of the bc1 complex measured using multiwavelength cell spectroscopy and show that the correct stoichiometry for complex I is 4H+/2e− in mouse and human cells at high and physiological proton motive force. PMID:23306206

Solid-phase synthesis of graphene quantum dots from the food additive citric acid under microwave irradiation and their use in live-cell imaging.

PubMed

Zhuang, Qianfen; Wang, Yong; Ni, Yongnian

2016-05-01

The work demonstrated that solid citric acid, one of the most common food additives, can be converted to graphene quantum dots (GQDs) under microwave heating. The as-prepared GQDs were further characterized by various analytical techniques like transmission electron microscopy, atomic force microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, fluorescence and UV-visible spectroscopy. Cytotoxicity of the GQDs was evaluated using HeLa cells. The result showed that the GQDs almost did not exhibit cytotoxicity at concentrations as high as 1000 µg mL(-1). In addition, it was found that the GQDs showed good solubility, excellent photostability, and excitation-dependent multicolor photoluminescence. Subsequently, the multicolor GQDs were successfully used as a fluorescence light-up probe for live-cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.

Resolving the molecular mechanism of cadherin catch bond formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manibog, Kristine; Li, Hui; Rakshit, Sabyasachi

2014-06-02

Classical cadherin Ca(2+)-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical force. Here we use single-molecule force-clamp spectroscopy with an atomic force microscope along with molecular dynamics and steered molecular dynamics simulations to resolve the molecular mechanisms underlying catch bond formation and the role of Ca(2+) ions in this process. Our data suggest that tensile force bends the cadherin extracellular region such that theymore » form long-lived, force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca(2+) concentration is decreased, fewer de novo hydrogen bonds are formed and catch bond formation is eliminated« less

Covalent conjugation of graphene oxide with methotrexate and its antitumor activity

NASA Astrophysics Data System (ADS)

Wojtoniszak, M.; Urbas, K.; Perużyńska, M.; Kurzawski, M.; Droździk, M.; Mijowska, E.

2013-05-01

Here, we have functionalized graphene oxide with anticancer drug methotrexate through amide bonding. A kinetics of the drug release from graphene oxide in physiological solution - phosphate buffered saline (PBS) containing different biocompatible polymers have been investigated. Dispersion of MTX-GO in poly sodium-4-styrene sulfonate and poly ethylene glycol resulted in increase of the release time. The material was characterized with transmission electron microscopy, atomic force microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and UV-vis spectroscopy. Furthermore, antineoplastic action against human breast adenocarcinoma cell line MCF7 of MTX-GO and empty graphene oxide was explored.

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

PubMed Central

Neuman, Keir C.; Nagy, Attila

2012-01-01

Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. These techniques are described and illustrated with examples highlighting current capabilities and limitations. PMID:18511917

An in vitro evaluation of graphene oxide reduced by Ganoderma spp. in human breast cancer cells (MDA-MB-231).

PubMed

Gurunathan, Sangiliyandi; Han, Jaewoong; Park, Jung Hyun; Kim, Jin Hoi

2014-01-01

Recently, graphene and graphene-related materials have attracted much attention due their unique properties, such as their physical, chemical, and biocompatibility properties. This study aimed to determine the cytotoxic effects of graphene oxide (GO) that is reduced biologically using Ganoderma spp. mushroom extracts in MDA-MB-231 human breast cancer cells. Herein, we describe a facile and green method for the reduction of GO using extracts of Ganoderma spp. as a reducing agent. GO was reduced without any hazardous chemicals in an aqueous solution, and the reduced GO was characterized using a range of analytical procedures. The Ganoderma extract (GE)-reduced GO (GE-rGO) was characterized by ultraviolet-visible absorption spectroscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, dynamic light scattering, scanning electron microscopy, Raman spectroscopy, and atomic force microscopy. Furthermore, the toxicity of GE-rGO was evaluated using a sequence of assays such as cell viability, lactate dehydrogenase leakage, and reactive oxygen species generation in human breast cancer cells (MDA-MB-231). The preliminary characterization of reduction of GO was confirmed by the red-shifting of the absorption peak for GE-rGO to 265 nm from 230 nm. The size of GO and GE-rGO was found to be 1,880 and 3,200 nm, respectively. X-ray diffraction results confirmed that reduction processes of GO and the processes of removing intercalated water molecules and the oxide groups. The surface functionalities and chemical natures of GO and GE-rGO were confirmed using Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. The surface morphologies of the synthesized graphene were analyzed using high-resolution scanning electron microscopy. Raman spectroscopy revealed single- and multilayer properties of GE-rGO. Atomic force microscopy images provided evidence for the formation of graphene. Furthermore, the effect of GO and GE-rGO was examined using a series of assays, such as cell viability, membrane integrity, and reactive oxygen species generation, which are key molecules involved in apoptosis. The results obtained from cell viability and lactate dehydrogenase assay suggest that GO and GE-rGO cause dose-dependent toxicity in the cells. Interestingly, it was found that biologically derived GE-rGO is more toxic to cancer cells than GO. We describe a simple, green, nontoxic, and cost-effective approach to producing graphene using mushroom extract as a reducing and stabilizing agent. The proposed method could enable synthesis of graphene with potential biological and biomedical applications such as in cancer and angiogenic disorders. To our knowledge, this is the first report using mushroom extract as a reducing agent for the synthesis of graphene. Mushroom extract can be used as a biocatalyst for the production of graphene.

Hydroxyethylated graphene oxide as potential carriers for methotrexate delivery

NASA Astrophysics Data System (ADS)

Du, Libo; Suo, Siqingaowa; Luo, Dan; Jia, Hongying; Sha, Yinlin; Liu, Yang

2013-06-01

In this study, we presented a simple approach to prepare hydroxyethylated graphene oxide (HE-GO) with high water solubility and physiological stability. The successful synthesis of HE-GO was confirmed by UV-Vis spectroscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The loading of anticancer drug methotrexate (MTX) onto this nanocarrier (MTX/HE-GO) was investigated. The results of in vitro drug release experiment showed that the rate of MTX release from MTX/HE-GO was pH dependent. Moreover, cell viability assay demonstrated that HE-GO loaded with MTX exhibits higher anticancer activity against human lung adenocarcinoma epithelial cell line than non-vehicle MTX.

Atomic force microscopy and spectroscopy to probe single membrane proteins in lipid bilayers.

PubMed

Sapra, K Tanuj

2013-01-01

The atomic force microscope (AFM) has opened vast avenues hitherto inaccessible to the biological scientist. The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever stylus is aptly termed as a "lab on a tip" owing to its versatility as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples assert that the AFM can be used to study the mechanical properties and monitor processes of single proteins and single cells, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of membrane proteins. Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theoretical, and practical skills.

High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization

NASA Astrophysics Data System (ADS)

Andolfi, Laura; Murello, Anna; Cassese, Damiano; Ban, Jelena; Dal Zilio, Simone; Lazzarino, Marco

2017-04-01

Cell-cell and cell-matrix interactions are essential to the survival and proliferation of most cells, and are responsible for triggering a wide range of biochemical pathways. More recently, the biomechanical role of those interactions was highlighted, showing, for instance, that adhesion forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small size, high aspect ratio and anisotropic mechanical response represent a useful model to investigate the forces involved in the adhesion processes and their role in cellular development. In this work we explored and quantified, by single cell force spectroscopy (SCFS), the interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that the cell adhesion forces are comparable to those found on collagen and bare glass coverslip, analogously the membrane tether extraction forces are similar to that on collagen but stronger than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a reduced proliferation after 36 h was observed. On the contrary both cell morphology and cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was characterized by a large number of filopodia and a significant decrease of the cell mobility. The cytoskeleton organization was characterized by the absence of actin fibers, which were instead dominant on collagen and flat glass support. Such findings suggest that the mechanical properties of disordered Si NWs, and in particular their strong asymmetry, play a major role in the adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion measurements by SCFS provide out-of-plane forces values consistent with those measured on conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and migration processes.

High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization.

PubMed

Andolfi, Laura; Murello, Anna; Cassese, Damiano; Ban, Jelena; Dal Zilio, Simone; Lazzarino, Marco

2017-04-18

Cell-cell and cell-matrix interactions are essential to the survival and proliferation of most cells, and are responsible for triggering a wide range of biochemical pathways. More recently, the biomechanical role of those interactions was highlighted, showing, for instance, that adhesion forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small size, high aspect ratio and anisotropic mechanical response represent a useful model to investigate the forces involved in the adhesion processes and their role in cellular development. In this work we explored and quantified, by single cell force spectroscopy (SCFS), the interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that the cell adhesion forces are comparable to those found on collagen and bare glass coverslip, analogously the membrane tether extraction forces are similar to that on collagen but stronger than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a reduced proliferation after 36 h was observed. On the contrary both cell morphology and cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was characterized by a large number of filopodia and a significant decrease of the cell mobility. The cytoskeleton organization was characterized by the absence of actin fibers, which were instead dominant on collagen and flat glass support. Such findings suggest that the mechanical properties of disordered Si NWs, and in particular their strong asymmetry, play a major role in the adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion measurements by SCFS provide out-of-plane forces values consistent with those measured on conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and migration processes.

Discrimination of bladder cancer cells from normal urothelial cells with high specificity and sensitivity: combined application of atomic force microscopy and modulated Raman spectroscopy.

PubMed

Canetta, Elisabetta; Riches, Andrew; Borger, Eva; Herrington, Simon; Dholakia, Kishan; Adya, Ashok K

2014-05-01

Atomic force microscopy (AFM) and modulated Raman spectroscopy (MRS) were used to discriminate between living normal human urothelial cells (SV-HUC-1) and bladder tumour cells (MGH-U1) with high specificity and sensitivity. MGH-U1 cells were 1.5-fold smaller, 1.7-fold thicker and 1.4-fold rougher than normal SV-HUC-1 cells. The adhesion energy was 2.6-fold higher in the MGH-U1 cells compared to normal SV-HUC-1 cells, which possibly indicates that bladder tumour cells are more deformable than normal cells. The elastic modulus of MGH-U1 cells was 12-fold lower than SV-HUC-1 cells, suggesting a higher elasticity of the bladder cancer cell membranes. The biochemical fingerprints of cancer cells displayed a higher DNA and lipid content, probably due to an increase in the nuclear to cytoplasm ratio. Normal cells were characterized by higher protein contents. AFM studies revealed a decrease in the lateral dimensions and an increase in thickness of cancer cells compared to normal cells; these studies authenticate the observations from MRS. Nanostructural, nanomechanical and biochemical profiles of bladder cells provide qualitative and quantitative markers to differentiate between normal and cancerous cells at the single cellular level. AFM and MRS allow discrimination between adhesion energy, elasticity and Raman spectra of SV-HUC-1 and MGH-U1 cells with high specificity (83, 98 and 95%) and sensitivity (97, 93 and 98%). Such single-cell-level studies could have a pivotal impact on the development of AFM-Raman combined methodologies for cancer profiling and screening with translational significance. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Near-field photothermal microspectroscopy for adult stem-cell identification and characterization.

PubMed

Grude, Olaug; Hammiche, Azzedine; Pollock, Hubert; Bentley, Adam J; Walsh, Michael J; Martin, Francis L; Fullwood, Nigel J

2007-12-01

The identification of stem cells in adult tissue is a challenging problem in biomedicine. Currently, stem cells are identified by individual epitopes, which are generally tissue specific. The discovery of a stem-cell marker common to other adult tissue types could open avenues in the development of therapeutic stem-cell strategies. We report the use of the novel technique of Fourier transform infrared near-field photothermal microspectroscopy (FTIR-PTMS) for the characterization of stem cells, transit amplifying (TA) cells and terminally differentiated (TD) cells in the corneal epithelium. Principal component analysis (PCA) data demonstrate excellent discrimination of cell type by spectra. PCA in combination with linear discriminant analysis (PCA-LDA) shows that FTIR-PTMS very effectively discriminates between the three cell populations. Statistically significant differences above the 99% confidence level between IR spectra from stem cells and TA cells suggest that nucleic acid conformational changes are an important component of the differences between spectral data from the two cell types. FTIR-PTMS is a new addition to existing spectroscopy methods based on the concept of interfacing a conventional FTIR spectrometer with an atomic force microscope equipped with a near-field thermal sensing probe. FTIR-PTMS spectroscopy currently has spatial resolution that is similar to that of diffraction-limited optical detection FTIR spectroscopy techniques, but as a near-field probing technique has considerable potential for further improvement. Our work also suggests that FTIR-PTMS is potentially more sensitive than synchrotron radiation FTIR spectroscopy for some applications. Microspectroscopy techniques like FTIR-PTMS provide information about the entire molecular composition of cells, in contrast to epitope recognition that only considers the presence or absence of individual molecules. Our results with FTIR-PTMS on corneal stem cells are promising for the potential development of an IR spectral fingerprint for stem cells.

Hands-on Force Spectroscopy: Weird Springs and Protein Folding

ERIC Educational Resources Information Center

Euler, Manfred

2008-01-01

A force spectroscopy model experiment is presented using a low-cost tensile apparatus described earlier. Force-extension measurements of twisted rubber bands are obtained. They exhibit a complex nonlinear elastic behaviour that resembles atomic force spectroscopy investigations of molecules of titin, a muscle protein. The model experiments open up…

Energy level alignment in TiO2/metal sulfide/polymer interfaces for solar cell applications.

PubMed

Lindblad, Rebecka; Cappel, Ute B; O'Mahony, Flannan T F; Siegbahn, Hans; Johansson, Erik M J; Haque, Saif A; Rensmo, Håkan

2014-08-28

Semiconductor sensitized solar cell interfaces have been studied with photoelectron spectroscopy to understand the interfacial electronic structures. In particular, the experimental energy level alignment has been determined for complete TiO2/metal sulfide/polymer interfaces. For the metal sulfides CdS, Sb2S3 and Bi2S3 deposited from single source metal xanthate precursors, it was shown that both driving forces for electron injection into TiO2 and hole transfer to the polymer decrease for narrower bandgaps. The energy level alignment results were used in the discussion of the function of solar cells with the same metal sulfides as light absorbers. For example Sb2S3 showed the most favourable energy level alignment with 0.3 eV driving force for electron injection and 0.4 eV driving force for hole transfer and also the most efficient solar cells due to high photocurrent generation. The energy level alignment of the TiO2/Bi2S3 interface on the other hand showed no driving force for electron injection to TiO2, and the performance of the corresponding solar cell was very low.

Quantifying Young's moduli of protein fibrils and particles with bimodal force spectroscopy.

PubMed

Gilbert, Jay; Charnley, Mirren; Cheng, Christopher; Reynolds, Nicholas P; Jones, Owen G

2017-10-19

Force spectroscopy is a means of obtaining mechanical information of individual nanometer-scale structures in composite materials, such as protein assemblies for use in consumer films or gels. As a recently developed force spectroscopy technique, bimodal force spectroscopy relates frequency shifts in cantilevers simultaneously excited at multiple frequencies to the elastic properties of the contacted material, yet its utility for quantitative characterization of biopolymer assemblies has been limited. In this study, a linear correlation between experimental frequency shift and Young's modulus of polymer films was used to calibrate bimodal force spectroscopy and quantify Young's modulus of two protein nanostructures: β-lactoglobulin fibrils and zein nanoparticles. Cross-sectional Young's modulus of protein fibrils was determined to be 1.6 GPa while the modulus of zein nanoparticles was determined as 854 MPa. Parallel measurement of β-lactoglobulin fibril by a competing pulsed-force technique found a higher cross-sectional Young's modulus, highlighting the importance of comparative calibration against known standards in both pulsed and bimodal force spectroscopies. These findings demonstrate a successful procedure for measuring mechanical properties of individual protein assemblies with potential use in biological or packaging applications using bimodal force spectroscopy.

Yeast-assisted synthesis of polypyrrole: Quantification and influence on the mechanical properties of the cell wall.

PubMed

Andriukonis, Eivydas; Stirke, Arunas; Garbaras, Andrius; Mikoliunaite, Lina; Ramanaviciene, Almira; Remeikis, Vidmantas; Thornton, Barry; Ramanavicius, Arunas

2018-04-01

In this study, the metabolism of yeast cells (Saccharomyces cerevisiae) was utilized for the synthesis of the conducting polymer - polypyrrole (Ppy).Yeast cells were modified in situ by synthesized Ppy. The Ppy was formed in the cell wall by redox-cycling of [Fe(CN) 6 ] 3-/4- , performed by the yeast cells. Fluorescence microscopy, enzymatic digestions, atomic force microscopy and isotope ratio mass spectroscopy were applied to determine both the polymerization reaction itself and the polymer location in yeast cells. Ppy formation resulted in enhanced resistance to lytic enzymes, significant increase of elasticity and alteration of other mechanical cell wall properties evaluated by atomic force microscopy (AFM). The suggested method of polymer synthesis allows the introduction of polypyrrole structures within the cell wall, which is build up from polymers consisting of carbohydrates. This cell wall modification strategy could increase the usefulness of yeast as an alternative energy source in biofuel cells, and in cell based biosensors. Copyright © 2018 Elsevier B.V. All rights reserved.

A targeted drug delivery system based on dopamine functionalized nano graphene oxide

NASA Astrophysics Data System (ADS)

Masoudipour, Elham; Kashanian, Soheila; Maleki, Nasim

2017-01-01

The cellular targeting property of a biocompatible drug delivery system can widely increase the therapeutic effect against various diseases. Here, we report a dopamine conjugated nano graphene oxide (DA-nGO) carrier for cellular delivery of the anticancer drug, Methotrexate (MTX) into DA receptor positive human breast adenocarcinoma cell line. The material was characterized using scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy and UV-vis spectroscopy. Furthermore, the antineoplastic action of MTX loaded DA-nGO against DA receptor positive and negative cell lines were explored. The results presented in this article demonstrated that the application of DA functionalized GO as a targeting drug carrier can improve the drug delivery efficacy for DA receptor positive cancer cell lines and promise future designing of carrier conjugates based on it.

[Atomic force microscopy: a tool to analyze the viral cycle].

PubMed

Bernaud, Julien; Castelnovo, Martin; Muriaux, Delphine; Faivre-Moskalenko, Cendrine

2015-05-01

Each step of the HIV-1 life cycle frequently involves a change in the morphology and/or mechanical properties of the viral particle or core. The atomic force microscope (AFM) constitutes a powerful tool for characterizing these physical changes at the scale of a single virus. Indeed, AFM enables the visualization of viral capsids in a controlled physiological environment and to probe their mechanical properties by nano-indentation. Finally, AFM force spectroscopy allows to characterize the affinities between viral envelope proteins and cell receptors at the single molecule level. © 2015 médecine/sciences – Inserm.

An in vitro evaluation of graphene oxide reduced by Ganoderma spp. in human breast cancer cells (MDA-MB-231)

PubMed Central

Gurunathan, Sangiliyandi; Han, JaeWoong; Park, Jung Hyun; Kim, Jin Hoi

2014-01-01

Background Recently, graphene and graphene-related materials have attracted much attention due their unique properties, such as their physical, chemical, and biocompatibility properties. This study aimed to determine the cytotoxic effects of graphene oxide (GO) that is reduced biologically using Ganoderma spp. mushroom extracts in MDA-MB-231 human breast cancer cells. Methods Herein, we describe a facile and green method for the reduction of GO using extracts of Ganoderma spp. as a reducing agent. GO was reduced without any hazardous chemicals in an aqueous solution, and the reduced GO was characterized using a range of analytical procedures. The Ganoderma extract (GE)-reduced GO (GE-rGO) was characterized by ultraviolet-visible absorption spectroscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, dynamic light scattering, scanning electron microscopy, Raman spectroscopy, and atomic force microscopy. Furthermore, the toxicity of GE-rGO was evaluated using a sequence of assays such as cell viability, lactate dehydrogenase leakage, and reactive oxygen species generation in human breast cancer cells (MDA-MB-231). Results The preliminary characterization of reduction of GO was confirmed by the red-shifting of the absorption peak for GE-rGO to 265 nm from 230 nm. The size of GO and GE-rGO was found to be 1,880 and 3,200 nm, respectively. X-ray diffraction results confirmed that reduction processes of GO and the processes of removing intercalated water molecules and the oxide groups. The surface functionalities and chemical natures of GO and GE-rGO were confirmed using Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. The surface morphologies of the synthesized graphene were analyzed using high-resolution scanning electron microscopy. Raman spectroscopy revealed single- and multilayer properties of GE-rGO. Atomic force microscopy images provided evidence for the formation of graphene. Furthermore, the effect of GO and GE-rGO was examined using a series of assays, such as cell viability, membrane integrity, and reactive oxygen species generation, which are key molecules involved in apoptosis. The results obtained from cell viability and lactate dehydrogenase assay suggest that GO and GE-rGO cause dose-dependent toxicity in the cells. Interestingly, it was found that biologically derived GE-rGO is more toxic to cancer cells than GO. Conclusion We describe a simple, green, nontoxic, and cost-effective approach to producing graphene using mushroom extract as a reducing and stabilizing agent. The proposed method could enable synthesis of graphene with potential biological and biomedical applications such as in cancer and angiogenic disorders. To our knowledge, this is the first report using mushroom extract as a reducing agent for the synthesis of graphene. Mushroom extract can be used as a biocatalyst for the production of graphene. PMID:24741313

Synthesis and characterization of polyamidoamine dendrimer-coated multi-walled carbon nanotubes and their application in gene delivery systems

NASA Astrophysics Data System (ADS)

Pan, Bifeng; Cui, Daxiang; Xu, Ping; Ozkan, Cengiz; Feng, Gao; Ozkan, Mihri; Huang, Tuo; Chu, Bingfeng; Li, Qing; He, Rong; Hu, Guohan

2009-03-01

With the aim of improving the amount and delivery efficiency of genes taken by carbon nanotubes into human cancer cells, different generations of polyamidoamine dendrimer modified multi-walled carbon nanotubes (dMNTs) were fabricated, and characterized by high-resolution transmission electron microscopy, atomic force microscopy, x-ray photoelectron spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis, revealing the presence of dendrimer capped on the surface of carbon nanotubes. The dMNTs fully conjugated with FITC-labeled antisense c-myc oligonucleotides (asODN), those resultant asODN-dMNTs composites were incubated with human breast cancer cell line MCF-7 cells and MDA-MB-435 cells, and liver cancer cell line HepG2 cells, and confirmed to enter into tumor cells within 15 min by laser confocal microscopy. These composites inhibited the cell growth in time- and dose-dependent means, and down-regulated the expression of the c-myc gene and C-Myc protein. Compared with the composites of CNT-NH2-asODN and dendrimer-asODN, no. 5 generation of dendrimer-modified MNT-asODN composites exhibit maximal transfection efficiencies and inhibition effects on tumor cells. The intracellular gene transport and uptake via dMNTs should be generic for the mammalian cell lines. The dMNTs have potentials in applications such as gene or drug delivery for cancer therapy and molecular imaging.

Assembly of live micro-organisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments

NASA Astrophysics Data System (ADS)

Dague, E.; Jauvert, E.; Laplatine, L.; Viallet, B.; Thibault, C.; Ressier, L.

2011-09-01

Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.

Corrosive effects of fluoride on titanium: investigation by X-ray photoelectron spectroscopy, atomic force microscopy, and human epithelial cell culturing.

PubMed

Stájer, Anette; Ungvári, Krisztina; Pelsoczi, István K; Polyánka, Hilda; Oszkó, Albert; Mihalik, Erzsébet; Rakonczay, Zoltán; Radnai, Márta; Kemény, Lajos; Fazekas, András; Turzó, Kinga

2008-11-01

High fluoride (F(-)) concentrations and acidic pH impair the corrosion resistance of titanium (Ti). Effects of F(-)-containing caries-preventive prophylactic rinses, and gels on Ti were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Human epithelial cell attachment and proliferation were investigated by dimethylthiazol-diphenyl tetrazolium bromide (MTT) and protein content assays. Aqueous 1% NaF solution (3800 ppm F(-), pH 4.5) or high (12,500 ppm) F(-) content gel (pH 4.8) strongly corroded the surface and modified its composition. XPS revealed formation of a strongly bound F(-)-containing complex (Na(2)TiF(6)). AFM indicated an increase in roughness (R(a)) of the surfaces: 10-fold for the NaF solution and smaller for the gel or a mouthwash (250 ppm F(-), pH 4.4). MTT revealed that cell attachment was significantly increased by the gel, but was not disturbed by either the mouthwash or the NaF. Cell proliferation determined by MTT decreased significantly only for the NaF-treated samples; protein content assay experiments showed no such effect. This study indicates that epithelial cell culturing results can depend on the method used, and the adverse effects of a high F(-) concentration and low pH should be considered when prophylactic gels are applied by patients with Ti implants or other dental devices.

Opto-nanomechanical spectroscopic material characterization

DOE PAGES

Tetard, Laurene; Passian, Ali; Farahi, R. H.; ...

2015-08-10

Cellulosic ethanol is a biofuel of considerable potential in the search for sustainable and renewable bioenergy [1,2]. However, while rich in carbohydrates [3], the plant cell walls exhibit a natural resistance to complex phenotype treatments such as enzymatic microbial deconstruction, heat and acid treatments that can remove the lignin polymers from cellulose before hydrolysis [5]. Noninvasive physical and chemical characterization of the cell walls and the effect of such treatments on biomass are challenging but necessary to understand and overcome such resistance [6]. Although lacking chemical recognition in their traditional forms, the various emerging modalities of nano-mechanical [7] and opto-nano-mechanicalmore » [8] force microscopies [9,10] provide a superb window into the needed nanoscale material characterization [6]. Infrared absorption spectroscopy is a powerful, non- destructive and ultra-sensitive technique that can provide the needed molecular fingerprinting but the photothermal channel is delocalized and thus lacks spatial resolution. Utilizing the emerging dynamic concepts of mode synthesizing atomic force microscopy (MSAFM) [11] and virtual resonance [12], we introduce a hybrid photonic and nanomechanical force microscopy (hp-MSAFM) with molecular recognition and characterize the extraction, holopulping and acid treatment of biomass. We present spatially and spectrally resolved cell wall images that reveal both the morphological and the compositional alterations of the cell walls. The measured biomolecular traits are in agreement with chemical maps obtained with infrared and confocal Raman micro-spectroscopies of the same samples. The presented findings should prove highly relevant in fields such as cancer research [13], nanotoxicity [14], energy storage and production [15], where morphological, chemical and subsurface studies of nanocomposites [16], nanoparticle uptake by cells [14], and nanoscale quality control [17] are in demand.« less

Atomic force microscopy-based characterization and design of biointerfaces

NASA Astrophysics Data System (ADS)

Alsteens, David; Gaub, Hermann E.; Newton, Richard; Pfreundschuh, Moritz; Gerber, Christoph; Müller, Daniel J.

2017-03-01

Atomic force microscopy (AFM)-based methods have matured into a powerful nanoscopic platform, enabling the characterization of a wide range of biological and synthetic biointerfaces ranging from tissues, cells, membranes, proteins, nucleic acids and functional materials. Although the unprecedented signal-to-noise ratio of AFM enables the imaging of biological interfaces from the cellular to the molecular scale, AFM-based force spectroscopy allows their mechanical, chemical, conductive or electrostatic, and biological properties to be probed. The combination of AFM-based imaging and spectroscopy structurally maps these properties and allows their 3D manipulation with molecular precision. In this Review, we survey basic and advanced AFM-related approaches and evaluate their unique advantages and limitations in imaging, sensing, parameterizing and designing biointerfaces. It is anticipated that in the next decade these AFM-related techniques will have a profound influence on the way researchers view, characterize and construct biointerfaces, thereby helping to solve and address fundamental challenges that cannot be addressed with other techniques.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy.

PubMed

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

PubMed Central

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53. PMID:22162658

Nanoscale investigation of the interaction of colistin with model phospholipid membranes by Langmuir technique, and combined infrared and force spectroscopies.

PubMed

Freudenthal, Oona; Quilès, Fabienne; Francius, Grégory; Wojszko, Kamila; Gorczyca, Marcelina; Korchowiec, Beata; Rogalska, Ewa

2016-11-01

Colistin (Polymyxin E), an antimicrobial peptide, is increasingly put forward as salvage for severe multidrug-resistant infections. Unfortunately, colistin is potentially toxic to mammalian cells. A better understanding of the interaction with specific components of the cell membranes may be helpful in controlling the factors that may enhance toxicity. Here, we report a physico-chemical study of model phospholipid (PL) mono- and bilayers exposed to colistin at different concentrations by Langmuir technique, atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The effect of colistin on chosen PL monolayers was examined. Insights into the topographical and elastic changes in the PL bilayers within time after peptide injection are presented via AFM imaging and force spectra. Finally, changes in the PL bilayers' ATR-FTIR spectra as a function of time within three bilayer compositions, and the influence of colistin on their spectral fingerprint are examined together with the time-evolution of the Amide II and νCO band integrated intensity ratios. Our study reveals a great importance in the role of the PL composition as well as the peptide concentration on the action of colistin on PL model membranes. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of Staphylococcus aureus adhesion forces on various dental restorative materials using atomic force microscopy

NASA Astrophysics Data System (ADS)

Merghni, Abderrahmen; Kammoun, Dorra; Hentati, Hajer; Janel, Sébastien; Popoff, Michka; Lafont, Frank; Aouni, Mahjoub; Mastouri, Maha

2016-08-01

In the oral cavity dental restorative biomaterials can act as a reservoir for infection with opportunistic Staphylococcus aureus pathogen, which can lead to the occurrence of secondary caries and treatment failures. Our aim was to evaluate the adhesion forces by S. aureus on four dental restorative biomaterials and to correlate this finding to differences in specific surface characteristics. Additionally, the influence of salivary conditioning films in exerted adhesion forces was investigated. The substrate hydrophobicity was measured by goniometer and the surface free energy was calculated using the equilibrium advancing contact angle values of water, formamide, and diiodomethane on the tested surfaces. The surface roughness was determined using atomic force microscope (AFM). Additionally, cell force spectroscopy was achieved to quantify the forces that drive cell-substrate interactions. S. aureus bacterium exerted a considerable adhesion forces on various dental restorative materials, which decreased in the presence of saliva conditioning film. The influence of the surface roughness and free energy in initial adhesion appears to be more important than the effect of hydrophobicity, either in presence or absence of saliva coating. Hence, control of surface properties of dental restorative biomaterials is of crucial importance in preventing the attachment and subsequent the biofilm formation.

Biophotonics for imaging and cell manipulation: quo vadis?

NASA Astrophysics Data System (ADS)

Serafetinides, Alexandros A.; Makropoulou, Mirsini; Kotsifaki, Domna G.; Tsigaridas, Giorgos

2016-01-01

As one of the major health problems for mankind is cancer, any development for the early detection and effective treatment of cancer is crucial to saving lives. Worldwide, the dream for the anti-cancer procedure of attack is the development of a safe and efficient early diagnosis technique, the so called "optical biopsy". As early diagnosis of cancer is associated with improved prognosis, several laser based optical diagnostic methods were developed to enable earlier, non-invasive detection of human cancer, as Laser Induced Fluorescence spectroscopy (LIFs), Diffuse Reflectance spectroscopy (DRs), confocal microscopy, and Optical Coherence Tomography (OCT). Among them, Optical Coherence Tomography (OCT) imaging is considered to be a useful tool to differentiate healthy from malignant (e.g. basal cell carcinoma, squamous cell carcinoma) skin tissue. If the demand is to perform imaging in sub-tissular or even sub-cellular level, optical tweezers and atomic force microscopy have enabled the visualization of molecular events underlying cellular processes in live cells, as well as the manipulation and characterization of microscale or even nanoscale biostructures. In this work, we will present the latest advances in the field of laser imaging and manipulation techniques, discussing some representative experimental data focusing on the 21th century biophotonics roadmap of novel diagnostic and therapeutical approaches. As an example of a recently discussed health and environmental problem, we studied both experimentally and theoretically the optical trapping forces exerted on yeast cells and modified with estrogen-like acting compounds yeast cells, suspended in various buffer media.

Enhanced adhesion of Streptococcus mutans to hydroxyapatite after exposure to saliva.

PubMed

Spengler, Christian; Thewes, Nicolas; Nolle, Friederike; Faidt, Thomas; Umanskaya, Natalia; Hannig, Matthias; Bischoff, Markus; Jacobs, Karin

2017-07-01

Streptococcus mutans cells form robust biofilms on human teeth and are strongly related to caries incidents. Hence, understanding the adhesion of S. mutans in the human oral cavity is of major interest for preventive dentistry. In this study, we report on atomic force microscopy-based single-cell force spectroscopy measurements of S. mutans cells to hydroxyapatite surfaces. We observe for almost all measurements a significant difference in adhesion strength for S. mutans as well as for Staphylococcus carnosus cells. However, the increase in adhesion strength after saliva exposure is much higher for S. mutans cells compared to S. carnosus cells. Our results demonstrate that S. mutans cells are well adapted to their natural environment, the oral cavity. This ability promotes the biofilm-forming capability of that species and hence the production of caries-provoking acids. In consequence, understanding the fundamentals of this mechanism may pave a way towards more effective caries-reducing techniques. Copyright © 2017 John Wiley & Sons, Ltd.

Nanomechanics of Yeast Surfaces Revealed by AFM

NASA Astrophysics Data System (ADS)

Dague, Etienne; Beaussart, Audrey; Alsteens, David

Despite the large and well-documented characterization of the microbial cell wall in terms of chemical composition, the determination of the mechanical properties of surface molecules in relation to their function remains a key challenge in cell biology.The emergence of powerful tools allowing molecular manipulations has already revolutionized our understanding of the surface properties of fungal cells. At the frontier between nanophysics and molecular biology, atomic force microscopy (AFM), and more specifically single-molecule force spectroscopy (SMFS), has strongly contributed to our current knowledge of the cell wall organization and nanomechanical properties. However, due to the complexity of the technique, measurements on live cells are still at their infancy.In this chapter, we describe the cell wall composition and recapitulate the principles of AFM as well as the main current methodologies used to perform AFM measurements on live cells, including sample immobilization and tip functionalization.The current status of the progress in probing nanomechanics of the yeast surface is illustrated through three recent breakthrough studies. Determination of the cell wall nanostructure and elasticity is presented through two examples: the mechanical response of mannoproteins from brewing yeasts and elasticity measurements on lacking polysaccharide mutant strains. Additionally, an elegant study on force-induced unfolding and clustering of adhesion proteins located at the cell surface is also presented.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatinmore » for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.« less

Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA

NASA Astrophysics Data System (ADS)

Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

2016-01-01

A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA.

PubMed

Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

2016-01-15

A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of laser tweezers Raman spectroscopy techniques to the monitoring of single cell response to stimuli

NASA Astrophysics Data System (ADS)

Chan, James W.; Liu, Rui; Matthews, Dennis L.

2012-06-01

Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

Pulling-induced rupture of ligand-receptor bonds between a spherically shaped bionanoparticle and the support

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.

2018-04-01

Contacts of biological or biologically-inspired spherically shaped nanoparticles (e.g., virions or lipid nanoparticles used for intracellular RNA delivery) with a lipid membrane of cells are often mediated by multiple relatively weak ligand-receptor bonds. Such contacts can be studied at a supported lipid bilayer. The rupture of bonds can be scrutinized by using force spectroscopy. Bearing a supported lipid bilayer in mind, the author shows analytically that the corresponding dependence of the force on the nanoparticle displacement and the effect of the force on the bond-rupture activation energy are qualitatively different compared to what is predicted by the conventional Bell approximation.

Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

PubMed

Lin, Juqiang; Xu, Han; Wu, Yangzhe; Tang, Mingjie; McEwen, Gerald D; Liu, Pin; Hansen, Dane R; Gilbertson, Timothy A; Zhou, Anhong

2013-02-05

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

Idiosyncrasies of Physical Vapor Deposition Processes from Various Knudsen Cells for Quinacridone Thin Film Growth on Silicon Dioxide

PubMed Central

2015-01-01

Thin films of quinacridone deposited by physical vapor deposition on silicon dioxide were investigated by thermal desorption spectroscopy (TDS), mass spectrometry (MS), atomic force microscopy (AFM), specular and grazing incidence X-ray diffraction (XRD, GIXD), and Raman spectroscopy. Using a stainless steel Knudsen cell did not allow the preparation of a pure quinacridone film. TDS and MS unambiguously showed that in addition to quinacridone, desorbing at about 500 K (γ-peak), significant amounts of indigo desorbed at about 420 K (β-peak). The existence of these two species on the surface was verified by XRD, GIXD, and Raman spectroscopy. The latter spectroscopies revealed that additional species are contained in the films, not detected by TDS. In the film mainly composed of indigo a species was identified which we tentatively attribute to carbazole. The film consisting of mainly quinacridone contained in addition p-sexiphenyl. The reason for the various decomposition species effusing from the metal Knudsen cell is the comparably high sublimation temperature of the hydrogen bonded quinacridone. With special experimental methods and by using glass Knudsen-type cells we were able to prepare films which exclusively consist of molecules either corresponding to the β-peak or the γ-peak. These findings are of relevance for choosing the proper deposition techniques in the preparation of quinacridone films in the context of organic electronic devices. PMID:26401189

Binding analysis of carbon nanoparticles to human immunoglobulin G: Elucidation of the cytotoxicity of CNPs and perturbation of immunoglobulin conformations

NASA Astrophysics Data System (ADS)

Zhang, Shengrui; Yang, Haitao; Ji, Xiaohui; Wang, Qin

2016-02-01

The chemical compositions, sizes and fluorescent properties of synthesized carbon nanoparticles (CNPs) were characterized. Escherichia coli (E. coli) cells were used as a model to study the cytotoxicity of CNPs, and the results of the cellular uptake of CNPs yielded excellent results: the CNPs demonstrated good biocompatibility and were non-toxic to the growth of the E. coli cells. Moreover, to assess the potential toxicity of CNPs to human health, the binding behavior of CNPs with human immunoglobulin G (HIgG) was examined by fluorescence quenching spectroscopy, synchronous fluorescence spectroscopy and circular dichroism spectroscopy under physiological conditions. The fluorescence quenching constants and parameters for the interaction at different temperatures had been calculated according to Scatchard. The thermodynamic parameters, such as enthalpy change (ΔH), entropy change (ΔS) and free energy change (ΔG), were calculated, and the results indicated strong static quenching and showed that van der Waals forces, hydrogen bonds and hydrophobic interactions were the predominant intermolecular forces stabilizing the CNP-HIgG complex. Synchronous fluorescence and circular dichroism spectra provided information regarding the conformational alteration of HIgG in the presence of CNPs. These findings help to characterize the interactions between CNPs and HIgG, which may clarify the potential risks and undesirable health effects of CNPs, as well as the related cellular trafficking and systemic translocation.

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy*

PubMed Central

Neundlinger, Isabel; Puntheeranurak, Theeraporn; Wildling, Linda; Rankl, Christian; Wang, Lai-Xi; Gruber, Hermann J.; Kinne, Rolf K. H.; Hinterdorfer, Peter

2014-01-01

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2′-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations. PMID:24962566

Flexible magnetic polyurethane/Fe2O3 nanoparticles as organic-inorganic nanocomposites for biomedical applications: Properties and cell behavior.

PubMed

Shahrousvand, Mohsen; Hoseinian, Monireh Sadat; Ghollasi, Marzieh; Karbalaeimahdi, Ali; Salimi, Ali; Tabar, Fatemeh Ahmadi

2017-05-01

Nowadays, the discovery of cell behaviors and their responses in communication with the stem cell niches and/or microenvironments are one of the major topics in tissue engineering and regenerative medicine. In this study, incorporated organic-inorganic polyurethane (PU) nanocomposites were prepared for better understanding of cell signaling and the effect of magnetite nanoparticles on cell proliferation and cell responses. The properties of PU-IONs were evaluated by fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), atomic-force microscopy (AFM), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and electrochemical impedance spectroscopy (EIS). The presence of the iron oxide nanoparticles (IONs) affects on the properties of polyurethane nanocomposites such as bulk morphology, mechanical, electrochemical, and biological properties. The electrical conductivity and hydrophilicity of PU-IONs were improved by increasing the magnetite nanoparticles; therefore water absorption, biodegradation and cell viability were changed. The biocompatibility of PU-IONs was investigated by MTT assay, cell attachment and cell staining. According to the results, the magnetite polyurethane nanocomposites could be a potential choice for cell therapy and tissue engineering, especially nerve repair. Copyright © 2016 Elsevier B.V. All rights reserved.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane.

PubMed

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

2016-08-15

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge. Copyright © 2016 Elsevier Inc. All rights reserved.

The effect of cigarette smoke extract on thrombomodulin-thrombin binding: an atomic force microscopy study.

PubMed

Wei, Yujie; Zhang, Xuejie; Xu, Li; Yi, Shaoqiong; Li, Yi; Fang, Xiaohong; Liu, Huiliang

2012-10-01

Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of how smoking promotes thrombosis is not fully understood. Thrombosis is associated with the imbalance of the coagulant system due to endothelial dysfunction. As a vital anticoagulation cofactor, thrombomodulin (TM) located on the endothelial cell surface is able to regulate intravascular coagulation by binding to thrombin, and the binding results in thrombosis inhibition. This work focused on the effects of cigarette smoke extract (CSE) on TM-thrombin binding by atomic force microscopy (AFM) based single-molecule force spectroscopy. The results from both in vitro and live-cell experiments indicated that CSE could notably reduce the binding probability of TM and thrombin. This study provided a new approach and new evidence for studying the mechanism of thrombosis triggered by cigarette smoking.

AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC-SIGN.

PubMed

te Riet, Joost; Reinieren-Beeren, Inge; Figdor, Carl G; Cambi, Alessandra

2015-11-01

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C-type lectin dendritic cell-specific intracellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), because a detailed characterization at the structural level is lacking. DC-SIGN recognizes specific Candida-associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan-branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope-based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC-SIGN. We demonstrate that slight differences in the N-mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC-SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 kB T. The single-bond affinity of tetrameric DC-SIGN for wild-type C. albicans is ~10.7 kB T and a dissociation constant kD of 23 μM, which is relatively strong compared with other carbohydrate-protein interactions described in the literature. In conclusion, this study shows that DC-SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC-SIGN and its pathogenic ligands will lead to a better understanding of how fungal-associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti-fungal drugs. Copyright © 2015 John Wiley & Sons, Ltd.

Quantitative nanoscale electrostatics of viruses

NASA Astrophysics Data System (ADS)

Hernando-Pérez, M.; Cartagena-Rivera, A. X.; Lošdorfer Božič, A.; Carrillo, P. J. P.; San Martín, C.; Mateu, M. G.; Raman, A.; Podgornik, R.; de Pablo, P. J.

2015-10-01

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed φ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed φ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04274g

Dielectrophoresis-Assisted Raman Spectroscopy of Intravesicular Analytes on Metallic Pyramids.

PubMed

Barik, Avijit; Cherukulappurath, Sudhir; Wittenberg, Nathan J; Johnson, Timothy W; Oh, Sang-Hyun

2016-02-02

Chemical analysis of membrane-bound containers such as secretory vesicles, organelles, and exosomes can provide insights into subcellular biology. These containers are loaded with a range of important biomolecules, which further underscores the need for sensitive and selective analysis methods. Here we present a metallic pyramid array for intravesicular analysis by combining site-selective dielectrophoresis (DEP) and Raman spectroscopy. Sharp pyramidal tips act as a gradient force generator to trap nanoparticles or vesicles from the solution, and the tips are illuminated by a monochromatic light source for concurrent spectroscopic detection of trapped analytes. The parameters suitable for DEP trapping were optimized by fluorescence microscopy, and the Raman spectroscopy setup was characterized by a nanoparticle based model system. Finally, vesicles loaded with 4-mercaptopyridine were concentrated at the tips and their Raman spectra were detected in real time. These pyramidal tips can perform large-area array-based trapping and spectroscopic analysis, opening up possibilities to detect molecules inside cells or cell-derived vesicles.

Force-detected nanoscale absorption spectroscopy in water at room temperature using an optical trap

NASA Astrophysics Data System (ADS)

Parobek, Alexander; Black, Jacob W.; Kamenetska, Maria; Ganim, Ziad

2018-04-01

Measuring absorption spectra of single molecules presents a fundamental challenge for standard transmission-based instruments because of the inherently low signal relative to the large background of the excitation source. Here we demonstrate a new approach for performing absorption spectroscopy in solution using a force measurement to read out optical excitation at the nanoscale. The photoinduced force between model chromophores and an optically trapped gold nanoshell has been measured in water at room temperature. This photoinduced force is characterized as a function of wavelength to yield the force spectrum, which is shown to be correlated to the absorption spectrum for four model systems. The instrument constructed for these measurements combines an optical tweezer with frequency domain absorption spectroscopy over the 400-800 nm range. These measurements provide proof-of-principle experiments for force-detected nanoscale spectroscopies that operate under ambient chemical conditions.

Cytocompatibility of amorphous hydrogenated carbon nitride films deposited by CH4/N2 dielectric barrier discharge plasmas with respect to cell lines

NASA Astrophysics Data System (ADS)

Majumdar, Abhijit; Schröder, Karsten; Hippler, Rainer

2008-10-01

Special amorphous hydrogenated carbon nitride (a-H-CNx) films have been prepared on glass substrates for the investigation of adhesion and proliferation of different mammalian cell lines. CH4/N2 dielectric barrier discharge plasmas were applied to deposit a-H-CNx coatings at half of the atmospheric pressure. Film quality was modified by varying the CH4:N2 ratio and deposition duration. Chemical composition was determined by x-ray photoelectron spectroscopy and Fourier transformed infrared spectroscopy. The N/C ratio was in the range of 0.20-0.55. A very small surface roughness was verified by atomic force microscopy. Human embryonic kidney (HEK) and rat adrenal pheochromocytoma (PC12) cells were cultivated on the a-H-CNx films to investigate the cytocompatibility of these surfaces. The microscopic images show that both kinds of cells lines were unable to survive. The cells did not adhere to the surfaces, and most of the cells died after certain time spans. Increased amounts of nitrogen in the film induce an accelerated cell death. It is concluded, that the deposited CNx film behaves cytotoxic to HEK and PC12 cell lines.

Single cell assessment of yeast metabolic engineering for enhanced lipid production using Raman and AFM-IR imaging.

PubMed

Kochan, Kamila; Peng, Huadong; Wood, Bayden R; Haritos, Victoria S

2018-01-01

Biodiesel is a valuable renewable fuel made from derivatized fatty acids produced in plants, animals, and oleaginous microbes. Of the latter, yeasts are of special interest due to their wide use in biotechnology, ability to synthesize fatty acids and store large amounts of triacylglycerols while utilizing non-food carbon sources. While yeast efficiently produce lipids, genetic modification and indeed, lipid pathway metabolic engineering, is usually required for cost-effective production. Traditionally, gas chromatography (GC) is used to measure fatty acid production and to track the success of a metabolic engineering strategy in a microbial culture; here we have employed vibrational spectroscopy approaches at population and single cell level of engineered yeast while simultaneously investigating metabolite levels in subcellular structures. Firstly, a strong correlation ( r 2  > 0.99) was established between Fourier transform infrared (FTIR) lipid in intact cells and GC analysis of fatty acid methyl esters in the differently engineered strains. Confocal Raman spectroscopy of individual cells carrying genetic modifications to enhance fatty acid synthesis and lipid accumulation revealed changes to the lipid body (LB), the storage organelle for lipids in yeast, with their number increasing markedly (up to tenfold higher); LB size was almost double in the strain that also expressed a LB stabilizing gene but considerable variation was also noted between cells. Raman spectroscopy revealed a clear trend toward reduced unsaturated fatty acid content in lipids of cells carrying more complex metabolic engineering. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of individual cells indicated large differences in subcellular constituents between strains: cells of the most highly engineered strain had elevated lipid and much reduced carbohydrate in their cytoplasm compared with unmodified cells. Vibrational spectroscopy analysis allowed the simultaneous measurement of strain variability in metabolite production and impact on cellular structures as a result of different gene introductions or knockouts, within a lipid metabolic engineering strategy and these inform the next steps in comprehensive lipid engineering. Additionally, single cell spectroscopic analysis measures heterogeneity in metabolite production across microbial cultures under genetic modification, an emerging issue for efficient biotechnological production.

Frequency modulation atomic force microscopy: a dynamic measurement technique for biological systems

NASA Astrophysics Data System (ADS)

Higgins, Michael J.; Riener, Christian K.; Uchihashi, Takayuki; Sader, John E.; McKendry, Rachel; Jarvis, Suzanne P.

2005-03-01

Frequency modulation atomic force microscopy (FM-AFM) has been modified to operate in a liquid environment within an atomic force microscope specifically designed for investigating biological samples. We demonstrate the applicability of FM-AFM to biological samples using the spectroscopy mode to measure the unbinding forces of a single receptor-ligand (biotin-avidin) interaction. We show that quantitative adhesion force measurements can only be obtained provided certain modifications are made to the existing theory, which is used to convert the detected frequency shifts to an interaction force. Quantitative force measurements revealed that the unbinding forces for the biotin-avidin interaction were greater than those reported in previous studies. This finding was due to the use of high average tip velocities, which were calculated to be two orders of magnitude greater than those typically used in unbinding receptor-ligand experiments. This study therefore highlights the potential use of FM-AFM to study a range of biological systems, including living cells and/or single biomolecule interactions.

Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman, Atomic Force and Surface Plasmon Resonance spectroscopies.

PubMed

Signorelli, Sara; Santini, Simona; Yamada, Tohru; Bizzarri, Anna Rita; Beattie, Craig W; Cannistraro, Salvatore

2017-04-01

Mutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in >50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding. Molecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28. We show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge. These results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function. Raman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and biological applications of optical tweezers and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Xie, Chang'an

Optical tweezers is a three-dimensional manipulation tool that employs a gradient force that originates from the single highly focused laser beam. Raman spectroscopy is a molecular analytical tool that can give a highly unique "fingerprint" for each substance by measuring the unique vibrations of its molecules. The combination of these two optical techniques offers a new tool for the manipulation and identification of single biological cells and microscopic particles. In this thesis, we designed and implemented a Laser-Tweezers-Raman-Spectroscopy (LTRS) system, also called the Raman-tweezers, for the simultaneous capture and analysis of both biological particles and non-biological particles. We show that microparticles can be conveniently captured at the focus of a laser beam and the Raman spectra of trapped particles can be acquired with high quality. The LTRS system overcomes the intrinsic Brownian motion and cell motility of microparticles in solution and provides a promising tool for in situ identifying suspicious agents. In order to increase the signal to noise ratio, several schemes were employed in LTRS system to reduce the blank noise and the fluorescence signal coming from analytes and the surrounding background. These techniques include near-infrared excitation, optical levitation, confocal microscopy, and frequency-shifted Raman difference. The LTRS system has been applied for the study in cell biology at the single cell level. With the built Raman-tweezers system, we studied the dynamic physiological processes of single living cells, including cell cycle, the transcription and translation of recombinant protein in transgenic yeast cells and the T cell activation. We also studied cell damage and associated biochemical processes in optical traps, UV radiations, and evaluated heating by near-infrared Raman spectroscopy. These studies show that the Raman-tweezers system is feasible to provide rapid and reliable diagnosis of cellular disorders and can be used as a valuable tool to study cellular processes within single living cells or intracellular organelles and may aid research in molecular and cellular biology.

Fluorinated poly(ether sulfone) ionomers with disulfonated naphthyl pendants for proton exchange membrane applications

NASA Astrophysics Data System (ADS)

Hu, Zhaoxia; Lu, Yao; Zhang, Xulve; Yan, Xiaobo; Li, Na; Chen, Shouwen

2018-06-01

Proton exchange membranes based on fluorinated poly(ether sulfone)s with disulfonated naphthyl pendants (sSPFES) have been successfully prepared by post functionalization through polymeric SNAr reaction. Copolymer structure was confirmed by H-nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy, the physico-chemical properties of the sSPFES membranes were evaluated by thermogravimetric analysis, gel permeation chromatography, electro-chemical impedance spectroscopy, atomic force microscopy, Fenton, water-swelling and fuel cell test. The pendant grafting degree was controlled by varying the feeding amount of the disulfonaphthols, resulting in the ion exchange capacity about 1.28-1.73 mmol/g. The obtained sSPFES membranes were thermal stable, mechanical ductile, and exhibited dimensional change less than 17%, water uptake below 70%, and proton conductivity as high as 0.17-0.28 S/cm at 90°C in water. In a single H2/O2 fuel cell test at 80°C, the sSPFES-B-3.2 membrane (1.61 mmol/g) showed the maximum power output of 593-658 mW/cm2 at 60%-80% relative humidity, indicating their rather promising potential for fuel cell applications.

Fluorinated poly(ether sulfone) ionomers with disulfonated naphthyl pendants for proton exchange membrane applications

NASA Astrophysics Data System (ADS)

Hu, Zhaoxia; Lu, Yao; Zhang, Xulve; Yan, Xiaobo; Li, Na; Chen, Shouwen

2018-05-01

Proton exchange membranes based on fluorinated poly(ether sulfone)s with disulfonated naphthyl pendants (sSPFES) have been successfully prepared by post functionalization through polymeric SNAr reaction. Copolymer structure was confirmed by H-nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy, the physico-chemical properties of the sSPFES membranes were evaluated by thermogravimetric analysis, gel permeation chromatography, electro-chemical impedance spectroscopy, atomic force microscopy, Fenton, water-swelling and fuel cell test. The pendant grafting degree was controlled by varying the feeding amount of the disulfonaphthols, resulting in the ion exchange capacity about 1.28-1.73 mmol/g. The obtained sSPFES membranes were thermal stable, mechanical ductile, and exhibited dimensional change less than 17%, water uptake below 70%, and proton conductivity as high as 0.17-0.28 S/cm at 90°C in water. In a single H2/O2 fuel cell test at 80°C, the sSPFES-B-3.2 membrane (1.61 mmol/g) showed the maximum power output of 593-658 mW/cm2 at 60%-80% relative humidity, indicating their rather promising potential for fuel cell applications.

SU-8 hollow cantilevers for AFM cell adhesion studies

NASA Astrophysics Data System (ADS)

Martinez, Vincent; Behr, Pascal; Drechsler, Ute; Polesel-Maris, Jérôme; Potthoff, Eva; Vörös, Janos; Zambelli, Tomaso

2016-05-01

A novel fabrication method was established to produce flexible, transparent, and robust tipless hollow atomic force microscopy (AFM) cantilevers made entirely from SU-8. Channels of 3 μm thickness and several millimeters length were integrated into 12 μm thick and 40 μm wide cantilevers. Connected to a pressure controller, the devices showed high sealing performance with no leakage up to 6 bars. Changing the cantilever lengths from 100 μm to 500 μm among the same wafer allowed the targeting of various spring constants ranging from 0.5 to 80 N m-1 within a single fabrication run. These hollow polymeric AFM cantilevers were operated in the optical beam deflection configuration. To demonstrate the performance of the device, single-cell force spectroscopy experiments were performed with a single probe detaching in a serial protocol more than 100 Saccharomyces cerevisiae yeast cells from plain glass and glass coated with polydopamine while measuring adhesion forces in the sub-nanoNewton range. SU-8 now offers a new alternative to conventional silicon-based hollow cantilevers with more flexibility in terms of complex geometric design and surface chemistry modification.

Analysis of dispersive interactions at polymer/TiAlN interfaces by means of dynamic force spectroscopy.

PubMed

Wiesing, M; de Los Arcos, T; Gebhard, M; Devi, A; Grundmeier, G

2017-12-20

The structural and electronic origins of the interactions between polycarbonate and sputter deposited TiAlN were analysed using a combined electron and force spectroscopic approach. Interaction forces were measured by means of dynamic force spectroscopy and the surface polarizability was analysed by X-ray photoelectron valence band spectroscopy. It could be shown that the adhesive interactions between polycarbonate and TiAlN are governed by van der Waals forces. Different surface cleansing and oxidizing treatments were investigated and the effect of the surface chemistry on the force interactions was analysed. Intense surface oxidation resulted in a decreased adhesion force by a factor of two due to the formation of a 2 nm thick Ti 0.21 Al 0.45 O surface oxide layer. The origin of the residual adhesion forces caused by the mixed Ti 0.21 Al 0.45 O surface oxide was clarified by considering the non-retarded Hamaker coefficients as calculated by Lifshitz theory, based on optical data from Reflection Electron Energy Loss Spectroscopy. This disclosed increased dispersion forces of Ti 0.21 Al 0.45 O due to the presence of Ti(iv) ions and related Ti 3d band optical transitions.

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro-dielectric barrier discharge plasma.

PubMed

Choi, Hyeongwon; Choi, Eun Ha; Kim, Kyung Sook

2017-10-01

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro-dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro-DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro-DBD plasma for various exposure times. The elasticity of a single cell was determined by force-distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma-treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro-DBD plasma were related to cell mechanical changes. © 2017 Wiley Periodicals, Inc.

α-Helix Unwinding as Force Buffer in Spectrins.

PubMed

Takahashi, Hirohide; Rico, Felix; Chipot, Christophe; Scheuring, Simon

2018-03-27

Spectrins are cytoskeletal proteins located at the inner face of the plasma membrane, making connections between membrane anchors and the actin cortex, and between actin filaments. Spectrins share a common structure forming a bundle of 3 α-helices and play a major role during cell deformation. Here, we used high-speed force spectroscopy and steered molecular dynamics simulations to understand the mechanical stability of spectrin, revealing a molecular force buffering function. We find that spectrin acts as a soft spring at short extensions (70-100 Å). Under continuous external stretching, its α-helices unwind, leading to a viscous mechanical response over larger extensions (100-300 Å), represented by a constant-force plateau in force/extension curves. This viscous force buffering emerges from a quasi-equilibrium competition between disruption and re-formation of α-helical hydrogen bonds. Our results suggest that, in contrast to β-sheet proteins, which unfold in a catastrophic event, α-helical spectrins dominantly unwind, providing a viscous force buffer over extensions about 5 times their folded length.

Dynamic-force spectroscopy measurement with precise force control using atomic-force microscopy probe

NASA Astrophysics Data System (ADS)

Takeuchi, Osamu; Miyakoshi, Takaaki; Taninaka, Atsushi; Tanaka, Katsunori; Cho, Daichi; Fujita, Machiko; Yasuda, Satoshi; Jarvis, Suzanne P.; Shigekawa, Hidemi

2006-10-01

The accuracy of dynamic-force spectroscopy (DFS), a promising technique of analyzing the energy landscape of noncovalent molecular bonds, was reconsidered in order to justify the use of an atomic-force microscopy (AFM) cantilever as a DFS force probe. The advantages and disadvantages caused, for example, by the force-probe hardness were clarified, revealing the pivotal role of the molecular linkage between the force probe and the molecular bonds. It was shown that the feedback control of the loading rate of tensile force enables us a precise DFS measurement using an AFM cantilever as the force probe.

Elucidating How Wood Adhesives Bond to Wood Cell Walls using High-Resolution Solution-State NMR Spectroscopy

Treesearch

Daniel J. Yelle

2013-01-01

Some extensively used wood adhesives, such as pMDI (polymeric methylene diphenyl diisocyanate) and PF (phenol formaldehyde) have shown excellent adhesion properties with wood. However, distinguishing whether the strength is due to physical bonds (i.e., van der Waals, London, or hydrogen bond forces) or covalent bonds between the adherend and the adhesive is not fully...

Silicon solar cell performance deposited by diamond like carbon thin film ;Atomic oxygen effects;

NASA Astrophysics Data System (ADS)

Aghaei, Abbas Ail; Eshaghi, Akbar; Karami, Esmaeil

2017-09-01

In this research, a diamond-like carbon thin film was deposited on p-type polycrystalline silicon solar cell via plasma-enhanced chemical vapor deposition method by using methane and hydrogen gases. The effect of atomic oxygen on the functioning of silicon coated DLC thin film and silicon was investigated. Raman spectroscopy, field emission scanning electron microscopy, atomic force microscopy and attenuated total reflection-Fourier transform infrared spectroscopy were used to characterize the structure and morphology of the DLC thin film. Photocurrent-voltage characteristics of the silicon solar cell were carried out using a solar simulator. The results showed that atomic oxygen exposure induced the including oxidation, structural changes, cross-linking reactions and bond breaking of the DLC film; thus reducing the optical properties. The photocurrent-voltage characteristics showed that although the properties of the fabricated thin film were decreased after being exposed to destructive rays, when compared with solar cell without any coating, it could protect it in atomic oxygen condition enhancing solar cell efficiency up to 12%. Thus, it can be said that diamond-like carbon thin layer protect the solar cell against atomic oxygen exposure.

Following aptamer-ricin specific binding by single molecule recognition and force spectroscopy measurements

USDA-ARS?s Scientific Manuscript database

The atomic force microscope (AFM) recognition and dynamic force spectroscopy (DFS) experiments provide both morphology and interaction information of the aptamer and protein, which can be used for the future study on the thermodynamics and kinetics properties of ricin-aptamer/antibody interactions. ...

pH-sensitive nanocargo based on smart polymer functionalized graphene oxide for site-specific drug delivery.

PubMed

Kavitha, Thangavelu; Abdi, Syed Izhar Haider; Park, Soo-Young

2013-04-14

Graphene oxide (GO) was functionalized covalently with pH-sensitive poly(2-(diethylamino) ethyl methacrylate) (PDEA) by surface-initiated in situ atom transfer radical polymerization. The structure of the PDEA-grafted GO (GO-PDEA) were examined by Fourier-transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis and atomic force microscopy. The grafted PDEA endowed the GO sheets with good solubility and stability in physiological solutions. Simple physisorption by π-π stacking and hydrophobic interactions on GO-PDEA can be used to load camptothecin (CPT), a widely used water-insoluble cancer drug. The loaded CPT was released only at the lower (acidic) pH normally found in a tumor environment but not in basic and neutral pH. GO-PDEA did not show practical toxicity to N2a cancer cells but the GO-PDEA-CPT complex exhibited high potency in killing N2a cancer cells in vitro. These results suggest that the GO-PDEA nanocargo carrier might be a promising material for site-specific anticancer drug delivery and controlled release.

Nanocarbon Allotropes-Graphene and Nanocrystalline Diamond-Promote Cell Proliferation.

PubMed

Verdanova, Martina; Rezek, Bohuslav; Broz, Antonin; Ukraintsev, Egor; Babchenko, Oleg; Artemenko, Anna; Izak, Tibor; Kromka, Alexander; Kalbac, Martin; Hubalek Kalbacova, Marie

2016-05-01

Two profoundly different carbon allotropes - nanocrystalline diamond and graphene - are of considerable interest from the viewpoint of a wide range of biomedical applications including implant coating, drug and gene delivery, cancer therapy, and biosensing. Osteoblast adhesion and proliferation on nanocrystalline diamond and graphene are compared under various conditions such as differences in wettability, topography, and the presence or absence of protein interlayers between cells and the substrate. The materials are characterized in detail by means of scanning electron microscopy, atomic force microscopy, photoelectron spectroscopy, Raman spectroscopy, and contact angle measurements. In vitro experiments have revealed a significantly higher degree of cell proliferation on graphene than on nanocrystalline diamond and a tissue culture polystyrene control material. Proliferation is promoted, in particular, by hydrophobic graphene with a large number of nanoscale wrinkles independent of the presence of a protein interlayer, i.e., substrate fouling is not a problematic issue in this respect. Nanowrinkled hydrophobic graphene, thus, exhibits superior characteristics for those biomedical applications where high cell proliferation is required under differing conditions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Topographical and Chemical Imaging of a Phase Separated Polymer Using a Combined Atomic Force Microscopy/Infrared Spectroscopy/Mass Spectrometry Platform

DOE PAGES

Tai, Tamin; Karácsony, Orsolya; Bocharova, Vera; ...

2016-02-18

This article describes how the use of a hybrid atomic force microscopy/infrared spectroscopy/mass spectrometry imaging platform was demonstrated for the acquisition and correlation of nanoscale sample surface topography and chemical images based on infrared spectroscopy and mass spectrometry.

Optical trapping for analytical biotechnology.

PubMed

Ashok, Praveen C; Dholakia, Kishan

2012-02-01

We describe the exciting advances of using optical trapping in the field of analytical biotechnology. This technique has opened up opportunities to manipulate biological particles at the single cell or even at subcellular levels which has allowed an insight into the physical and chemical mechanisms of many biological processes. The ability of this technique to manipulate microparticles and measure pico-Newton forces has found several applications such as understanding the dynamics of biological macromolecules, cell-cell interactions and the micro-rheology of both cells and fluids. Furthermore we may probe and analyse the biological world when combining trapping with analytical techniques such as Raman spectroscopy and imaging. Copyright © 2011 Elsevier Ltd. All rights reserved.

Influence of layer-by-layer assembled electrospun poly (L-lactic acid) nanofiber mats on the bioactivity of endothelial cells

NASA Astrophysics Data System (ADS)

Wu, Keke; Zhang, Xiazhi; Yang, Wufeng; Liu, Xiaoyan; Jiao, Yanpeng; Zhou, Changren

2016-12-01

Electrospun poly(L-lactic acid) (PLLA) nanofiber mats were successfully modified by deposition of multilayers with chitosan (CS), heparin (Hep) and graphene oxide (GO) through electrostatic layer-by-layer (LBL) self-assembly method. In this study, the surface properties of PLLA nanofiber mats before and after modification were investigated via scanning electron microscope (SEM), atomic force microscopy (AFM), attenuated total reflectance fourier transformation infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angle measurement. In addition, the cytocompatibility of the modified PLLA nanofiber mats were investigated by testing endothelial cells compatibility, including cell attachment, cell proliferation and cell cycle. The results revealed that the surfaces of modified PLLA nanofiber mats become much rougher, stifiness and the hydrophilicity of the LBL modified PLLA nanofiber mats were improved compared to original PLLA one. Moreover, the modified PLLA nanofiber mats had promoted the endothelial cells viability attachment significantly. Besides, we studied the PLLA nanofiber mats on the expression of necrosis factor (TNF-α), interleukine-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. The results showed that modified PLLA nanofiber mats had inhibited the inflammatory response to some extent.

Investigation of biochemical property changes in activation-induced CD 8 + T cell apoptosis using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Lee, Young Ju; Ahn, Hyung Joon; Lee, Gi-Ja; Jung, Gyeong Bok; Lee, Gihyun; Kim, Dohyun; Shin, Jae-Ho; Jin, Kyung-Hyun; Park, Hun-Kuk

2015-07-01

The study was to investigate the changes in biochemical properties of activated mature CD8+ T cells related to apoptosis at a molecular level. We confirmed the activation and apoptosis of CD8+ T cells by fluorescence-activated cell sorting and atomic force microscopy and then performed Raman spectral measurements on activated mature CD8+ T cells and cellular deoxyribose nucleic acid (DNA). In the activated mature CD8+ T cells, there were increases in protein spectra at 1002 and 1234 cm-1. In particular, to assess the apoptosis-related DNA spectral signatures, we investigated the spectra of the cellular DNA isolated from resting and activated mature CD8+ T cells. Raman spectra at 765 to 786 cm-1 and 1053 to 1087 cm-1 were decreased in activated mature DNA. In addition, we analyzed Raman spectrum using the multivariate statistical method including principal component analysis. Raman spectra of activated mature DNA are especially well-discriminated from those of resting DNA. Our findings regarding the biochemical and structural changes associated with apoptosis in activated mature T cells and cellular DNA according to Raman spectroscopy provide important insights into allospecific immune responses generated after organ transplantation, and may be useful for therapeutic manipulation of the immune response.

Scanning electron and atomic force microscopy, and raman and x-ray photoelectron spectroscopy characterization of near-isogenic soft and hard wheat kernels and corresponding flours

USDA-ARS?s Scientific Manuscript database

Atomic force microscopy (AFM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) are used to investigate vitreous (hard) and non-vitreous (soft) wheat kernels and their corresponding wheat flours. AFM data reveal two different microstructures. The vitreous kernel reveals a granular text...

Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

NASA Astrophysics Data System (ADS)

Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

2008-09-01

Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

Probing the influence of cell surface polysaccharides on nanodendrimer binding to Gram-negative and Gram-positive bacteria using single-nanoparticle force spectroscopy.

PubMed

Beaussart, Audrey; Beloin, Christophe; Ghigo, Jean-Marc; Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius; Duval, Jérôme F L

2018-06-27

The safe use and design of nanoparticles (NPs) ask for a comprehensive interpretation of their potentially adverse effects on (micro)organisms. In this respect, the prior assessment of the interactions experienced by NPs in the vicinity of - and in contact with - complex biological surfaces is mandatory. It requires the development of suitable techniques for deciphering the processes that govern nano-bio interactions when a single organism is exposed to an extremely low dose of NPs. Here, we used atomic force spectroscopy (AFM)-based force measurements to investigate at the nanoscale the interactions between carboxylate-terminated polyamidoamine (PAMAM) nanodendrimers (radius ca. 4.5 nm) and two bacteria with very distinct surface properties, Escherichia coli and Lactococcus lactis. The zwitterionic nanodendrimers exhibit a negative peripheral surface charge and/or a positive intraparticulate core depending on the solution pH and salt concentration. Following an original strategy according to which a single dendrimer NP is grafted at the very apex of the AFM tip, the density and localization of NP binding sites are probed at the surface of E. coli and L. lactis mutants expressing different cell surface structures (presence/absence of the O-antigen of the lipopolysaccharides (LPS) or of a polysaccharide pellicle). In line with electrokinetic analysis, AFM force measurements evidence that adhesion of NPs onto pellicle-decorated L. lactis is governed by their underlying electrostatic interactions as controlled by the pH-dependent charge of the peripheral and internal NP components, and the negatively-charged cell surface. In contrast, the presence of the O-antigen on E. coli systematically suppresses the adhesion of nanodendrimers onto cells, may the apparent NP surface charge be determined by the peripheral carboxylate groups or by the internal amine functions. Altogether, this work highlights the differentiated roles played by surface polysaccharides in mediating NP attachment to Gram-positive and Gram-negative bacteria. It further demonstrates that the assessment of NP bioadhesion features requires a critical analysis of the electrostatic contributions stemming from the various structures composing the stratified cell envelope, and those originating from the bulk and surface NP components. The joint use of electrokinetics and AFM provides a valuable option for rapidly addressing the binding propensity of NPs to microorganisms, as urgently needed in NP risk assessments.

Absorption Spectroscopy of Rubidium in an Alkali Metal Dispenser Cell and Bleached Wave Analysis

DTIC Science & Technology

2015-03-26

Department of Engineering Physics Graduate School of Engineering and Management Air Force Institute of Technology Air University Air Education and...at atmospheric temperatures and pressures, so none of the safety measures needed with pure solid alkali metal would be required. AMDs can also be...Institute of Technology Graduate School of Engineering and Management (AFIT/ENP) 2950 Hobson Way WPAFB OH 45433-7765 8. PERFORMING ORGANIZATION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; University of Chinese Academy of Sciences, Beijing 100049; Liu, Lianqing, E-mail: lqliu@sia.cn

Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measuremore » the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.« less

Optical tweezers reveal force plateau and internal friction in PEG-induced DNA condensation.

PubMed

Ojala, Heikki; Ziedaite, Gabija; Wallin, Anders E; Bamford, Dennis H; Hæggström, Edward

2014-03-01

The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force-extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force-extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.

Atomic force microscopy as an advanced tool in neuroscience

PubMed Central

Jembrek, Maja Jazvinšćak; Šimić, Goran; Hof, Patrick R.; Šegota, Suzana

2015-01-01

This review highlights relevant issues about applications and improvements of atomic force microscopy (AFM) toward a better understanding of neurodegenerative changes at the molecular level with the hope of contributing to the development of effective therapeutic strategies for neurodegenerative illnesses. The basic principles of AFM are briefly discussed in terms of evaluation of experimental data, including the newest PeakForce Quantitative Nanomechanical Mapping (QNM) and the evaluation of Young’s modulus as the crucial elasticity parameter. AFM topography, revealed in imaging mode, can be used to monitor changes in live neurons over time, representing a valuable tool for high-resolution detection and monitoring of neuronal morphology. The mechanical properties of living cells can be quantified by force spectroscopy as well as by new AFM. A variety of applications are described, and their relevance for specific research areas discussed. In addition, imaging as well as non-imaging modes can provide specific information, not only about the structural and mechanical properties of neuronal membranes, but also on the cytoplasm, cell nucleus, and particularly cytoskeletal components. Moreover, new AFM is able to provide detailed insight into physical structure and biochemical interactions in both physiological and pathophysiological conditions. PMID:28123795

Forced-rupture of cell-adhesion complexes reveals abrupt switch between two brittle states

NASA Astrophysics Data System (ADS)

Toan, Ngo Minh; Thirumalai, D.

2018-03-01

Cell adhesion complexes (CACs), which are activated by ligand binding, play key roles in many cellular functions ranging from cell cycle regulation to mediation of cell extracellular matrix adhesion. Inspired by single molecule pulling experiments using atomic force spectroscopy on leukocyte function-associated antigen-1 (LFA-1), expressed in T-cells, bound to intercellular adhesion molecules (ICAM), we performed constant loading rate (rf) and constant force (F) simulations using the self-organized polymer model to describe the mechanism of ligand rupture from CACs. The simulations reproduce the major experimental finding on the kinetics of the rupture process, namely, the dependence of the most probable rupture forces (f*s) on ln rf (rf is the loading rate) exhibits two distinct linear regimes. The first, at low rf, has a shallow slope, whereas the slope at high rf is much larger, especially for a LFA-1/ICAM-1 complex with the transition between the two occurring over a narrow rf range. Locations of the two transition states (TSs) extracted from the simulations show an abrupt change from a high value at low rf or constant force, F, to a low value at high rf or F. This unusual behavior in which the CACs switch from one brittle (TS position is a constant over a range of forces) state to another brittle state is not found in forced-rupture in other protein complexes. We explain this novel behavior by constructing the free energy profiles, F(Λ)s, as a function of a collective reaction coordinate (Λ), involving many key charged residues and a critical metal ion (Mg2+). The TS positions in F(Λ), which quantitatively agree with the parameters extracted using the Bell-Evans model, change abruptly at a critical force, demonstrating that it, rather than the molecular extension, is a good reaction coordinate. Our combined analyses using simulations performed in both the pulling modes (constant rf and F) reveal a new mechanism for the two loading regimes observed in the rupture kinetics in CACs.

Antibody-Unfolding and Metastable-State Binding in Force Spectroscopy and Recognition Imaging

PubMed Central

Kaur, Parminder; Qiang-Fu; Fuhrmann, Alexander; Ros, Robert; Kutner, Linda Obenauer; Schneeweis, Lumelle A.; Navoa, Ryman; Steger, Kirby; Xie, Lei; Yonan, Christopher; Abraham, Ralph; Grace, Michael J.; Lindsay, Stuart

2011-01-01

Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance—possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics. PMID:21190677

Local probing and stimulation of neuronal cells by optical manipulation

NASA Astrophysics Data System (ADS)

Cojoc, Dan

2014-09-01

During development and in the adult brain, neurons continuously explore the environment searching for guidance cues, leading to the appropriate connections. Elucidating these mechanisms represents a gold goal in neurobiology. Here, I discuss our recent achievements developing new approaches to locally probe the growth cones and stimulate neuronal cell compartments with high spatial and temporal resolution. Optical tweezers force spectroscopy applied in conjunction with metabolic inhibitors reveals new properties of the cytoskeleton dynamics. On the other hand, using optically manipulated microvectors as functionalized beads or filled liposomes, we demonstrate focal stimulation of neurons by small number of signaling molecules.

Quantitative nanoscale electrostatics of viruses.

PubMed

Hernando-Pérez, M; Cartagena-Rivera, A X; Lošdorfer Božič, A; Carrillo, P J P; San Martín, C; Mateu, M G; Raman, A; Podgornik, R; de Pablo, P J

2015-11-07

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed ϕ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.

AFM-IR: Technology and Applications in Nanoscale Infrared Spectroscopy and Chemical Imaging.

PubMed

Dazzi, Alexandre; Prater, Craig B

2016-12-13

Atomic force microscopy-based infrared spectroscopy (AFM-IR) is a rapidly emerging technique that provides chemical analysis and compositional mapping with spatial resolution far below conventional optical diffraction limits. AFM-IR works by using the tip of an AFM probe to locally detect thermal expansion in a sample resulting from absorption of infrared radiation. AFM-IR thus can provide the spatial resolution of AFM in combination with the chemical analysis and compositional imaging capabilities of infrared spectroscopy. This article briefly reviews the development and underlying technology of AFM-IR, including recent advances, and then surveys a wide range of applications and investigations using AFM-IR. AFM-IR applications that will be discussed include those in polymers, life sciences, photonics, solar cells, semiconductors, pharmaceuticals, and cultural heritage. In the Supporting Information , the authors provide a theoretical section that reviews the physics underlying the AFM-IR measurement and detection mechanisms.

Molecular and Nanoscale Engineering of High Efficiency Excitonic Solar Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenekhe, Samson A.; Ginger, David S.; Cao, Guozhong

We combined the synthesis of new polymers and organic-inorganic hybrid materials with new experimental characterization tools to investigate bulk heterojunction (BHJ) polymer solar cells and hybrid organic-inorganic solar cells during the 2007-2010 period (phase I) of this project. We showed that the bulk morphology of polymer/fullerene blend solar cells could be controlled by using either self-assembled polymer semiconductor nanowires or diblock poly(3-alkylthiophenes) as the light-absorbing and hole transport component. We developed new characterization tools in-house, including photoinduced absorption (PIA) spectroscopy, time-resolved electrostatic force microscopy (TR-EFM) and conductive and photoconductive atomic force microscopy (c-AFM and pc-AFM), and used them to investigatemore » charge transfer and recombination dynamics in polymer/fullerene BHJ solar cells, hybrid polymer-nanocrystal (PbSe) devices, and dye-sensitized solar cells (DSSCs); we thus showed in detail how the bulk photovoltaic properties are connected to the nanoscale structure of the BHJ polymer solar cells. We created various oxide semiconductor (ZnO, TiO 2) nanostructures by solution processing routes, including hierarchical aggregates and nanorods/nanotubes, and showed that the nanostructured photoanodes resulted in substantially enhanced light-harvesting and charge transport, leading to enhanced power conversion efficiency of dye-sensitized solar cells.« less

UHV AFM based colloidal probe studies of adhesive properties of VAlN hard coatings

NASA Astrophysics Data System (ADS)

Wiesing, M.; de los Arcos, T.; Grundmeier, G.

2018-01-01

The adhesion of polystyrene (PS) on V0.27Al0.29N0.44 and the related influence of the oxidation states of both surfaces was investigated using X-Ray Photoelectron Spectroscopy (XPS) and Colloidal Force Spectroscopy (CFS) in Ultra-High Vacuum (UHV). Complementary, the intimate relation between the adhesion force, the chemical structure and surface polarizability was investigated by XPS valence band spectroscopy and the calculation of non-retarded Hamaker coefficients using Lifshitz theory based on optical data as derived from Reflection Electron Energy Loss Spectroscopy (REELS) spectra. The combined electron and force spectroscopic analysis of the interaction forces disclosed quantitatively the separation of the adhesion force in van der Waals and Lewis acid-base contributions. Further, the surface polarizability of VAlN was shown to be unaffected by oxygen incorporation due to the formation of an only gradually oxidized surface comprising a range of vanadium oxidation states. In contrast, the adhesion force analysis revealed additional Lewis acid-base interactions between the oxidized and non-oxidized VAlN surfaces and carboxyl groups present in the surface of PS after an oxidative oxygen beam treatment.

Atomic force microscopy and force spectroscopy on the assessment of protein folding and functionality.

PubMed

Carvalho, Filomena A; Martins, Ivo C; Santos, Nuno C

2013-03-01

Atomic force microscopy (AFM) applied to biological systems can, besides generating high-quality and well-resolved images, be employed to study protein folding via AFM-based force spectroscopy. This approach allowed remarkable advances in the measurement of inter- and intramolecular interaction forces with piconewton resolution. The detection of specific interaction forces between molecules based on the AFM sensitivity and the manipulation of individual molecules greatly advanced the understanding of intra-protein and protein-ligand interactions. Apart from the academic interest in the resolution of basic scientific questions, this technique has also key importance on the clarification of several biological questions of immediate biomedical relevance. Force spectroscopy is an especially appropriate technique for "mechanical proteins" that can provide crucial information on single protein molecules and/or domains. Importantly, it also has the potential of combining in a single experiment spatial and kinetic measurements. Here, the main principles of this methodology are described, after which the ability to measure interactions at the single-molecule level is discussed, in the context of relevant protein-folding examples. We intend to demonstrate the potential of AFM-based force spectroscopy in the study of protein folding, especially since this technique is able to circumvent some of the difficulties typically encountered in classical thermal/chemical denaturation studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Accurate formulas for interaction force and energy in frequency modulation force spectroscopy

NASA Astrophysics Data System (ADS)

Sader, John E.; Jarvis, Suzanne P.

2004-03-01

Frequency modulation atomic force microscopy utilizes the change in resonant frequency of a cantilever to detect variations in the interaction force between cantilever tip and sample. While a simple relation exists enabling the frequency shift to be determined for a given force law, the required complementary inverse relation does not exist for arbitrary oscillation amplitudes of the cantilever. In this letter we address this problem and present simple yet accurate formulas that enable the interaction force and energy to be determined directly from the measured frequency shift. These formulas are valid for any oscillation amplitude and interaction force, and are therefore of widespread applicability in frequency modulation dynamic force spectroscopy.

Fabrication and electrochemical properties of insoluble fullerene-diamine adduct thin-films as buffer layer by alternate immersion process

NASA Astrophysics Data System (ADS)

Saito, Jo; Akiyama, Tsuyoshi; Suzuki, Atsushi; Oku, Takeo

2017-01-01

Insoluble fullerene-diamine adduct thin-films consisting of C60 and 1,2-diaminoethane were easily fabricated on an electrode by an alternate immersion process. Formation of the C60-diamine adduct films were confirmed using transmission absorption spectroscopy and atomic force microscopy. An inverted-type organic solar cells were fabricated by using the C60-diamine adduct film as the electron transport layer. The resultant photoelectric conversation performance of the solar cells suggested that photocurrent is generated via the photoexcitation of polythiophene. The result suggests that the present insoluble fullerene-diamine adduct films worked as buffer layer for organic thin-film solar cells.

Carbon-Supported Pd and PdFe Alloy Catalysts for Direct Methanol Fuel Cell Cathodes.

PubMed

Rivera Gavidia, Luis M; Sebastián, David; Pastor, Elena; Aricò, Antonino S; Baglio, Vincenzo

2017-05-25

Direct methanol fuel cells (DMFCs) are electrochemical devices that efficiently produce electricity and are characterized by a large flexibility for portable applications and high energy density. Methanol crossover is one of the main obstacles for DMFC commercialization, forcing the search for highly electro-active and methanol tolerant cathodes. In the present work, carbon-supported Pd and PdFe catalysts were synthesized using a sodium borohydride reduction method and physico-chemically characterized using transmission electron microscopy (TEM) and X-ray techniques such as photoelectron spectroscopy (XPS), diffraction (XRD) and energy dispersive spectroscopy (EDX). The catalysts were investigated as DMFC cathodes operating at different methanol concentrations (up to 10 M) and temperatures (60 °C and 90 °C). The cell based on PdFe/C cathode presented the best performance, achieving a maximum power density of 37.5 mW·cm -2 at 90 °C with 10 M methanol, higher than supported Pd and Pt commercial catalysts, demonstrating that Fe addition yields structural changes to Pd crystal lattice that reduce the crossover effects in DMFC operation.

Carbon-Supported Pd and PdFe Alloy Catalysts for Direct Methanol Fuel Cell Cathodes

PubMed Central

Rivera Gavidia, Luis M.; Sebastián, David; Pastor, Elena; Aricò, Antonino S.; Baglio, Vincenzo

2017-01-01

Direct methanol fuel cells (DMFCs) are electrochemical devices that efficiently produce electricity and are characterized by a large flexibility for portable applications and high energy density. Methanol crossover is one of the main obstacles for DMFC commercialization, forcing the search for highly electro-active and methanol tolerant cathodes. In the present work, carbon-supported Pd and PdFe catalysts were synthesized using a sodium borohydride reduction method and physico-chemically characterized using transmission electron microscopy (TEM) and X-ray techniques such as photoelectron spectroscopy (XPS), diffraction (XRD) and energy dispersive spectroscopy (EDX). The catalysts were investigated as DMFC cathodes operating at different methanol concentrations (up to 10 M) and temperatures (60 °C and 90 °C). The cell based on PdFe/C cathode presented the best performance, achieving a maximum power density of 37.5 mW·cm−2 at 90 °C with 10 M methanol, higher than supported Pd and Pt commercial catalysts, demonstrating that Fe addition yields structural changes to Pd crystal lattice that reduce the crossover effects in DMFC operation. PMID:28772937

Self-assembled supramolecular hetero-bimetallacycles for anticancer potency via intracellular release

PubMed Central

Mishra, Anurag; Lee, Seung Chang; Kaushik, Neha; Cook, Timothy R.; Choi, Eun Ha

2015-01-01

Two new tetracationic hetero-bimetallacycles, 4 and 5, have been constructed from an N,N′-bis(4-(pyridin-4-ylethynyl)phenyl)pyridine-2,6-dicarboxamide ligand, 1, and cis-blocked complexes [M(dppf)](OTf)2 (dppf = 1,1′-bis(diphenylphosphino)ferrocene; M = Pd (2), Pt (3)) in CH3NO2/CH2Cl2 (1:1) solvent. Both complexes were isolated with adequate yields as triflate salts and were then characterized using 1H, 13C, and 31P NMR spectroscopy, elemental analysis, UV-Vis spectroscopy, and high-resolution electrospray mass spectrometry (HR-ESMS). The molecular structure of 4 was determined by molecular mechanics force field calculations. The cytotoxic effect of both new complexes were analyzed against T98G (brain tumor), KB (head and neck cancer), SNU-80 (thyroid cancer), and HEK-293 non-malignant cell lines. The cytotoxicity of complexes 4 and 5 were found to be considerably more effective against cancer cells than reference drug cisplatin. Annexin-V/PI staining, caspase-3/-7 activity, reduction in mitochondrial membrane potential justify a significant level of apoptosis in complex treated cells. PMID:25209962

Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

PubMed

Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

2012-08-08

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Reduced graphene oxide growth on 316L stainless steel for medical applications

NASA Astrophysics Data System (ADS)

Cardenas, L.; MacLeod, J.; Lipton-Duffin, J.; Seifu, D. G.; Popescu, F.; Siaj, M.; Mantovani, D.; Rosei, F.

2014-07-01

We report a new method for the growth of reduced graphene oxide (rGO) on the 316L alloy of stainless steel (SS) and its relevance for biomedical applications. We demonstrate that electrochemical etching increases the concentration of metallic species on the surface and enables the growth of rGO. This result is supported through a combination of Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM), density functional theory (DFT) calculations and static water contact angle measurements. Raman spectroscopy identifies the G and D bands for oxidized species of graphene at 1595 cm-1 and 1350 cm-1, respectively, and gives an ID/IG ratio of 1.2, indicating a moderate degree of oxidation. XPS shows -OH and -COOH groups in the rGO stoichiometry and static contact angle measurements confirm the wettability of rGO. SEM and AFM measurements were performed on different substrates before and after coronene treatment to confirm rGO growth. Cell viability studies reveal that these rGO coatings do not have toxic effects on mammalian cells, making this material suitable for biomedical and biotechnological applications.

Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

PubMed

Angely, Christelle; Nguyen, Ngoc-Minh; Andre Dias, Sofia; Planus, Emmanuelle; Pelle, Gabriel; Louis, Bruno; Filoche, Marcel; Chenal, Alexandre; Ladant, Daniel; Isabey, Daniel

2017-08-01

The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure <1 h. At 30 min of exposure, CyaA toxin has a minimal effect on cell viability (>95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 μm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in intracellular cAMP in these target cells. These results suggest that mechanical and adhesion properties of the cells appear as pertinent markers of cytotoxicity of CyaA toxin. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Surface Characterization.

ERIC Educational Resources Information Center

Fulghum, J. E.; And Others

1989-01-01

This review is divided into the following analytical methods: ion spectroscopy, electron spectroscopy, scanning tunneling microscopy, atomic force microscopy, optical spectroscopy, desorption techniques, and X-ray techniques. (MVL)

Matrix Isolation Spectroscopy Applied to Positron Moderatioin in Cryogenic Solids

DTIC Science & Technology

2011-07-01

Current Positron Applications • 2-γ decay exploited in Positron Emission Tomography (PET) scanners. • Positrons localize & annihilate preferentially at...Air Force  Eglin Air Force Base AFRL-RW-EG-TP-2011-024 Matrix Isolation Spectroscopy Applied to Positron Moderation in Cryogenic Solids Distribution... Spectroscopy Applied to Positron Moderation in Cryogenic Solids 5a. CONTRACT NUMBER 5b. GRANT NUMBER 62602F 5c. PROGRAM ELEMENT NUMBER 6

Atomic force microscope-assisted scanning tunneling spectroscopy under ambient conditions.

PubMed

Vakhshouri, Amin; Hashimoto, Katsushi; Hirayama, Yoshiro

2014-12-01

We have developed a method of atomic force microscopy (AFM)-assisted scanning tunneling spectroscopy (STS) under ambient conditions. An AFM function is used for rapid access to a selected position prior to performing STS. The AFM feedback is further used to suppress vertical thermal drift of the tip-sample distance during spectroscopy, enabling flexible and stable spectroscopy measurements at room temperature. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A pre-therapeutic coating for medical devices that prevents the attachment of Candida albicans.

PubMed

Vargas-Blanco, Diego; Lynn, Aung; Rosch, Jonah; Noreldin, Rony; Salerni, Anthony; Lambert, Christopher; Rao, Reeta P

2017-05-19

Hospital acquired fungal infections are defined as "never events"-medical errors that should never have happened. Systemic Candida albicans infections results in 30-50% mortality rates. Typically, adhesion to abiotic medical devices and implants initiates such infections. Efficient adhesion initiates formation of aggressive biofilms that are difficult to treat. Therefore, inhibitors of adhesion are important for drug development and likely to have a broad spectrum efficacy against many fungal pathogens. In this study we further the development of a small molecule, Filastatin, capable of preventing C. albicans adhesion. We explored the potential of Filastatin as a pre-therapeutic coating of a diverse range of biomaterials. Filastatin was applied on various biomaterials, specifically bioactive glass (cochlear implants, subcutaneous drug delivery devices and prosthetics); silicone (catheters and other implanted devices) and dental resin (dentures and dental implants). Adhesion to biomaterials was evaluated by direct visualization of wild type C. albicans or a non-adherent mutant edt1 -/- that were stained or fluorescently tagged. Strains grown overnight at 30 °C were harvested, allowed to attach to surfaces for 4 h and washed prior to visualization. The adhesion force of C. albicans cells attached to surfaces treated with Filastatin was measured using Atomic Force Microscopy. Effectiveness of Filastatin was also demonstrated under dynamic conditions using a flow cell bioreactor. The effect of Filastatin under microfluidic flow conditions was quantified using electrochemical impedance spectroscopy. Experiments were typically performed in triplicate. Treatment with Filastatin significantly inhibited the ability of C. albicans to adhere to bioactive glass (by 99.06%), silicone (by 77.27%), and dental resin (by 60.43%). Atomic force microcopy indicated that treatment with Filastatin decreased the adhesion force of C. albicans from 0.23 to 0.017 nN. Electrochemical Impedance Spectroscopy in a microfluidic device that mimic physiological flow conditions in vivo showed lower impedance for C. albicans when treated with Filastatin as compared to untreated control cells, suggesting decreased attachment. The anti-adhesive properties were maintained when Filastatin was included in the preparation of silicone materials. We demonstrate that Filastatin treated medical devices prevented adhesion of Candida, thereby reducing nosocomial infections.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

PubMed Central

Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

2015-01-01

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence. PMID:26038837

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

PubMed

Ghamrawi, Sarah; Gastebois, Amandine; Zykwinska, Agata; Vandeputte, Patrick; Marot, Agnès; Mabilleau, Guillaume; Cuenot, Stéphane; Bouchara, Jean-Philippe

2015-01-01

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Optical tweezers for single molecule force spectroscopy on bacterial adhesion organelles

NASA Astrophysics Data System (ADS)

Andersson, Magnus; Axner, Ove; Uhlin, Bernt Eric; Fällman, Erik

2006-08-01

Instrumentation and methodologies for single molecule force spectroscopy on bacterial adhesion organelles by the use of force measuring optical tweezers have been developed. A thorough study of the biomechanical properties of fimbrial adhesion organelles expressed by uropathogenic E. coli, so-called pili, is presented. Steady-state as well as dynamic force measurements on P pili, expressed by E. coli causing pyelonephritis, have revealed, among other things, various unfolding and refolding properties of the helical structure of P pili, the PapA rod. Based on these properties an energy landscape model has been constructed by which specific biophysical properties of the PapA rod have been extracted, e.g. the number of subunits, the length of a single pilus, bond lengths and activation energies for bond opening and closure. Moreover, long time repetitive measurements have shown that the rod can be unfolded and refolded repetitive times without losing its intrinsic properties. These properties are believed to be of importance for the bacteria's ability to maintain close contact with host cells during initial infections. The results presented are considered to be of importance for the field of biopolymers in general and the development of new pharmaceuticals towards urinary tract infections in particular. The results show furthermore that the methodology can be used to gain knowledge of the intrinsic biomechanical function of adhesion organelles. The instrumentation is currently used for characterization of type 1 pili, expressed by E. coli causing cystitis, i.e. infections in the bladder. The first force spectrometry investigations of these pili will be presented.

Athermalization in atomic force microscope based force spectroscopy using matched microstructure coupling.

PubMed

Torun, H; Finkler, O; Degertekin, F L

2009-07-01

The authors describe a method for athermalization in atomic force microscope (AFM) based force spectroscopy applications using microstructures that thermomechanically match the AFM probes. The method uses a setup where the AFM probe is coupled with the matched structure and the displacements of both structures are read out simultaneously. The matched structure displaces with the AFM probe as temperature changes, thus the force applied to the sample can be kept constant without the need for a separate feedback loop for thermal drift compensation, and the differential signal can be used to cancel the shift in zero-force level of the AFM.

On artifacts in single-molecule force spectroscopy

PubMed Central

Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

2015-01-01

In typical force spectroscopy experiments, a small biomolecule is attached to a soft polymer linker that is pulled with a relatively large bead or cantilever. At constant force, the total extension stochastically changes between two (or more) values, indicating that the biomolecule undergoes transitions between two (or several) conformational states. In this paper, we consider the influence of the dynamics of the linker and mesoscopic pulling device on the force-dependent rate of the conformational transition extracted from the time dependence of the total extension, and the distribution of rupture forces in force-clamp and force-ramp experiments, respectively. For these different experiments, we derive analytic expressions for the observables that account for the mechanical response and dynamics of the pulling device and linker. Possible artifacts arise when the characteristic times of the pulling device and linker become comparable to, or slower than, the lifetimes of the metastable conformational states, and when the highly anharmonic regime of stretched linkers is probed at high forces. We also revisit the problem of relating force-clamp and force-ramp experiments, and identify a linker and loading rate-dependent correction to the rates extracted from the latter. The theory provides a framework for both the design and the quantitative analysis of force spectroscopy experiments by highlighting, and correcting for, factors that complicate their interpretation. PMID:26540730

Rapid internal contraction boosts DNA friction.

PubMed

Otto, Oliver; Sturm, Sebastian; Laohakunakorn, Nadanai; Keyser, Ulrich F; Kroy, Klaus

2013-01-01

Macroscopic objects are usually manipulated by force and observed with light. On the nanoscale, however, this is often done oppositely: individual macromolecules are manipulated by light and monitored with force. This procedure, which is the basis of single-molecule force spectroscopy, has led to much of our quantitative understanding of how DNA works, and is now routinely applied to explore molecular structure and interactions, DNA-protein reactions and protein folding. Here we develop the technique further by introducing a dynamic force spectroscopy set-up for a non-invasive inspection of the tension dynamics in a taut strand of DNA. The internal contraction after a sudden release of the molecule is shown to give rise to a drastically enhanced viscous friction, as revealed by the slow relaxation of an attached colloidal tracer. Our systematic theory explains the data quantitatively and provides a powerful tool for the rational design of new dynamic force spectroscopy assays.

Probing fibronectin–antibody interactions using AFM force spectroscopy and lateral force microscopy

PubMed Central

Kulik, Andrzej J; Lee, Kyumin; Pyka-Fościak, Grazyna; Nowak, Wieslaw

2015-01-01

Summary The first experiment showing the effects of specific interaction forces using lateral force microscopy (LFM) was demonstrated for lectin–carbohydrate interactions some years ago. Such measurements are possible under the assumption that specific forces strongly dominate over the non-specific ones. However, obtaining quantitative results requires the complex and tedious calibration of a torsional force. Here, a new and relatively simple method for the calibration of the torsional force is presented. The proposed calibration method is validated through the measurement of the interaction forces between human fibronectin and its monoclonal antibody. The results obtained using LFM and AFM-based classical force spectroscopies showed similar unbinding forces recorded at similar loading rates. Our studies verify that the proposed lateral force calibration method can be applied to study single molecule interactions. PMID:26114080

Detection and Classification of Live and Dead Escherichia coli by Laser-Induced Breakdown Spectroscopy

PubMed Central

Sivakumar, P.; Fernández-Bravo, A.; Taleh, L.; Biddle, J.F.

2015-01-01

Abstract A common goal for astrobiology is to detect organic materials that may indicate the presence of life. However, organic materials alone may not be representative of currently living systems. Thus, it would be valuable to have a method with which to determine the health of living materials. Here, we present progress toward this goal by reporting on the application of laser-induced breakdown spectroscopy (LIBS) to study characteristics of live and dead cells using Escherichia coli (E. coli) strain K12 cells as a model organism since its growth and death in the laboratory are well understood. Our goal is to determine whether LIBS, in its femto- and/or nanosecond forms, could ascertain the state of a living organism. E. coli strain K12 cells were grown, collected, and exposed to one of two types of inactivation treatments: autoclaving and sonication. Cells were also kept alive as a control. We found that LIBS yields key information that allows for the discrimination of live and dead E. coli bacteria based on ionic shifts reflective of cell membrane integrity. Key Words: E. coli—Trace elements—Live and dead cells—Laser-induced breakdown spectroscopy—Atomic force microscopy. Astrobiology 15, 144–153. PMID:25683088

Minimizing pulling geometry errors in atomic force microscope single molecule force spectroscopy.

PubMed

Rivera, Monica; Lee, Whasil; Ke, Changhong; Marszalek, Piotr E; Cole, Daniel G; Clark, Robert L

2008-10-01

In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies.

Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells.

PubMed

Millaku, Agron; Drobne, Damjana; Torkar, Matjaz; Novak, Sara; Remškar, Maja; Pipan-Tkalec, Živa

2013-09-15

We provide data obtained by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) on the interaction of ingested tungsten nanofibers with epithelial cells of the digestive tubes of a test organism Porcellio scaber. Conventional toxicity endpoints including feeding behaviour, weight loss and mortality were also measured in each investigated animal. No toxicity was detected in any of exposed animals after 14 days of feeding on tungsten nanofiber dosed food, but when nanofibers enter the digestive system they can react with epithelial cells of the digestive tubes, becoming physically inserted into the cells. In this way, nanofibers can injure the epithelial cells of digestive gland tubes when they are ingested with food. Our SEM data suggest that peristaltic forces may have an important role, not predicted by in vitro experiments, in the interactions of nanomaterials with digestive intestinal cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis*

PubMed Central

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M.

2010-01-01

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed. PMID:20382731

Single-molecule force spectroscopy approach to enzyme catalysis.

PubMed

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M

2010-06-18

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.

Analysis of acid-base interactions at Al2O3 (11-20) interfaces by means of single molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Mosebach, Bastian; Ozkaya, Berkem; Giner, Ignacio; Keller, Adrian; Grundmeier, Guido

2017-10-01

Single molecule force spectroscopy (SMFS) was employed to investigate the interaction forces between aliphatic amino, hydroxyl and ether groups and aluminum oxide single crystal surfaces in an aqueous electrolyte at pH = 6. The force studies were based on the variation of the terminal group of polyethylene glycol which was bound via a Ssbnd Au bond to the gold coated AFM tip. X-ray Photoelectron Spectroscopy (XPS) was performed to characterize the surface chemistry of the substrate. Force distance curves were measured between the PEG-NH2, sbnd OH and sbnd OCH3 functionalized atomic force microscope (AFM) tip and the non-polar single crystalline Al2O3(11-20) surface. The experimental results exhibit non-equilibrium desorption events which hint at acid-base interactions of the electron donating hydroxyl and amino groups with Al-ions in the surface of the oxide. The observed desorption forces for the sbnd NH2, sbnd OH/Al2O3(11-20) were in the range of 100-200 pN.

Acidic Extracellular pH Promotes Activation of Integrin αvβ3

PubMed Central

Paradise, Ranjani K.; Lauffenburger, Douglas A.; Van Vliet, Krystyn J.

2011-01-01

Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin αvβ3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the αvβ3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin αvβ3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer. PMID:21283814

Mechanical properties of complex biological systems using AFM-based force spectroscopy

NASA Astrophysics Data System (ADS)

Graham, John Stephen

An atomic force microscope (AFM) was designed and built to study the mechanical properties of small collagen fibrils and the plasma membrane of living cells. Collagen is a major component of bone, skin and connective tissues, and is abundant in the extracellular matrix (ECM). Because of its abundance, an understanding of how disease affects collagen mechanics is crucial in disease prevention efforts. Two levels of type I collagen structure were investigated, subfibrils (on the order of 1 mum in length) and longer fibrils. Comparisons were made between measurements of wild-type (wt) collagen and collagen from the mouse model of osteogenesis imperfecta (OI). Significant differences between OI and wt collagen were observed, primarily that intermolecular bonds in OI collagen fibrils are weaker than in wt, or not ruptured, as in the case of OI subfibrils. As cells interact with collagen in the ECM, the mechanical properties of the plasma membrane are also of great interest. Membrane tethers were extracted from living cells under varied conditions in order to assess the contributions of membrane-associated macromolecules such as the actin cytoskeleton and the glycocalyx, and intracellular signaling. Tether extraction force was found to be sensitive to all of these altered conditions, suggesting that tether extraction may be used to monitor various cellular processes.

Enhanced electrical properties in sub-10-nm WO3 nanoflakes prepared via a two-step sol-gel-exfoliation method

PubMed Central

2014-01-01

The morphology and electrical properties of orthorhombic β-WO3 nanoflakes with thickness of ~7 to 9 nm were investigated at the nanoscale with a combination of scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), current sensing force spectroscopy atomic force microscopy (CSFS-AFM, or PeakForce TUNA™), Fourier transform infra-red absorption spectroscopy (FTIR), linear sweep voltammetry (LSV) and Raman spectroscopy techniques. CSFS-AFM analysis established good correlation between the topography of the developed nanostructures and various features of WO3 nanoflakes synthesized via a two-step sol-gel-exfoliation method. It was determined that β-WO3 nanoflakes annealed at 550°C possess distinguished and exceptional thickness-dependent properties in comparison with the bulk, micro and nanostructured WO3 synthesized at alternative temperatures. PMID:25221453

Effect of small peptide (P-15) on HJMSCs adhesion to hydroxyap-atite

NASA Astrophysics Data System (ADS)

Cheng, Wei; Tong, Xin; Hu, QinGang; Mou, YongBin; Qin, HaiYan

2016-02-01

P-15, a synthetic peptide of 15-amino acids, has been tested in clinical trials to enhance cell adhesion and promote osseointe- gration. This feature of P-15 has also inspired the development of designing new bone substitute materials. Despite the increasing applications of P-15 in bone graft alternatives, few studies focus on the mechanism of cell adhesion promoted by P-15 and the mechanical property changes of the cells interacting with P-15. In this article, we used atomic force microscope (AFM) based single cell indentation force spectroscopy to study the impact of P-15 on the stiffness and the adhesion ability of human jaw bone mesenchymal stem cells (HJMSCs) to hydroxyapatite (HA). We found that the stiffness of HJMSCs increases as the concentration of P-15 grows in short culture intervals and that the adhesion forces between HJMSCs and HA particles in both the presence and absence of P-15 are all around 30pN. Moreover, by calculating the binding energy of HJMSCs to HA particles mixed with and without P-15, we proved that P-15 could increase the adhesion energy by nearly four times. Scanning electron microscope (SEM) was also exploited to study the morphology of HJMSCs cultured in the presence and absence of P-15 on HA disc surface for a short term. Apparent morphological differences were observed between the cells cultured with and without P-15. These results explain the probable underlying adhesion mechanism of HJMSC promoted by P-15 and can serve as the bases for the design of bone graft substitute materials.

Determination of the nano-scaled contact area of staphylococcal cells.

PubMed

Spengler, Christian; Thewes, Nicolas; Jung, Philipp; Bischoff, Markus; Jacobs, Karin

2017-07-20

Bacterial adhesion is a crucial step during the development of infections as well as the formation of biofilms. Hence, fundamental research of bacterial adhesion mechanisms is of utmost importance. So far, less is known about the size of the contact area between bacterial cells and a surface. This gap will be filled by this study using a single-cell force spectroscopy-based method to investigate the contact area between a single bacterial cell of Staphylococcus aureus and a solid substrate. The technique relies on the strong influence of the hydrophobic interaction on bacterial adhesion: by incrementally crossing a very sharp hydrophobic/hydrophilic interface while performing force-distance curves with a single bacterial probe, the bacterial contact area can be determined. Assuming circular contact areas, their radii - determined in our experiments - are in the range from tens of nanometers to a few hundred nanometers. The contact area can be slightly enlarged by a larger load force, yet does not resemble a Hertzian contact, rather, the enlargement is a property of the individual bacterial cell. Additionally, Staphylococcus carnosus has been probed, which is less adherent than S. aureus, yet both bacteria exhibit a similar contact area size. This corroborates the notion that the adhesive strength of bacteria is not a matter of contact area, but rather a matter of which and how many molecules of the bacterial species' cell wall form the contact. Moreover, our method of determining the contact area can be applied to other microorganisms and the results might also be useful for studies using nanoparticles covered with soft, macromolecular coatings.

High-permeability functionalized silicone magnetic microspheres with low autofluorescence for biomedical applications

PubMed Central

Evans, Benjamin A.; Ronecker, Julia C.; Han, David T.; Glass, Daniel R.; Train, Tonya L.; Deatsch, Alison E.

2017-01-01

Functionalized magnetic microspheres are widely used for cell separations, isolation of proteins and other biomolecules, in vitro diagnostics, tissue engineering, and microscale force spectroscopy. We present here the synthesis and characterization of a silicone magnetic microsphere which can be produced in diameters ranging from 0.5 to 50 μm via emulsion polymerization of a silicone ferrofluid precursor. This bottom-up approach to synthesis ensures a uniform magnetic concentration across all sizes, leading to significant advances in magnetic force generation. We demonstrate that in a size range of 5–20 μm, these spheres supply a full order of magnitude greater magnetic force than leading commercial products. In addition, the unique silicone matrix exhibits autofluorescence two orders of magnitude lower than polystyrene microspheres. Finally, we demonstrate the ability to chemically functionalize our silicone microspheres using a standard EDC reaction, and show that our folate-functionalized silicone microspheres specifically bind to targeted HeLa and Jurkat cells. These spheres show tremendous potential for replacing magnetic polystyrene spheres in applications which require either large magnetic forces or minimal autofluorescence, since they represent order-of-magnitude improvements in each. In addition, the unique silicone matrix and proven biocompatibility suggest that they may be useful for encapsulation and targeted delivery of lipophilic pharmaceuticals. PMID:26952493

Characterization of new drug delivery nanosystems using atomic force microscopy

NASA Astrophysics Data System (ADS)

Spyratou, Ellas; Mourelatou, Elena A.; Demetzos, C.; Makropoulou, Mersini; Serafetinides, A. A.

2015-01-01

Liposomes are the most attractive lipid vesicles for targeted drug delivery in nanomedicine, behaving also as cell models in biophotonics research. The characterization of the micro-mechanical properties of drug carriers is an important issue and many analytical techniques are employed, as, for example, optical tweezers and atomic force microscopy. In this work, polyol hyperbranched polymers (HBPs) have been employed along with liposomes for the preparation of new chimeric advanced drug delivery nanosystems (Chi-aDDnSs). Aliphatic polyester HBPs with three different pseudogenerations G2, G3 and G4 with 16, 32, and 64 peripheral hydroxyl groups, respectively, have been incorporated in liposomal formulation. The atomic force microscopy (AFM) technique was used for the comparative study of the morphology and the mechanical properties of Chi-aDDnSs and conventional DDnS. The effects of both the HBPs architecture and the polyesters pseudogeneration number in the stability and the stiffness of chi-aDDnSs were examined. From the force-distance curves of AFM spectroscopy, the Young's modulus was calculated.

Highly enhanced compatibility of human brain vascular pericyte cells on monolayer graphene

PubMed Central

Kim, Jangheon; Kim, Soohyun; Jung, Wonsuk

2017-01-01

ABSTRACT We introduce a method for increasing the compatibility of human brain vascular pericyte (HBVP) cells on a glass substrate, based on wet transferred monolayer graphene without any treatment. As a novel material, graphene has key properties for incubating cells, such as chemical stability, transparency, appropriate roughness, hydrophobicity and high electrical conductivity. These outstanding properties of graphene were examined by Raman spectroscopy, water contact angle measurements and atomic force microscopy. The performance of this graphene-based implant was investigated by a cell compatibility test, comparing the growth rate of cells on the graphene surface and that on a bare glass substrate. After an incubation period of 72 h, the number of live HBVP cells on a graphene surface with an area of 1×1 mm2 was 1.83 times greater than that on the glass substrate. PMID:27689961

Preparation and biocompatibility of crosslinked poly(3-hydroxyundecenoate).

PubMed

Chung, Chungwook; Chung, Kyudon; Kim, Do Young; Lee, Sun Hee; Kim, Jong-Sik; Rhee, Young Ha

2018-02-01

A sticky polymer, poly(3-hydroxyundecenoate) (PHU), was produced by Pseudomonas oleovorans when nonanoate and undecenoate were used as carbon sources. Crosslinked PHU (CL-PHU) was prepared by heating using benzoyl peroxide as a crosslinker. According to the degree of crosslinking in the polymer, three types of CL-PHU were prepared: CL-PHU50, CL-PHU60 and CL-PHU70. Fourier transform-infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry results suggested that crosslinking of PHU was successfully achieved by heat, which increased the crosslinking density and decreased stiffness and flexibility of the polymer. Water contact angle measurements revealed no differences of hydrophilicity as the crosslinking density. Slight morphological changes of CL-PHU film surfaces were observed by atomic force microscopy. Chinese hamster ovary cells were used to investigate the biocompatibility of CL-PHU films using poly(l-lactide) surfaces as control. Surface properties of the film, such as roughness and adhesive force, enhanced the adhesion and proliferation of cells on the films. CL-PHU might be useful for cell compatible biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Probing Cytoskeletal Structures by Coupling Optical Superresolution and AFM Techniques for a Correlative Approach

PubMed Central

Chacko, Jenu Varghese; Zanacchi, Francesca Cella; Diaspro, Alberto

2013-01-01

In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Young's modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution-AFM working is a clear example of correlative multimodal microscopy. PMID:24027190

Atomic force microscopy studies on molybdenum disulfide flakes as sodium-ion anodes.

PubMed

Lacey, Steven D; Wan, Jiayu; von Wald Cresce, Arthur; Russell, Selena M; Dai, Jiaqi; Bao, Wenzhong; Xu, Kang; Hu, Liangbing

2015-02-11

A microscale battery comprised of mechanically exfoliated molybdenum disulfide (MoS2) flakes with copper connections and a sodium metal reference was created and investigated as an intercalation model using in situ atomic force microscopy in a dry room environment. While an ethylene carbonate-based electrolyte with a low vapor pressure allowed topographical observations in an open cell configuration, the planar microbattery was used to conduct in situ measurements to understand the structural changes and the concomitant solid electrolyte interphase (SEI) formation at the nanoscale. Topographical observations demonstrated permanent wrinkling behavior of MoS2 electrodes upon sodiation at 0.4 V. SEI formation occurred quickly on both flake edges and planes at voltages before sodium intercalation. Force spectroscopy measurements provided quantitative data on the SEI thickness for MoS2 electrodes in sodium-ion batteries for the first time.

Spatial and temporal control of the diazonium modification of sp2 carbon surfaces.

PubMed

Kirkman, Paul M; Güell, Aleix G; Cuharuc, Anatolii S; Unwin, Patrick R

2014-01-08

Interest in the controlled chemical functionalization of sp(2) carbon materials using diazonium compounds has been recently reignited, particularly as a means to generate a band gap in graphene. We demonstrate local diazonium modification of pristine sp(2) carbon surfaces, with high control, at the micrometer scale through the use of scanning electrochemical cell microscopy (SECCM). Electrochemically driven diazonium patterning is investigated at a range of driving forces, coupled with surface analysis using atomic force microscopy (AFM) and Raman spectroscopy. We highlight how the film density, level of sp(2)/sp(3) rehybridization and the extent of multilayer formation can be controlled, paving the way for the use of localized electrochemistry as a route to controlled diazonium modification.

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy.

PubMed

Das, Priyadip; Duanias-Assaf, Tal; Reches, Meital

2017-03-06

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Unraveling protein-protein interactions in clathrin assemblies via atomic force spectroscopy.

PubMed

Jin, Albert J; Lafer, Eileen M; Peng, Jennifer Q; Smith, Paul D; Nossal, Ralph

2013-03-01

Atomic force microscopy (AFM), single molecule force spectroscopy (SMFS), and single particle force spectroscopy (SPFS) are used to characterize intermolecular interactions and domain structures of clathrin triskelia and clathrin-coated vesicles (CCVs). The latter are involved in receptor-mediated endocytosis (RME) and other trafficking pathways. Here, we subject individual triskelia, bovine-brain CCVs, and reconstituted clathrin-AP180 coats to AFM-SMFS and AFM-SPFS pulling experiments and apply novel analytics to extract force-extension relations from very large data sets. The spectroscopic fingerprints of these samples differ markedly, providing important new information about the mechanism of CCV uncoating. For individual triskelia, SMFS reveals a series of events associated with heavy chain alpha-helix hairpin unfolding, as well as cooperative unraveling of several hairpin domains. SPFS of clathrin assemblies exposes weaker clathrin-clathrin interactions that are indicative of inter-leg association essential for RME and intracellular trafficking. Clathrin-AP180 coats are energetically easier to unravel than the coats of CCVs, with a non-trivial dependence on force-loading rate. Published by Elsevier Inc.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy.

PubMed

Anselmetti, Dario; Bartels, Frank Wilco; Becker, Anke; Decker, Björn; Eckel, Rainer; McIntosh, Matthew; Mattay, Jochen; Plattner, Patrik; Ros, Robert; Schäfer, Christian; Sewald, Norbert

2008-02-19

Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

PubMed

Carrion-Vazquez, M; Oberhauser, A F; Fisher, T E; Marszalek, P E; Li, H; Fernandez, J M

2000-01-01

Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).

Fabrication of bi-layer graphene and theoretical simulation for its possible application in thin film solar cell.

PubMed

Behura, Sanjay K; Mahala, Pramila; Nayak, Sasmita; Yang, Qiaoqin; Mukhopadhyay, Indrajit; Janil, Omkar

2014-04-01

High quality graphene film is fabricated using mechanical exfoliation of highly-oriented pyrolytic graphite. The graphene films on glass substrates are characterized using field-emission scanning electron microscopy, atomic force microscopy, Raman spectroscopy, UV-vis spectroscopy and Fourier transform infrared spectroscopy. A very high intensity ratio of 2D to G-band (to approximately 1.67) and narrow 2D-band full-width at half maximum (to approximately 40 cm(-1)) correspond to the bi-layer graphene formation. The bi-layer graphene/p-GaN/n-InGaN/n-GaN/GaN/sAl2O3 system is studied theoretically using TCAD Silvaco software, in which the properties of exfoliated bi-layer graphene are used as transparent and conductive film, and the device exhibits an efficiency of 15.24% compared to 13.63% for ITO/p-GaN/n-InGaN/n-GaN/GaN/Al2O3 system.

Preparation and characterization of green graphene using grape seed extract for bioapplications.

PubMed

Yaragalla, Srinivasarao; Rajendran, Rajakumari; Jose, Jiya; AlMaadeed, Mariam A; Kalarikkal, Nandakumar; Thomas, Sabu

2016-08-01

The development of functionalized graphene materials concerning health and environmental aspects via green approaches is currently the most recent topic in the field of nanoscience and nanotechnology. Herein, we report the green reduction of graphene oxide (GO) to reduced graphene oxide (RGO) using grape seed extract (GSE). Structural properties of the prepared RGO were investigated using Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, thermogravimetric analysis (TGA), UV-Visible spectroscopy and X-ray diffraction analysis. These all characterization techniques clearly revealed that the RGO has been successfully prepared. Moreover, the average thickness (4.2nm) of RGO layers was also confirmed by transmission electron microscopy (TEM). Optical properties such as band gap and photoluminescence of the synthesized RGO were evaluated. The band gap of RGO was found to be 3.84eV and it showed emission in the visible region. Efficient antimicrobial activity against Escherichia coli and Staphylococcus aureus was observed with 4μgml(-1) & 5μgml(-1) of RGO and also the cell wall damage of these strains has been proved by atomic force microscopy (AFM). The in vitro study of RGO (500μg) disclosed the effective anti-proliferative activity (88%) against HCT-116 cell lines. Copyright © 2016 Elsevier B.V. All rights reserved.

In vitro biomechanical properties, fluorescence imaging, surface-enhanced Raman spectroscopy, and photothermal therapy evaluation of luminescent functionalized CaMoO4:Eu@Au hybrid nanorods on human lung adenocarcinoma epithelial cells

PubMed Central

Li, Qifei; Parchur, Abdul K.; Zhou, Anhong

2016-01-01

Abstract Highly dispersible Eu3+-doped CaMoO4@Au-nanorod hybrid nanoparticles (HNPs) exhibit optical properties, such as plasmon resonances in the near-infrared region at 790 nm and luminescence at 615 nm, offering multimodal capabilities: fluorescence imaging, surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT). HNPs were conjugated with a Raman reporter (4-mercaptobenzoic acid), showing a desired SERS signal (enhancement factor 5.0 × 105). The HNPs have a heat conversion efficiency of 25.6%, and a hyperthermia temperature of 42°C could be achieved by adjusting either concentration of HNPs, or laser power, or irradiation time. HNPs were modified with antibody specific to cancer biomarker epidermal growth factor receptor, then applied to human lung cancer (A549) and mouse hepatocyte cells (AML12), and in vitro PTT effect was studied. In addition, the biomechanical properties of A549 cells were quantified using atomic force microscopy. This study shows the potential applications of these HNPs in fluorescence imaging, SERS detection, and PTT with good photostability and biocompatibility. PMID:27877887

A surface-mediated siRNA delivery system developed with chitosan/hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly

NASA Astrophysics Data System (ADS)

Wu, Lijuan; Wu, Changlin; Liu, Guangwan; Liao, Nannan; Zhao, Fang; Yang, Xuxia; Qu, Hongyuan; Peng, Bo; Chen, Li; Yang, Guang

2016-12-01

siRNA delivery remains highly challenging because of its hydrophilic and anionic nature and its sensitivity to nuclease degradation. Effective siRNA loading and improved transfection efficiency into cells represents a key problem. In our study, we prepared Chitosan/Hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly, in which siRNAs can be effectively loaded and protected. The construction process was characterized by FTIR, 13C NMR (CP/MAS), UV-vis spectroscopy, and atomic force microscopy (AFM). We presented the controlled-release performance of the films during incubation in 1 M NaCl solution for several days through UV-vis spectroscopy and polyacrylamide gel electrophoresis (PAGE). Additionally, we verified the stability and integrity of the siRNA loaded on multilayer films. Finally, the biological efficacy of the siRNA delivery system was evaluated via cells adhesion and gene silencing analyses in eGFP-HEK 293T cells. This new type of surface-mediated non-viral multilayer films may have considerable potential in the localized and controlled-release delivery of siRNA in mucosal tissues, and tissue engineering application.

Microwave-assisted green synthesis of superparamagnetic nanoparticles using fruit peel extracts: surface engineering, T 2 relaxometry, and photodynamic treatment potential.

PubMed

Bano, Shazia; Nazir, Samina; Nazir, Alia; Munir, Saeeda; Mahmood, Tariq; Afzal, Muhammad; Ansari, Farzana Latif; Mazhar, Kehkashan

2016-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have the potential to be used as multimodal imaging and cancer therapy agents due to their excellent magnetism and ability to generate reactive oxygen species when exposed to light. We report the synthesis of highly biocompatible SPIONs through a facile green approach using fruit peel extracts as the biogenic reductant. This green synthesis protocol involves the stabilization of SPIONs through coordination of different phytochemicals. The SPIONs were functionalized with polyethylene glycol (PEG)-6000 and succinic acid and were extensively characterized by X-ray diffraction analysis, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy, Rutherford backscattering spectrometry, diffused reflectance spectroscopy, fluorescence emission, Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, and magnetization analysis. The developed SPIONs were found to be stable, almost spherical with a size range of 17-25 nm. They exhibited excellent water dispersibility, colloidal stability, and relatively high R 2 relaxivity (225 mM(-1) s(-1)). Cell viability assay data revealed that PEGylation or carboxylation appears to significantly shield the surface of the particles but does not lead to improved cytocompatibility. A highly significant increase of reactive oxygen species in light-exposed samples was found to play an important role in the photokilling of human cervical epithelial malignant carcinoma (HeLa) cells. The bio-SPIONs developed are highly favorable for various biomedical applications without risking interference from potentially toxic reagents.

Microwave-assisted green synthesis of superparamagnetic nanoparticles using fruit peel extracts: surface engineering, T2 relaxometry, and photodynamic treatment potential

PubMed Central

Bano, Shazia; Nazir, Samina; Nazir, Alia; Munir, Saeeda; Mahmood, Tariq; Afzal, Muhammad; Ansari, Farzana Latif; Mazhar, Kehkashan

2016-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have the potential to be used as multimodal imaging and cancer therapy agents due to their excellent magnetism and ability to generate reactive oxygen species when exposed to light. We report the synthesis of highly biocompatible SPIONs through a facile green approach using fruit peel extracts as the biogenic reductant. This green synthesis protocol involves the stabilization of SPIONs through coordination of different phytochemicals. The SPIONs were functionalized with polyethylene glycol (PEG)-6000 and succinic acid and were extensively characterized by X-ray diffraction analysis, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy, Rutherford backscattering spectrometry, diffused reflectance spectroscopy, fluorescence emission, Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, and magnetization analysis. The developed SPIONs were found to be stable, almost spherical with a size range of 17–25 nm. They exhibited excellent water dispersibility, colloidal stability, and relatively high R2 relaxivity (225 mM−1 s−1). Cell viability assay data revealed that PEGylation or carboxylation appears to significantly shield the surface of the particles but does not lead to improved cytocompatibility. A highly significant increase of reactive oxygen species in light-exposed samples was found to play an important role in the photokilling of human cervical epithelial malignant carcinoma (HeLa) cells. The bio-SPIONs developed are highly favorable for various biomedical applications without risking interference from potentially toxic reagents. PMID:27570452

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

PubMed

Wang, Congzhou; Jin, Yingzi; Desai, Umesh R; Yadavalli, Vamsi K

2015-06-01

The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Reconstructing Folding Energy Landscapes by Single-Molecule Force Spectroscopy

PubMed Central

Woodside, Michael T.; Block, Steven M.

2015-01-01

Folding may be described conceptually in terms of trajectories over a landscape of free energies corresponding to different molecular configurations. In practice, energy landscapes can be difficult to measure. Single-molecule force spectroscopy (SMFS), whereby structural changes are monitored in molecules subjected to controlled forces, has emerged as a powerful tool for probing energy landscapes. We summarize methods for reconstructing landscapes from force spectroscopy measurements under both equilibrium and nonequilibrium conditions. Other complementary, but technically less demanding, methods provide a model-dependent characterization of key features of the landscape. Once reconstructed, energy landscapes can be used to study critical folding parameters, such as the characteristic transition times required for structural changes and the effective diffusion coefficient setting the timescale for motions over the landscape. We also discuss issues that complicate measurement and interpretation, including the possibility of multiple states or pathways and the effects of projecting multiple dimensions onto a single coordinate. PMID:24895850

Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

PubMed

Dutta, Samrat; Armitage, Bruce A; Lyubchenko, Yuri L

2016-03-15

Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

Molecular Mechanistic Insights into the Endothelial Receptor Mediated Cytoadherence of Plasmodium falciparum-Infected Erythrocytes

PubMed Central

Li, Ang; Lim, Tong Seng; Shi, Hui; Yin, Jing; Tan, Swee Jin; Li, Zhengjun; Low, Boon Chuan; Tan, Kevin Shyong Wei; Lim, Chwee Teck

2011-01-01

Cytoadherence or sequestration is essential for the pathogenesis of the most virulent human malaria species, Plasmodium falciparum (P. falciparum). Similar to leukocyte-endothelium interaction in response to inflammation, cytoadherence of P. falciparum infected red blood cells (IRBCs) to endothelium occurs under physiological shear stresses in blood vessels and involves an array of molecule complexes which cooperate to form stable binding. Here, we applied single-molecule force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involving two endothelial receptor proteins: thrombospondin (TSP) and CD36. It was shown that CD36 mediated interaction was much more stable than that mediated by TSP at single molecule level, although TSP-IRBC interaction appeared stronger than CD36-IRBC interaction in the high pulling rate regime. This suggests that TSP-mediated interaction may initiate cell adhesion by capturing the fast flowing IRBCs whereas CD36 functions as the ‘holder’ for providing stable binding. PMID:21437286

A Simultaneously Antimicrobial, Protein-Repellent, and Cell-Compatible Polyzwitterion Network.

PubMed

Kurowska, Monika; Eickenscheidt, Alice; Guevara-Solarte, Diana-Lorena; Widyaya, Vania Tanda; Marx, Franziska; Al-Ahmad, Ali; Lienkamp, Karen

2017-04-10

A simultaneously antimicrobial, protein-repellent, and cell-compatible surface-attached polymer network is reported, which reduces the growth of bacterial biofilms on surfaces through its multifunctionality. The coating was made from a poly(oxonorbornene)-based zwitterion (PZI), which was surface-attached and cross-linked in one step by simultaneous UV-activated CH insertion and thiol-ene reaction. The process was applicable to both laboratory surfaces like silicon, glass, and gold and real-life surfaces like polyurethane foam wound dressings. The chemical structure and physical properties of the PZI surface and the two reference surfaces SMAMP ("synthetic mimic of an antimicrobial peptide"), an antimicrobial but protein-adhesive polymer coating, and PSB (poly(sulfobetaine)), a protein-repellent but not antimicrobial polyzwitterion coating were characterized by Fourier transform infrared spectroscopy, ellipsometry, contact angle measurements, photoelectron spectroscopy, swellability measurements (using surface plasmon resonance spectroscopy, SPR), zeta potential measurements, and atomic force microscopy. The time-dependent antimicrobial activity assay (time-kill assay) confirmed the high antimicrobial activity of the PZI; SPR was used to demonstrate that it was also highly protein-repellent. Biofilm formation studies showed that the material effectively reduced the growth of Escherichia coli and Staphylococcus aureus biofilms. Additionally, it was shown that the PZI was highly compatible with immortalized human mucosal gingiva keratinocytes and human red blood cells using the Alamar Blue assay, the live-dead stain, and the hemolysis assay. PZI thus may be an attractive coating for biomedical applications, particularly for the fight against bacterial biofilms on medical devices and in other applications.

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy.

PubMed

Huang, Xun; He, Jiexiang; Zhang, Huan-Tian; Sun, Kai; Yang, Jie; Wang, Huajun; Zhang, Hongxin; Guo, Zhenzhao; Zha, Zhen-Gang; Zhou, Changren

2017-01-01

CD44 ligand-receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand-receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand-receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process.

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy

PubMed Central

Huang, Xun; He, Jiexiang; Zhang, Huan-tian; Sun, Kai; Yang, Jie; Wang, Huajun; Zhang, Hongxin; Guo, Zhenzhao; Zha, Zhen-gang; Zhou, Changren

2017-01-01

CD44 ligand–receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand–receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand–receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process. PMID:29296081

In situ biosensing of the nanomechanical property and electrochemical spectroscopy of Streptococcus mutans-containing biofilms

NASA Astrophysics Data System (ADS)

Haochih Liu, Bernard; Li, Kun-Lin; Kang, Kai-Li; Huang, Wen-Ke; Liao, Jiunn-Der

2013-07-01

This work presents in situ biosensing approaches to study the nanomechanical and electrochemical behaviour of Streptococcus mutans biofilms under different cultivation conditions and microenvironments. The surface characteristics and sub-surface electrochemistry of the cell wall of S. mutans were measured by atomic force microscopy (AFM) based techniques to monitor the in situ biophysical status of biofilms under common anti-pathogenic procedures such as ultraviolet (UV) radiation and alcohol treatment. The AFM nanoindentation suggested a positive correlation between nanomechanical strength and the level of UV radiation of S. mutans; scanning impedance spectroscopy of dehydrated biofilms revealed reduced electrical resistance that is distinctive from that of living biofilms, which can be explained by the discharge of cytoplasm after alcohol treatment. Furthermore, the localized elastic moduli of four regions of the biofilm were studied: septum (Z-ring), cell wall, the interconnecting area between two cells and extracellular polymeric substance (EPS) area. The results indicated that cell walls exhibit the highest elastic modulus, followed by Z-ring, interconnect and EPS. Our approach provides an effective alternative for the characterization of the viability of living cells without the use of biochemical labelling tools such as fluorescence dyeing, and does not rely on surface binding or immobilization for detection. These AFM-based techniques can be very promising approaches when the conventional methods fall short.

Tunable Stoichiometry of BCxNy Thin Films Through Multitarget Pulsed Laser Deposition Monitored via In Situ Ellipsometry (Postprint)

DTIC Science & Technology

2014-02-05

X - ray photoelectron spectroscopy (XPS), Raman spectroscopy , and atomic ...calculate thickness, n and k. X - ray photoelectron spectroscopy (XPS), Raman spectroscopy , and atomic force microscopy (AFM) were all performed on each of the... X - ray photoelectron spectroscopy (XPS) and Raman spectroscopy were used to measure and compare the composition of the films.6 In this paper,

Optical trapping, pulling, and Raman spectroscopy of airborne absorbing particles based on negative photophoretic force

NASA Astrophysics Data System (ADS)

Chen, Gui-hua; He, Lin; Wu, Mu-ying; Yang, Guang; Li, Y. Q.

2017-08-01

Optical pulling is the attraction of objects back to the light source by the use of optically induced "negative forces". The light-induced photophoretic force is generated by the momentum transfer between the heating particles and surrounding gas molecules and can be several orders of magnitude larger than the radiation force and gravitation force. Here, we demonstrate that micron-sized absorbing particles can be optically pulled and manipulated towards the light source over a long distance in air with a collimated Gaussian laser beam based on a negative photophoretic force. A variety of airborne absorbing particles can be pulled by this optical pipeline to the region where they are optically trapped with another focused laser beam and their chemical compositions are characterized with Raman spectroscopy. We found that micron-sized particles are pulled over a meter-scale distance in air with a pulling speed of 1-10 cm/s in the optical pulling pipeline and its speed can be controlled by changing the laser intensity. When an aerosol particle is optically trapped with a focused Gaussian beam, we measured its rotation motion around the laser propagation direction and measured its Raman spectroscopy for chemical identification by molecular fingerprints. The centripetal acceleration of the trapped particle as high as 20 times the gravitational acceleration was observed. Optical pulling over large distances with lasers in combination with Raman spectroscopy opens up potential applications for the collection and identification of atmospheric particles.

Thz Spectroscopy and DFT Modeling of Intermolecular Vibrations in Hydrophobic Amino Acids

NASA Astrophysics Data System (ADS)

Williams, michael R. C.; Aschaffenburg, Daniel J.; Schmuttenmaer, Charles A.

2013-06-01

Vibrations that involve intermolecular displacements occur in molecular crystals at frequencies in the 0.5-5 THz range (˜15-165 cm^{-1}), and these motions are direct indicators of the interaction potential between the molecules. The intermolecular potential energy surface of crystalline hydrophobic amino acids is inherently interesting simply because of the wide variety of forces (electrostatic, dipole-dipole, hydrogen-bonding, van der Waals) that are present. Furthermore, an understanding of these particular interactions is immediately relevant to important topics like protein conformation and pharmaceutical polymorphism. We measured the low-frequency absorption spectra of several polycrystalline hydrophobic amino acids using THz time-domain spectroscopy, and in addition we carried out DFT calculations using periodic boundary conditions and an exchange-correlation functional that accounts for van der Waals dispersion forces. We chose to investigate a series of similar amino acids with closely analogous unit cells (leucine, isoleucine, and allo-isoleucine, in racemic or pseudo-racemic mixtures). This allows us to consider trends in the vibrational spectra as a function of small changes in molecular arrangement and/or crystal geometry. In this way, we gain confidence that peak assignments are not based on serendipitous similarities between calculated and observed features.

Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells

DOE PAGES

Yu M. Zhong; Nam, Chang -Yong; Trinh, M. Tuan; ...

2015-09-18

Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealedmore » both electron and hole transfer processes at the donor–acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. As a result, this study describes a new motif for designing highly efficient acceptors for organic solar cells.« less

Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells.

PubMed

Zhong, Yu; Trinh, M Tuan; Chen, Rongsheng; Purdum, Geoffrey E; Khlyabich, Petr P; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; Zhang, Boyuan; Wang, Wei; Nam, Chang-Yong; Sfeir, Matthew Y; Black, Charles T; Steigerwald, Michael L; Loo, Yueh-Lin; Ng, Fay; Zhu, X-Y; Nuckolls, Colin

2015-09-18

Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealed both electron and hole transfer processes at the donor-acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. This study describes a new motif for designing highly efficient acceptors for organic solar cells.

A Simple Bioconjugate Attachment Protocol for Use in Single Molecule Force Spectroscopy Experiments Based on Mixed Self-Assembled Monolayers

PubMed Central

Attwood, Simon J.; Simpson, Anna M. C.; Stone, Rachael; Hamaia, SamirW.; Roy, Debdulal; Farndale, RichardW.; Ouberai, Myriam; Welland, Mark E.

2012-01-01

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin. PMID:23202965

Evaluation of synthetic linear motor-molecule actuation energetics

PubMed Central

Brough, Branden; Northrop, Brian H.; Schmidt, Jacob J.; Tseng, Hsian-Rong; Houk, Kendall N.; Stoddart, J. Fraser; Ho, Chih-Ming

2006-01-01

By applying atomic force microscope (AFM)-based force spectroscopy together with computational modeling in the form of molecular force-field simulations, we have determined quantitatively the actuation energetics of a synthetic motor-molecule. This multidisciplinary approach was performed on specifically designed, bistable, redox-controllable [2]rotaxanes to probe the steric and electrostatic interactions that dictate their mechanical switching at the single-molecule level. The fusion of experimental force spectroscopy and theoretical computational modeling has revealed that the repulsive electrostatic interaction, which is responsible for the molecular actuation, is as high as 65 kcal·mol−1, a result that is supported by ab initio calculations. PMID:16735470

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid.

PubMed

Long, Xi; Parks, Joseph W; Stone, Michael D

2016-08-01

Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules. Copyright © 2016 Elsevier Inc. All rights reserved.

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid

PubMed Central

Long, Xi; Parks, Joseph W.; Stone, Michael D.

2017-01-01

Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules. PMID:27320203

Tunneling mechanism and contact mechanics of colloidal nanoparticle assemblies.

PubMed

Biaye, Moussa; Zbydniewska, Ewa; Mélin, Thierry; Deresmes, Dominique; Copie, Guillaume; Cleri, Fabrizio; Sangeetha, Neralagatta; Decorde, Nicolas; Viallet, Benoit; Grisolia, Jérémie; Ressier, Laurence; Diesinger, Heinrich

2016-11-25

Nanoparticle assemblies with thiol-terminated alkyl chains are studied by conducting atomic force microscopy (c-AFM) regarding their use as strain gauges for touch-sensitive panels. Current-force spectroscopy is used as a characterization tool complementary to the macroscopic setup since it allows a bias to be applied to a limited number of junctions, overcoming the Coulomb blockade energy and focusing on the contact electromechanics and the transport mechanism across the ligand. First, transition voltage spectroscopy is applied with varying force to target the underlying tunneling mechanism by observing whether the transition between the ohmic and exponential current-voltage behavior is force-dependent. Secondly, current-force spectroscopy in the ohmic range below the transition voltage is performed. The current-force behavior of the AFM probe in contact with a nanoparticle multilayer is associated with the spread of force and current within the nanoparticle lattice and at the level of adjacent particles by detailed contact mechanics treatment. The result is twofold: concerning the architecture of sensors, this work is a sample case of contact electromechanics at scales ranging from the device scale down to the individual ligand molecule. Regarding transport across the molecule, the vacuum tunneling mechanism is favored over the conduction by coherent molecular states, which is a decision-making aid for the choice of ligand in applications.

Ambient CdCl{sub 2} treatment on CdS buffer layer for improved performance of Sb{sub 2}Se{sub 3} thin film photovoltaics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Liang; Luo, Miao; Qin, Sikai

2015-10-05

Antimony selenide (Sb{sub 2}Se{sub 3}) is appealing as a promising light absorber because of its intrinsically benign grain boundaries, suitable band gap (∼1.1 eV), strong absorption coefficient, and relatively environmentally friendly constituents. Recently, we achieved a certified 5.6% efficiency Sb{sub 2}Se{sub 3} thin film solar cell with the assistance of ambient CdCl{sub 2} treatment on the CdS buffer layer. Here, we focused on investigating the underlying mechanism from a combined materials and device physics perspective applying current density-voltage (J-V) fitting analysis, atomic force microscope, X-ray photoelectron spectroscopy, fluorescence, and UV–Vis transmission spectroscopy. Our results indicated that ambient CdCl{sub 2} treatment onmore » CdS film not only improved CdS grain size and quality, but also incorporated Cl and more O into the film, both of which can significantly improve the heterojunction quality and device performance of CdS/Sb{sub 2}Se{sub 3} solar cells.« less

Improvement of in vitro corrosion and cytocompatibility of biodegradable Fe surface modified by Zn ion implantation

NASA Astrophysics Data System (ADS)

Wang, Henan; Zheng, Yang; Li, Yan; Jiang, Chengbao

2017-05-01

Pure Fe was surface-modified by Zn ion implantation to improve the biodegradable behavior and cytocompatibility. Surface topography, chemical composition, corrosion resistance and cytocompatibility were investigated. Atomic force microscopy, auger electron spectroscopy and X-ray photoelectron spectroscopy results showed that Zn was implanted into the surface of pure Fe in the depth of 40-60 nm and Fe2O3/ZnO oxides were formed on the outmost surface. Electrochemical measurements and immersion tests revealed an improved degradable behavior for the Zn-implanted Fe samples. An approximately 12% reduction in the corrosion potential (Ecorr) and a 10-fold increase in the corrosion current density (icorr) were obtained after Zn ion implantation with a moderate incident ion dose, which was attributed to the enhanced pitting corrosion. The surface free energy of pure Fe was decreased by Zn ion implantation. The results of direct cell culture indicated that the short-term (4 h) cytocompatibility of MC3T3-E1 cells was promoted by the implanted Zn on the surface.

Novel amphiphilic poly(dimethylsiloxane) based polyurethane networks tethered with carboxybetaine and their combined antibacterial and anti-adhesive property

NASA Astrophysics Data System (ADS)

Jiang, Jingxian; Fu, Yuchen; Zhang, Qinghua; Zhan, Xiaoli; Chen, Fengqiu

2017-08-01

The traditional nonfouling materials are powerless against bacterial cells attachment, while the hydrophobic bactericidal surfaces always suffer from nonspecific protein adsorption and dead bacterial cells accumulation. Here, amphiphilic polyurethane (PU) networks modified with poly(dimethylsiloxane) (PDMS) and cationic carboxybetaine diol through simple crosslinking reaction were developed, which had an antibacterial efficiency of 97.7%. Thereafter, the hydrolysis of carboxybetaine ester into zwitterionic groups brought about anti-adhesive properties against bacteria and proteins. The surface chemical composition and wettability performance of the PU network surfaces were investigated by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and contact angle analysis. The surface distribution of PDMS and zwitterionic segments produced an obvious amphiphilic heterogeneous surface, which was demonstrated by atomic force microscopy (AFM). Enzyme-linked immunosorbent assays (ELISA) were used to test the nonspecific protein adsorption behaviors. With the advantages of the transition from excellent bactericidal performance to anti-adhesion and the combination of fouling resistance and fouling release property, the designed PDMS-based amphiphilic PU network shows great application potential in biomedical devices and marine facilities.

Time-dependent efficiency measurements of donor-acceptor, dye-sensitized polymer solar cells

NASA Astrophysics Data System (ADS)

Bandaccari, Kyle; Chesmore, Grace; Tajalli-Tehrani Valverde, Parisa; Bugaj, Mitchel; McNelis, Brian; Barber, Richard, Jr.

The fullerene/polymer active layer pairing of PCBM/P3HT has become the model system within the field of polymer solar cell research. A large body of work concerned with reporting improved efficiencies for this system exists, but truly quantitative studies of device lifetime and long-term degradation tendencies are much rarer. Here, we report the effects of two donor-acceptor diazo dye sensitizers on efficiency and lifetime upon addition into the PCBM/P3HT active layer at varied concentrations. The electrical and efficiency measurements were supplemented by time-dependent UV-visible spectroscopy studies and morphology investigations via atomic-force microscopy (AFM). This pairing with spectroscopy offers an internal check on the data as the rate of change in absorbance of the active layer correlates almost exactly to the rate of power conversion efficiency decrease. Additionally, AFM imaging reveals different morphology patterns when dye concentrations and functionalities change. Such observations suggest that such small-molecule sensitizers exert yet undetermined effects on the organization of components within the active layer at the molecular level.

Dielectrophoretic positioning of single nanoparticles on atomic force microscope tips for tip-enhanced Raman spectroscopy.

PubMed

Leiterer, Christian; Deckert-Gaudig, Tanja; Singh, Prabha; Wirth, Janina; Deckert, Volker; Fritzsche, Wolfgang

2015-05-01

Tip-enhanced Raman spectroscopy, a combination of Raman spectroscopy and scanning probe microscopy, is a powerful technique to detect the vibrational fingerprint of molecules at the nanometer scale. A metal nanoparticle at the apex of an atomic force microscope tip leads to a large enhancement of the electromagnetic field when illuminated with an appropriate wavelength, resulting in an increased Raman signal. A controlled positioning of individual nanoparticles at the tip would improve the reproducibility of the probes and is quite demanding due to usually serial and labor-intensive approaches. In contrast to commonly used submicron manipulation techniques, dielectrophoresis allows a parallel and scalable production, and provides a novel approach toward reproducible and at the same time affordable tip-enhanced Raman spectroscopy tips. We demonstrate the successful positioning of an individual plasmonic nanoparticle on a commercial atomic force microscope tip by dielectrophoresis followed by experimental proof of the Raman signal enhancing capabilities of such tips. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-performance polymeric photovoltaic cells with a gold chloride-treated polyacrylonitrile hole extraction interlayer

NASA Astrophysics Data System (ADS)

Jeong, Ji-Ho; Noh, Yong-Jin; Kim, Seok-Soon; Kwon, Sung-Nam; Na, Seok-In

2018-03-01

We introduce a high efficiency polymeric photovoltaic cell (PPV) to be obtained by polyacrylonitrile (PAN) hole extraction layer (HEL) modification with gold chloride (AuCl3). The role of PAN HELs with AuCl3 and their effects on solar cell performances were studied with ultraviolet photoemission spectroscopy, atomic force microscopy, internal resistances in PPVs, and current-voltage power curves. The resultant PPVs with AuCl3-treated PAN HELs showed improved cell efficiency compared to PSCs with no interlayer and PAN without AuCl3. Furthermore, with AuCl3-treated PAN, we finally achieved a high efficiency of 6.91%, and a desirable PPV-stability in poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b‧]dithiophe-ne-2,6-diyl][3-fluoro-2-[(2-thylhexyl)carbonyl]-thieno[3,4-b]thiophenediyl

Pore spanning lipid bilayers on silanised nanoporous alumina membranes

NASA Astrophysics Data System (ADS)

Md Jani, Abdul M.; Zhou, Jinwen; Nussio, Matthew R.; Losic, Dusan; Shapter, Joe G.; Voelcker, Nicolas H.

2008-12-01

The preparation of bilayer lipid membranes (BLMs) on solid surfaces is important for many studies probing various important biological phenomena including the cell barrier properties, ion-channels, biosensing, drug discovery and protein/ligand interactions. In this work we present new membrane platforms based on suspended BLMs on nanoporous anodic aluminium oxide (AAO) membranes. AAO membranes were prepared by electrochemical anodisation of aluminium foil in 0.3 M oxalic acid using a custom-built etching cell and applying voltage of 40 V, at 1oC. AAO membranes with controlled diameter of pores from 30 - 40 nm (top of membrane) and 60 -70 nm (bottom of membrane) were fabricated. Pore dimensions have been confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). AAO membranes were chemically functionalised with 3-aminopropyltriethoxysilane (APTES). Confirmation of the APTES attachment to the AAO membrane was achieved by means of infrared spectroscopy, X-ray photoelectron spectroscopy and contact angle measurements. The Fourier transform infrared (FTIR) spectra of functionalised membranes show several peaks from 2800 to 3000 cm-1 which were assigned to symmetric and antisymmetric CH2 bands. XPS data of the membrane showed a distinct increase in C1s (285 eV), N1s (402 eV) and Si2p (102 eV) peaks after silanisation. The water contact angle of the functionalised membrane was 80o as compared to 20o for the untreated membrane. The formation of BLMs comprising dioleoyl-phosphatidylserine (DOPS) on APTESmodified AAO membranes was carried using the vesicle spreading technique. AFM imaging and force spectroscopy was used to characterise the structural and nanomechanical properties of the suspended membrane. This technique also confirmed the stability of bilayers on the nanoporous alumina support for several days. Fabricated suspended BLMs on nanoporous AAO hold promise for the construction of biomimetic membrane architectures with embedded transmembrane proteins.

An All-vanadium Continuous-flow Photoelectrochemical Cell for Extending State-of-charge in Solar Energy Storage.

PubMed

Wei, Zi; Shen, Yi; Liu, Dong; Liu, Fuqiang

2017-04-04

Greater levels of solar energy storage provide an effective solution to the inherent nature of intermittency, and can substantially improve reliability, availability, and quality of the renewable energy source. Here we demonstrated an all-vanadium (all-V) continuous-flow photoelectrochemical storage cell (PESC) to achieve efficient and high-capacity storage of solar energy, through improving both photocurrent and photocharging depth. It was discovered that forced convective flow of electrolytes greatly enhanced the photocurrent by 5 times comparing to that with stagnant electrolytes. Electrochemical impedance spectroscopy (EIS) study revealed a great reduction of charge transfer resistance with forced convective flow of electrolytes as a result of better mass transport at U-turns of the tortuous serpentine flow channel of the cell. Taking advantage of the improved photocurrent and diminished charge transfer resistance, the all-V continuous-flow PESC was capable of producing ~20% gain in state of charge (SOC) under AM1.5 illumination for ca. 1.7 hours without any external bias. This gain of SOC was surprisingly three times more than that with stagnant electrolytes during a 25-hour period of photocharge.

Protein Adsorption Alters Hydrophobic Surfaces Used for Suspension Culture of Pluripotent Stem Cells.

PubMed

Jonas, Steven J; Stieg, Adam Z; Richardson, Wade; Guo, Shuling; Powers, David N; Wohlschlegel, James; Dunn, Bruce

2015-02-05

This Letter examines the physical and chemical changes that occur at the interface of methyl-terminated alkanethiol self-assembled monolayers (SAMs) after exposure to cell culture media used to derive embryoid bodies (EBs) from pluripotent stem cells. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy analysis of the SAMs indicates that protein components within the EB cell culture medium preferentially adsorb at the hydrophobic interface. In addition, we examined the adsorption process using surface plasmon resonance and atomic force microscopy. These studies identify the formation of a porous, mat-like adsorbed protein film with an approximate thickness of 2.5 nm. Captive bubble contact angle analysis reveals a shift toward superhydrophilic wetting behavior at the cell culture interface due to adsorption of these proteins. These results show how EBs are able to remain in suspension when derived on hydrophobic materials, which carries implications for the rational design of suspension culture interfaces for lineage specific stem-cell differentiation.

Endothelial cell behaviour on gas-plasma-treated PLA surfaces: the roles of surface chemistry and roughness.

PubMed

Shah, Amita; Shah, Sarita; Mani, Gopinath; Wenke, Joseph; Agrawal, Mauli

2011-04-01

Glow-discharge gas-plasma (GP) treatment has been shown to induce surface modifications such that cell adhesion and growth are enhanced. However, it is not known which gas used in GP treatment is optimal for endothelial cell function. Polylactic acid (PLA) films treated oxygen, argon, or nitrogen GP were characterized using contact angles, scanning electron microscopy, atomic force microscopy, optical profilometry, and x-ray photoelectron spectroscopy. All three GP treatments decreased the carbon atomic concentration and surface roughness and increased the oxygen atomic concentration. Human umbilical vein endothelial cells were cultured on the PLA films for up to 7 days. Based on proliferation and live/dead assays, surface chemistry was shown to have the greatest effect on the attachment, proliferation, and viability of these cells, while roughness did not have a significant influence. Of the different gases, endothelial cell viability, attachment and proliferation were most significantly increased on PLA surfaces treated with oxygen and argon gas plasma. Copyright © 2010 John Wiley & Sons, Ltd.

pH dependent growth of poly( L-lysine)/poly( L-glutamic) acid multilayer films and their cell adhesion properties

NASA Astrophysics Data System (ADS)

Richert, Ludovic; Arntz, Youri; Schaaf, Pierre; Voegel, Jean-Claude; Picart, Catherine

2004-10-01

The short-term interaction of chondrosarcoma cells with (PGA/PLL) polyelectrolyte multilayers was investigated in a serum-containing medium for films built at different pHs and subsequently exposed to the culture medium. The buildup of the films and their stability was first investigated by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, streaming potential measurements and atomic force microscopy. While film growth is linear at all pHs, after a few layers have been deposited the growth is much larger for the films built at basic pH and even more pronounced for those built at acidic pH. However, these latter films remain stable in the culture medium only if they have been crosslinked prior to the ionic strength and pH jumps. The films built at acidic pH were found to swell in water by about 200% whereas those built at other pHs did not swell in a physiological buffer. For thin films (≈20 nm) built at pH = 7.4, the detachment forces were dependent on the outermost layer, the forces being significantly higher on PLL-ending films than on PGA-ending ones. In contrast, for the thick films built at pH = 4.4 and at pH = 10.4 (thickness of the order of few hundred of nanometers), the detachment forces were independent of the outermost layer of the film. The films built at pH = 10.4, which shrink in contact with salt containing solutions, were highly cell adhesive whereas those built at acidic pH were highly cell resistant. Protein adsorption and film roughness (as measured by AFM) could not explain these striking differences. The high adhesion observed on the film built at pH 10.4 may rather be related to the secondary structure of the film and to its relatively low swellability in water, whereas the cell resistance of the films built at pH 4.4 may be linked to their high swellability. Therefore, for the PGA/PLL films, the cell adhesion properties can be tuned depending on the deposition pH of the polyelectrolyte solutions. This study reveals the importance of the multilayer structure and architecture to control the detachment force of cells onto such films.

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry

PubMed Central

Sedlak, Steffen M.; Bauer, Magnus S.; Kluger, Carleen; Schendel, Leonard C.; Milles, Lukas F.; Pippig, Diana A.

2017-01-01

The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin’s tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10−6 s-1 range. PMID:29206886

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

PubMed

Sedlak, Steffen M; Bauer, Magnus S; Kluger, Carleen; Schendel, Leonard C; Milles, Lukas F; Pippig, Diana A; Gaub, Hermann E

2017-01-01

The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.

Synthesis, Characterization, Cytotoxic Activity, and Interactions with CT-DNA and BSA of Cationic Ruthenium(II) Complexes Containing Dppm and Quinoline Carboxylates

PubMed Central

da Silva, Edinaldo N.; da Silva, Paulo A. B.; Graminha, Angélica E.; de Oliveira, Pollyanna F.; Damasceno, Jaqueline L.; Tavares, Denise C.; Batista, Alzir A.

2017-01-01

The complexes cis-[Ru(quin)(dppm)2]PF6 and cis-[Ru(kynu)(dppm)2]PF6 (quin = quinaldate; kynu = kynurenate; dppm = bis(diphenylphosphino)methane) were prepared and characterized by elemental analysis, electronic, FTIR, 1H, and 31P{1H} NMR spectroscopies. Characterization data were consistent with a cis arrangement for the dppm ligands and a bidentate coordination through carboxylate oxygens of the quin and kynu anions. These complexes were not able to intercalate CT-DNA as shown by circular dichroism spectroscopy. On the other hand, bovine serum albumin (BSA) binding constants and thermodynamic parameters suggest spontaneous interactions with this protein by hydrogen bonds and van der Waals forces. Cytotoxicity assays were carried out on a panel of human cancer cell lines including HepG2, MCF-7, and MO59J and one normal cell line GM07492A. In general, the new ruthenium(II) complexes displayed a moderate to high cytotoxicity in all the assayed cell lines with IC50 ranging from 10.1 to 36 µM and were more cytotoxic than the precursor cis-[RuCl2(dppm)2]. The cis-[Ru(quin)(dppm)2]PF6 were two to three times more active than the reference metallodrug cisplatin in the MCF-7 and MO59J cell lines. PMID:28814948

A nu-space for image correlation spectroscopy: characterization and application to measure protein transport in live cells

NASA Astrophysics Data System (ADS)

Potvin-Trottier, Laurent; Chen, Lingfeng; Horwitz, Alan Rick; Wiseman, Paul W.

2013-08-01

We introduce a new generalized theoretical framework for image correlation spectroscopy (ICS). Using this framework, we extend the ICS method in time-frequency (ν, nu) space to map molecular flow of fluorescently tagged proteins in individual living cells. Even in the presence of a dominant immobile population of fluorescent molecules, nu-space ICS (nICS) provides an unbiased velocity measurement, as well as the diffusion coefficient of the flow, without requiring filtering. We also develop and characterize a tunable frequency-filter for spatio-temporal ICS (STICS) that allows quantification of the density, the diffusion coefficient and the velocity of biased diffusion. We show that the techniques are accurate over a wide range of parameter space in computer simulation. We then characterize the retrograde flow of adhesion proteins (α6- and αLβ2-GFP integrins and mCherry-paxillin) in CHO.B2 cells plated on laminin and intercellular adhesion molecule 1 (ICAM-1) ligands respectively. STICS with a tunable frequency filter, in conjunction with nICS, measures two new transport parameters, the density and transport bias coefficient (a measure of the diffusive character of a flow/biased diffusion), showing that molecular flow in this cell system has a significant diffusive component. Our results suggest that the integrin-ligand interaction, along with the internal myosin-motor generated force, varies for different integrin-ligand pairs, consistent with previous results.

NMR-driven identification of anti-amyloidogenic compounds in green and roasted coffee extracts.

PubMed

Ciaramelli, Carlotta; Palmioli, Alessandro; De Luigi, Ada; Colombo, Laura; Sala, Gessica; Riva, Chiara; Zoia, Chiara Paola; Salmona, Mario; Airoldi, Cristina

2018-06-30

To identify food and beverages that provide the regular intake of natural compounds capable of interfering with toxic amyloidogenic aggregates, we developed an experimental protocol that combines NMR spectroscopy and atomic force microscopy, in vitro biochemical and cell assays to detect anti-Aβ molecules in natural edible matrices. We applied this approach to investigate the potential anti-amyloidogenic properties of coffee and its molecular constituents. Our data showed that green and roasted coffee extracts and their main components, 5-O-caffeoylquinic acid and melanoidins, can hinder Aβ on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Coffee extracts and melanoidins also counteract hydrogen peroxide- and rotenone-induced cytotoxicity and modulate some autophagic pathways in the same cell line. Copyright © 2018 Elsevier Ltd. All rights reserved.

Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells.

PubMed

Bai, Ding-Ping; Zhang, Xi-Feng; Zhang, Guo-Liang; Huang, Yi-Fan; Gurunathan, Sangiliyandi

2017-01-01

Zinc oxide nanoparticles (ZnO NPs) are frequently used in industrial products such as paint, surface coating, and cosmetics, and recently, they have been explored in biologic and biomedical applications. Therefore, this study was undertaken to investigate the effect of ZnO NPs on cytotoxicity, apoptosis, and autophagy in human ovarian cancer cells (SKOV3). ZnO NPs with a crystalline size of 20 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays. Exposure of cells to ZnO NPs resulted in a dose-dependent loss of cell viability, and the characteristic apoptotic features such as rounding and loss of adherence, enhanced reactive oxygen species generation, and loss of mitochondrial membrane potential were observed in the ZnO NP-treated cells. Furthermore, the cells treated with ZnO NPs showed significant double-strand DNA breaks, which are gained evidences from significant number of γ-H 2 AX and Rad51 expressed cells. ZnO NP-treated cells showed upregulation of p53 and LC3, indicating that ZnO NPs are able to upregulate apoptosis and autophagy. Finally, the Western blot analysis revealed upregulation of Bax, caspase-9, Rad51, γ-H 2 AX, p53, and LC3 and downregulation of Bcl-2. The study findings demonstrated that the ZnO NPs are able to induce significant cytotoxicity, apoptosis, and autophagy in human ovarian cells through reactive oxygen species generation and oxidative stress. Therefore, this study suggests that ZnO NPs are suitable and inherent anticancer agents due to their several favorable characteristic features including favorable band gap, electrostatic charge, surface chemistry, and potentiation of redox cycling cascades.

Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evaluation of its antibacterial activity.

PubMed

Velmurugan, Palanivel; Iydroose, Mahudunan; Mohideen, Mohmed Hanifa Abdul Kader; Mohan, Thankiah Selva; Cho, Min; Oh, Byung-Taek

2014-08-01

This study highlights the ability of nitrate-reducing Bacillus subtilis EWP-46 cell-free extract used for preparation of silver nanoparticles (AgNPs) by reduction of silver ions into nano silver. The production of AgNPs was optimized with several parameters such as hydrogen ion concentration, temperature, silver ion (Ag(+) ion) and time. The maximum AgNPs production was achieved at pH 10.0, temperature 60 °C, 1.0 mM Ag(+) ion and 720 min. The UV-Vis spectrum showed surface plasmon resonance peak at 420 nm, energy-dispersive X-ray spectroscopy (SEM-EDX) spectra showed the presence of element silver in pure form. Atomic force microscopy (AFM) and transmission electron microscopy images illustrated the nanoparticle size, shape, and average particle size ranging from 10 to 20 nm. Fourier transform infrared spectroscopy provided the evidence for the presence of biomolecules responsible for the reduction of silver ion, and X-ray diffraction analysis confirmed that the obtained nanoparticles were in crystalline form. SDS-PAGE was performed to identify the proteins and its molecular mass in the purified nitrate reductase from the cell-free extract. In addition, the minimum inhibitory concentration and minimum bactericidal concentration of AgNPs were investigated against gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus aureus) bacteria.

Characterization and evaluation of 5-fluorouracil-loaded solid lipid nanoparticles prepared via a temperature-modulated solidification technique.

PubMed

Patel, Meghavi N; Lakkadwala, Sushant; Majrad, Mohamed S; Injeti, Elisha R; Gollmer, Steven M; Shah, Zahoor A; Boddu, Sai Hanuman Sagar; Nesamony, Jerry

2014-12-01

The aim of this research was to advance solid lipid nanoparticle (SLN) preparation methodology by preparing glyceryl monostearate (GMS) nanoparticles using a temperature-modulated solidification process. The technique was reproducible and prepared nanoparticles without the need of organic solvents. An anticancer agent, 5-fluorouracil (5-FU), was incorporated in the SLNs. The SLNs were characterized by particle size analysis, zeta potential analysis, differential scanning calorimetry (DSC), infrared spectroscopy, atomic force microscopy (AFM), transmission electron microscopy (TEM), drug encapsulation efficiency, in vitro drug release, and in vitro cell viability studies. Particle size of the SLN dispersion was below 100 nm, and that of redispersed lyophilizates was ~500 nm. DSC and infrared spectroscopy suggested that the degree of crystallinity did not decrease appreciably when compared to GMS. TEM and AFM images showed well-defined spherical to oval particles. The drug encapsulation efficiency was found to be approximately 46%. In vitro drug release studies showed that 80% of the encapsulated drug was released within 1 h. In vitro cell cultures were biocompatible with blank SLNs but demonstrated concentration-dependent changes in cell viability to 5-FU-loaded SLNs. The 5-FU-loaded SLNs can potentially be utilized in an anticancer drug delivery system.

Raman spectroscopy for bacterial identification and characterization

NASA Astrophysics Data System (ADS)

Bernatová, Silvie; Samek, Ota; Pilát, Zdeněk.; Šerý, Mojmír.; Ježek, Jan; Krzyžánek, Vladislav; Zemánek, Pavel; Ružička, Filip

2012-01-01

The main goal of our investigation is to use Raman tweezers technique so that the responce of Raman scattering on microorganisms suspended in liquid media (bacteria, algae and yeast cells in microfluidic chips) can be used to identify different species. The investigations presented here include identification of different bacteria strains (biofilm-positive and biofilm-negative) and yeast cells by using principal component analysis (PCA). The main driving force behind our investigation was a common problem in the clinical microbiology laboratory - how to distinguish between contaminant and invasive isolates. Invasive bacterial/yeast isolates can be assumed to form a biofilm, while isolates which do not form a biofilm can be treated as contaminant. Thus, the latter do not represent an important virulence factor.

Nanomechanical characterization of phospholipid bilayer islands on flat and porous substrates: a force spectroscopy study.

PubMed

Nussio, Matthew R; Oncins, Gerard; Ridelis, Ingrid; Szili, Endre; Shapter, Joseph G; Sanz, Fausto; Voelcker, Nicolas H

2009-07-30

In this study, we compare for the first time the nanomechanical properties of lipid bilayer islands on flat and porous surfaces. 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers were deposited on flat (silicon and mica) and porous silicon (pSi) substrate surfaces and examined using atomic force spectroscopy and force volume imaging. Force spectroscopy measurements revealed the effects of the underlying substrate and of the lipid phase on the nanomechanical properties of bilayers islands. For mica and silicon, significant differences in breakthrough force between the center and the edges of bilayer islands were observed for both phospolipids. These differences were more pronounced for DMPC than for DPPC, presumably due to melting effects at the edges of DMPC bilayers. In contrast, bilayer islands deposited on pSi yielded similar breakthrough forces in the central region and along the perimeter of the islands, and those values in turn were similar to those measured along the perimeter of bilayer islands deposited on the flat substrates. The study also demonstrates that pSi is suitable solid support for the formation of pore-spanning phospholipid bilayers with potential applications in transmembrane protein studies, drug delivery, and biosensing.

Preparation and performance evaluation of novel alkaline stable anion exchange membranes

NASA Astrophysics Data System (ADS)

Irfan, Muhammad; Bakangura, Erigene; Afsar, Noor Ul; Hossain, Md. Masem; Ran, Jin; Xu, Tongwen

2017-07-01

Novel alkaline stable anion exchange membranes are prepared from various amounts of N-methyl dipicolylamine (MDPA) and brominated poly (2,6-dimethyl-1,4-phenylene oxide) (BPPO). The dipicolylamine and MDPA are synthesized through condensation reaction and confirmed by 1H NMR spectroscopy. The morphologies of prepared membranes are investigated by atomic force microscopy (AFM), fourier transform infrared spectroscopy (FTIR), 1H NMR spectroscopy and scanning electron microscopy (SEM). The electrochemical and physical properties of AEMs are tested comprising water uptake (WU), ion exchange capacity (IEC), alkaline stability, linear expansion ratio (LER), thermal stability and mechanical stability. The obtained hydroxide conductivity of MDPA-4 is 66.5 mS/cm at 80 °C. The MDPA-4 membrane shows good alkaline stability, high hydroxide conductivity, low methanol permeability (3.43 × 10-7 cm2/s), higher selectivity (8.26 × 107 mS s/cm3), less water uptake (41.1%) and lower linear expansion (11.1%) despite of high IEC value (1.62 mmol/g). The results prove that MDPA membranes have great potential application in anion exchange membrane fuel cell.

Graphene oxide nanoplatforms to enhance catalytic performance of iron phthalocyanine for oxygen reduction reaction in bioelectrochemical systems

NASA Astrophysics Data System (ADS)

Costa de Oliveira, Maida Aysla; Mecheri, Barbara; D'Epifanio, Alessandra; Placidi, Ernesto; Arciprete, Fabrizio; Valentini, Federica; Perandini, Alessando; Valentini, Veronica; Licoccia, Silvia

2017-07-01

We report the development of electrocatalysts based on iron phthalocyanine (FePc) supported on graphene oxide (GO), obtained by electrochemical oxidation of graphite in aqueous solution of LiCl, LiClO4, and NaClO4. Structure, surface chemistry, morphology, and thermal stability of the prepared materials were investigated by Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, atomic force microscopy (AFM), thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS). The catalytic activity toward oxygen reduction reaction (ORR) at neutral pH was evaluated by cyclic voltammetry. The experimental results demonstrate that the oxidation degree of GO supports affects the overall catalytic activity of FePc/GO, due to a modulation effect of the interaction between FePc and the basal plane of GO. On the basis of electrochemical, spectroscopic, and morphological investigations, FePc/GO_LiCl was selected to be assembled at the cathode side of a microbial fuel cell prototype, demonstrating a good electrochemical performance in terms of voltage and power generation.

A measurement of the hysteresis loop in force-spectroscopy curves using a tuning-fork atomic force microscope

PubMed Central

van Vörden, Dennis; Möller, Rolf

2012-01-01

Summary Measurements of the frequency shift versus distance in noncontact atomic force microscopy (NC-AFM) allow measurements of the force gradient between the oscillating tip and a surface (force-spectroscopy measurements). When nonconservative forces act between the tip apex and the surface the oscillation amplitude is damped. The dissipation is caused by bistabilities in the potential energy surface of the tip–sample system, and the process can be understood as a hysteresis of forces between approach and retraction of the tip. In this paper, we present the direct measurement of the whole hysteresis loop in force-spectroscopy curves at 77 K on the PTCDA/Ag/Si(111) √3 × √3 surface by means of a tuning-fork-based NC-AFM with an oscillation amplitude smaller than the distance range of the hysteresis loop. The hysteresis effect is caused by the making and breaking of a bond between PTCDA molecules on the surface and a PTCDA molecule at the tip. The corresponding energy loss was determined to be 0.57 eV by evaluation of the force–distance curves upon approach and retraction. Furthermore, a second dissipation process was identified through the damping of the oscillation while the molecule on the tip is in contact with the surface. This dissipation process occurs mainly during the retraction of the tip. It reaches a maximum value of about 0.22 eV/cycle. PMID:22496993

Communication: atomic force detection of single-molecule nonlinear optical vibrational spectroscopy.

PubMed

Saurabh, Prasoon; Mukamel, Shaul

2014-04-28

Atomic Force Microscopy (AFM) allows for a highly sensitive detection of spectroscopic signals. This has been first demonstrated for NMR of a single molecule and recently extended to stimulated Raman in the optical regime. We theoretically investigate the use of optical forces to detect time and frequency domain nonlinear optical signals. We show that, with proper phase matching, the AFM-detected signals closely resemble coherent heterodyne-detected signals. Applications are made to AFM-detected and heterodyne-detected vibrational resonances in Coherent Anti-Stokes Raman Spectroscopy (χ((3))) and sum or difference frequency generation (χ((2))).

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

PubMed

Chen, Jiahuan; Ganguly, Anutosh; Mucsi, Ashley D; Meng, Junchen; Yan, Jiacong; Detampel, Pascal; Munro, Fay; Zhang, Zongde; Wu, Mei; Hari, Aswin; Stenner, Melanie D; Zheng, Wencheng; Kubes, Paul; Xia, Tie; Amrein, Matthias W; Qi, Hai; Shi, Yan

2017-02-01

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner. © 2017 Chen et al.

Force spectroscopy studies on protein-ligand interactions: a single protein mechanics perspective.

PubMed

Hu, Xiaotang; Li, Hongbin

2014-10-01

Protein-ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein-ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein-ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein-ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Optical Spectroscopy for Noninvasive Monitoring of Stem Cell Differentiation

PubMed Central

Downes, Andrew; Mouras, Rabah; Elfick, Alistair

2010-01-01

There is a requirement for a noninvasive technique to monitor stem cell differentiation. Several candidates based on optical spectroscopy are discussed in this review: Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy. These techniques are briefly described, and the ability of each to distinguish undifferentiated from differentiated cells is discussed. FTIR spectroscopy has demonstrated its ability to distinguish between stem cells and their derivatives. Raman spectroscopy shows a clear reduction in DNA and RNA concentrations during embryonic stem cell differentiation (agreeing with the well-known reduction in the nucleus to cytoplasm ratio) and also shows clear increases in mineral content during differentiation of mesenchymal stem cells. CARS microscopy can map these DNA, RNA, and mineral concentrations at high speed, and Mutliplex CARS spectroscopy/microscopy is highlighted as the technique with most promise for future applications. PMID:20182537

Identification of nanoparticles and nanosystems in biological matrices with scanning probe microscopy.

PubMed

Angeloni, Livia; Reggente, Melania; Passeri, Daniele; Natali, Marco; Rossi, Marco

2018-04-17

Identification of nanoparticles and nanosystems into cells and biological matrices is a hot research topic in nanobiotechnologies. Because of their capability to map physical properties (mechanical, electric, magnetic, chemical, or optical), several scanning probe microscopy based techniques have been proposed for the subsurface detection of nanomaterials in biological systems. In particular, atomic force microscopy (AFM) can be used to reveal stiff nanoparticles in cells and other soft biomaterials by probing the sample mechanical properties through the acquisition of local indentation curves or through the combination of ultrasound-based methods, like contact resonance AFM (CR-AFM) or scanning near field ultrasound holography. Magnetic force microscopy can detect magnetic nanoparticles and other magnetic (bio)materials in nonmagnetic biological samples, while electric force microscopy, conductive AFM, and Kelvin probe force microscopy can reveal buried nanomaterials on the basis of the differences between their electric properties and those of the surrounding matrices. Finally, scanning near field optical microscopy and tip-enhanced Raman spectroscopy can visualize buried nanostructures on the basis of their optical and chemical properties. Despite at a still early stage, these methods are promising for detection of nanomaterials in biological systems as they could be truly noninvasive, would not require destructive and time-consuming specific sample preparation, could be performed in vitro, on alive samples and in water or physiological environment, and by continuously imaging the same sample could be used to dynamically monitor the diffusion paths and interaction mechanisms of nanomaterials into cells and biological systems. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology. © 2018 Wiley Periodicals, Inc.

Surface grafting of a thermoplastic polyurethane with methacrylic acid by previous plasma surface activation and by ultraviolet irradiation to reduce cell adhesion.

PubMed

Alves, P; Pinto, S; Kaiser, Jean-Pierre; Bruinink, Arie; de Sousa, Hermínio C; Gil, M H

2011-02-01

The material performance, in a biological environment, is mainly mediated by its surface properties and by the combination of chemical, physical, biological, and mechanical properties required, for a specific application. In this study, the surface of a thermoplastic polyurethane (TPU) material (Elastollan(®)1180A50) was activated either by plasma or by ultra-violet (UV) irradiation. After surface activation, methacrylic acid (MAA) was linked to the surface of TPU in order to improve its reactivity and to reduce cell adhesion. Grafted surfaces were evaluated by X-ray photoelectron spectroscopy (XPS), by atomic force microscopy (AFM) and by contact angle measurements. Blood compatibility studies and cell adhesion tests with human bone marrow cells (HBMC) were also performed. If was found that UV grafting method led to better results than the plasma activation method, since cell adhesion was reduced when methacrylic acid was grafted to the TPU surface by UV. Copyright © 2010 Elsevier B.V. All rights reserved.

Adhesion of Chlamydomonas microalgae to surfaces is switchable by light

NASA Astrophysics Data System (ADS)

Kreis, Christian Titus; Le Blay, Marine; Linne, Christine; Makowski, Marcin Michal; Bäumchen, Oliver

2018-01-01

Microalgae are photoactive microbes that live in liquid-infused environments, such as soil, temporary pools and rocks, where they encounter and colonize a plethora of surfaces. Their photoactivity manifests itself in a variety of processes, including light-directed motility (phototaxis), the growth of microalgal populations, and their photosynthetic machinery. Although microbial responses to light have been widely recognized, any influence of light on cell-surface interactions remains elusive. Here, we reveal that the unspecific adhesion of microalgae to surfaces can be reversibly switched on and off by light. Using a micropipette force spectroscopy technique, we measured in vivo single-cell adhesion forces and show that the microalga's flagella provide light-switchable adhesive contacts with the surface. This light-induced adhesion to surfaces is an active and completely reversible process that occurs on a timescale of seconds. Our results suggest that light-switchable adhesiveness is a natural functionality of microalgae to regulate the transition between the planktonic and the surface-associated state, which yields an adhesive adaptation to optimize the photosynthetic efficiency in conjunction with phototaxis.

All-polymer solar cells with bulk heterojunction nanolayers of chemically doped electron-donating and electron-accepting polymers.

PubMed

Nam, Sungho; Shin, Minjung; Park, Soohyeong; Lee, Sooyong; Kim, Hwajeong; Kim, Youngkyoo

2012-11-21

We report the improved performance of all-polymer solar cells with bulk heterojunction nanolayers of an electron-donating polymer (poly(3-hexylthiophene) (P3HT)) and an electron-accepting polymer (poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT)), which were both doped with 4-ethylbenzenesulfonic acid (EBSA). To choose the doping ratio of P3HT for all-polymer solar cells, various EBSA doping ratios (0, 1, 3, 5, 10, 20 wt%) were tested by employing optical absorption spectroscopy, photoluminescence spectroscopy, photoelectron yield spectroscopy, and space-charge-limited current (SCLC) mobility measurement. The doping reaction of P3HT with EBSA was followed by observing the colour change in solutions. The final doping ratio for P3HT was chosen as 1 wt% from the best hole mobility measured in the thickness direction, while that for F8BT was fixed as 10 wt% (F8BT-EBSA). The polymer:polymer solar cells with bulk heterojunction nanolayers of P3HT-EBSA (EBSA-doped P3HT) and F8BT-EBSA (EBSA-doped F8BT) showed greatly improved short circuit current density (J(SC)) and open circuit voltage (V(OC)), compared to the undoped solar cells. As a result, the power conversion efficiency (PCE) was enhanced by ca. 300% for the 6 : 4 (P3HT-EBSA : F8BT-EBSA) composition and ca. 400% for the 8 : 2 composition. The synchrotron-radiation grazing incidence angle X-ray diffraction (GIXD) measurement revealed that the crystallinity of the doped nanolayers significantly increased by EBSA doping owing to the formation of advanced phase segregation morphology, as supported by the surface morphology change measured by atomic force microscopy. Thus the improved PCE can be attributed to the enhanced charge transport by the formation of permanent charges and better charge percolation paths by EBSA doping.

Effect of perfluorooctane sulfonate on the conformation of wheat germ acid phosphatase.

PubMed

Xu, Dongmei; Jin, Jianchang; Shen, Tong; Wang, Yanhua

2013-11-01

Fluorescence spectroscopy was used to study the quenching mechanism, the type of force and the binding sites of perfluorooctane sulfonate (PFOS) on wheat germ acid phosphatase (ACPase). The results showed that the quenching effect of PFOS on ACPase was mainly due to a static quenching mechanism that occurred via the formation of hydrogen bonds and van der Waals forces. The results from synchronous fluorescence spectroscopy demonstrated that PFOS interacts with ACPase close to the tryptophan residues. In addition, synchronous fluorescence spectroscopy also showed that PFOS increases the hydrophobicity of the microenvironment of the tyrosine residues, hence decreasing the local polarity.

Force spectroscopy of biomolecular folding and binding: theory meets experiment

NASA Astrophysics Data System (ADS)

Dudko, Olga

2015-03-01

Conformational transitions in biological macromolecules usually serve as the mechanism that brings biomolecules into their working shape and enables their biological function. Single-molecule force spectroscopy probes conformational transitions by applying force to individual macromolecules and recording their response, or ``mechanical fingerprints,'' in the form of force-extension curves. However, how can we decode these fingerprints so that they reveal the kinetic barriers and the associated timescales of a biological process? I will present an analytical theory of the mechanical fingerprints of macromolecules. The theory is suitable for decoding such fingerprints to extract the barriers and timescales. The application of the theory will be illustrated through recent studies on protein-DNA interactions and the receptor-ligand complexes involved in blood clot formation.

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy

PubMed Central

Mucsi, Ashley D.; Meng, Junchen; Yan, Jiacong; Zhang, Zongde; Wu, Mei; Hari, Aswin; Stenner, Melanie D.; Zheng, Wencheng; Kubes, Paul; Xia, Tie; Amrein, Matthias W.

2017-01-01

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell–DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1–dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin–cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1–dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell–mediated DC suppression in a contact-dependent manner. PMID:28082358

Fast and Forceful Refolding of Stretched α-Helical Solenoid Proteins

PubMed Central

Kim, Minkyu; Abdi, Khadar; Lee, Gwangrog; Rabbi, Mahir; Lee, Whasil; Yang, Ming; Schofield, Christopher J.; Bennett, Vann; Marszalek, Piotr E.

2010-01-01

Abstract Anfinsen's thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report investigations on the mechanical properties of individual molecules of ankyrin-R, β-catenin, and clathrin, which are representative examples of over 800 predicted human proteins composed of tightly packed α-helical repeats (termed ANK, ARM, or HEAT repeats, respectively) that form spiral-shaped protein domains. Using atomic force spectroscopy, we find that these polypeptides possess unprecedented stretch ratios on the order of 10–15, exceeding that of other proteins studied so far, and their extension and relaxation occurs with minimal energy dissipation. Their sequence-encoded elasticity is governed by stepwise unfolding of small repeats, which upon relaxation of the stretching force rapidly and forcefully refold, minimizing the hysteresis between the stretching and relaxing parts of the cycle. Thus, we identify a new class of proteins that behave as highly reversible nanosprings that have the potential to function as mechanosensors in cells and as building blocks in springy nanostructures. Our physical view of the protein component of cells as being comprised of predominantly inextensible structural elements under tension may need revision to incorporate springs. PMID:20550922

Gigantol from Dendrobium chrysotoxum Lindl. binds and inhibits aldose reductase gene to exert its anti-cataract activity: An in vitro mechanistic study.

PubMed

Wu, Jie; Li, Xue; Wan, Wencheng; Yang, Qiaohong; Ma, Weifeng; Chen, Dan; Hu, Jiangmiao; Chen, C-Y Oliver; Wei, Xiaoyong

2017-02-23

Dendrobium. chrysotoxum Lindl is a commonly used species of medicinal Dendrobium which belongs to the family of Orchidaceae, locally known as "Shihu" or "Huangcao". D. chrysotoxum Lindl is widely known for medicinal values in traditional Chinese medicine as it possesses anti-inflammatory, anti-hyperglycemic induction, antitumor and antioxidant properties. To characterize the interaction between gigantol extracted from D. chrysotoxum Lindl and the AR gene, and determine gigantol's efficacy against cataractogenesis. Human lens epithelial cells (HLECs) were induced by glucose as the model group. Reverse transcription polymerase chain reaction (RT-PCR) was used to assess AR gene expression. Then, the mode of interaction of gigantol with the AR gene was evaluated by UV-visible spectroscopy, atomic force microscope (AFM) and surface-enhanced Raman spectroscopy (SERS). The binding constant was determined by UV-visible. Gigantol depressed AR gene expression in HLECs. UV-visible spectra preliminarily indicated that interaction between the AR gene and gigantol may follow the groove mode, with a binding constant of 1.85×10 3 L/mol. Atomic force microscope (AFM) data indicated that gigantol possibly bound to insert AR gene base pairs of the double helix. Surface-enhanced Raman spectroscopy (SERS) studies further supported these observations. Gigantol extracted from D. chrysotoxum Lindl not only has inhibitory effects on aldose reductase, but also inhibits AR gene expression. These findings provide a more comprehensive theoretical basis for the use of Dendrobium for the treatment of diabetic cataract. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

The importance of hydration and DNA conformation in interpreting infrared spectra of cells and tissues.

PubMed

Wood, Bayden R

2016-04-07

Since Watson and Crick's historical papers on the structure and function of DNA based on Rosalind Franklin's and Maurice Wilkin's X-ray diffraction patterns tremendous scientific curiosity has been aroused by the unique and dynamic structure of the molecule of life. A-DNA and B-DNA represent different conformations of the DNA molecule, which is stabilised by hydrogen interactions between base pairs, stacking interactions between neighboring bases and long-range intra- and inter-backbone forces. This review highlights the contribution Fourier transform infrared (FTIR) spectroscopy has made to the understanding of DNA conformation in relation to hydration and its potential role in clinical diagnostics. The review will first begin by elucidating the main forms of DNA conformation found in nature and the general structures of the A, B and Z forms. This is followed by a detailed critique on infrared spectroscopy applied to DNA conformation highlighting pivotal studies on isolated DNA, polynucleotides, nucleoprotein and nucleohistone complexes. A discussion on the potential of diagnosing cancer using FTIR spectroscopy based on the detection of DNA bands in cells and tissues will ensue, highlighting the recent studies investigating the conformation of DNA in hydrated and dehydrated cells. The method of hydration as a way to facilitate DNA conformational band assignment will be discussed and the conformational change to the A-form upon dehydration will be used to explain the reason for the apparent lack of FTIR DNA signals observed in fixed or air-dried cells and tissues. The advantages of investigating B-DNA in the hydrated state, as opposed to A-DNA in the dehydrated state, are exemplified in a series of studies that show: (1) improved quantification of DNA in cells; (2) improved discrimination and reproducibility of FTIR spectra recorded of cells progressing through the cell cycle; (3) insights into the biological significance of A-DNA as evidenced by an interesting study on bacteria, which can survive desiccation and at the same time undergo the B-A-B transition. Finally, the importance of preserving the B-DNA conformation for the diagnosis of cancer is put forward as way to improve the sensitivity of this powerful technique.

Membrane-based actuation for high-speed single molecule force spectroscopy studies using AFM.

PubMed

Sarangapani, Krishna; Torun, Hamdi; Finkler, Ofer; Zhu, Cheng; Degertekin, Levent

2010-07-01

Atomic force microscopy (AFM)-based dynamic force spectroscopy of single molecular interactions involves characterizing unbinding/unfolding force distributions over a range of pulling speeds. Owing to their size and stiffness, AFM cantilevers are adversely affected by hydrodynamic forces, especially at pulling speeds >10 microm/s, when the viscous drag becomes comparable to the unbinding/unfolding forces. To circumvent these adverse effects, we have fabricated polymer-based membranes capable of actuating commercial AFM cantilevers at speeds >or=100 microm/s with minimal viscous drag effects. We have used FLUENT, a computational fluid dynamics (CFD) software, to simulate high-speed pulling and fast actuation of AFM cantilevers and membranes in different experimental configurations. The simulation results support the experimental findings on a variety of commercial AFM cantilevers and predict significant reduction in drag forces when membrane actuators are used. Unbinding force experiments involving human antibodies using these membranes demonstrate that it is possible to achieve bond loading rates >or=10(6) pN/s, an order of magnitude greater than that reported with commercial AFM cantilevers and systems.

AFM-based force spectroscopy on polystyrene brushes: effect of brush thickness on protein adsorption.

PubMed

Hentschel, Carsten; Wagner, Hendrik; Smiatek, Jens; Heuer, Andreas; Fuchs, Harald; Zhang, Xi; Studer, Armido; Chi, Lifeng

2013-02-12

Herein we present a study on nonspecific binding of proteins at highly dense packed hydrophobic polystyrene brushes. In this context, an atomic force microscopy tip was functionalized with concanavalin A to perform single-molecule force spectroscopy measurements on polystyrene brushes with thicknesses of 10 and 60 nm, respectively. Polystyrene brushes with thickness of 10 nm show an almost two times stronger protein adsorption than brushes with a thickness of 60 nm: 72 pN for the thinner and 38 pN for the thicker layer, which is in qualitative agreement with protein adsorption studies conducted macroscopically by fluorescence microscopy.

Probing Protein-Protein Interactions by Dynamic Force Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Barsegov, V.; Thirumalai, D.

2005-10-01

We develop a formalism for single molecule dynamic force spectroscopy to map the energy landscape of protein-protein complex (P1P2). The joint distribution P(τ1,τ2) of unbinding lifetimes τ1 and τ2, measurable in a compression-tension cycle, which accounts for the internal relaxation dynamics of the proteins under tension, shows that the histogram of τ1 is not Poissonian. The theory is applied to the forced unbinding of protein P1, modeled as a wormlike chain, from P1P2. We propose a new class of experiments which can resolve the effect of internal protein dynamics on the unbinding lifetimes.

Improved efficiency of organic solar cells using Au NPs incorporated into PEDOT:PSS buffer layer

NASA Astrophysics Data System (ADS)

Otieno, Francis; Shumbula, Ndivhuwo P.; Airo, Mildred; Mbuso, Mlambo; Moloto, Nosipho; Erasmus, Rudolph M.; Quandt, Alexander; Wamwangi, Daniel

2017-08-01

Au based plasmonic phenomenon inside the hole transport layer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) of an organic solar cell based on blend of poly(3-hexylthiophene) (P3HT) and [6:6]-phenyl-C61-butyric acid (PCBM) is investigated. The concentration of the Au nanoparticles synthesized by wet chemical reduction is one of the key factors to strong light trapping when the spherical gold nanoparticles are blended into the PEDOT:PSS solution. Studies of the influence of the concentration of nanoparticles distribution in the PEDOT:PSS were carried out using UV-Vis spectroscopy and atomic force microscopy. Electrical characteristics of the pristine device and of device with metallic nanostructures were analyzed from J -V characteristics to observe the plasmonic effects on the performance in the P3HT:PCBM organic solar cells. The origin of the photocurrent enhancements with varying Au nanoparticles concentrations on PEDOT:PSS are discussed.

Physicochemical modifications accompanying UV laser induced surface structures on poly(ethylene terephthalate) and their effect on adhesion of mesenchymal cells.

PubMed

Rebollar, Esther; Pérez, Susana; Hernández, Margarita; Domingo, Concepción; Martín, Margarita; Ezquerra, Tiberio A; García-Ruiz, Josefa P; Castillejo, Marta

2014-09-07

This work reports on the formation of different types of structures on the surface of polymer films upon UV laser irradiation. Poly(ethylene terephthalate) was irradiated with nanosecond UV pulses at 193 and 266 nm. The polarization of the laser beam and the irradiation angle of incidence were varied, giving rise to laser induced surface structures with different shapes and periodicities. The irradiated surfaces were topographically characterized by atomic force microscopy and the chemical modifications induced by laser irradiation were inspected via micro-Raman and fluorescence spectroscopies. Contact angle measurements were performed with different liquids, and the results evaluated in terms of surface free energy components. Finally, in order to test the influence of surface properties for a potential application, the modified surfaces were used for mesenchymal stem cell culture assays and the effect of nanostructure and surface chemistry on cell adhesion was evaluated.

Efficiency Improvement Using Molybdenum Disulphide Interlayers in Single-Wall Carbon Nanotube/Silicon Solar Cells.

PubMed

Alzahly, Shaykha; Yu, LePing; Shearer, Cameron J; Gibson, Christopher T; Shapter, Joseph G

2018-04-21

Molybdenum disulphide (MoS₂) is one of the most studied and widely applied nanomaterials from the layered transition-metal dichalcogenides (TMDs) semiconductor family. MoS₂ has a large carrier diffusion length and a high carrier mobility. Combining a layered structure of single-wall carbon nanotube (SWCNT) and MoS₂ with n-type silicon (n-Si) provided novel SWCNT/n-Si photovoltaic devices. The solar cell has a layered structure with Si covered first by a thin layer of MoS₂ flakes and then a SWCNT film. The films were examined using scanning electron microscopy, atomic force microscopy and Raman spectroscopy. The MoS₂ flake thickness ranged from 5 to 90 nm while the nanosheet’s lateral dimensions size ranged up to 1 μm². This insertion of MoS₂ improved the photoconversion efficiency (PCE) of the SWCNT/n-Si solar cells by approximately a factor of 2.

Inverted polymer solar cells with enhanced fill factor by inserting the potassium stearate interfacial modification layer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jiangsheng; Faculty of Materials Science and Chemical Engineering, Ningbo University, No. 818 Fenghua Road, Ningbo 315211; Jiu, Tonggang, E-mail: jiutonggang@nimte.ac.cn, E-mail: fangjf@nimte.ac.cn

2016-05-02

A thin potassium stearate (KSt) film combined with an optimized ZnO film was introduced to improve the fill factor (FF) of highly efficient inverted polymer solar cells (PSCs). Atomic force microscopy and contact angle measurements were used to show that the introduction of KSt did not change the morphology of interlayer. On the contrary, it is beneficial for the spread of the active layer on the interlayer. The origin of enhanced FF was systematically studied by the ideal current-voltage model for a single heterojunction solar cell and electrochemical impedance spectroscopy. On the basis of the data analysis, the reduced chargemore » recombination loss was responsible for this improved FF. At last, when KSt was replaced by sodium stearate (NaSt), the similar experiment phenomenon was observed. This indicates that inserting a metallic stearate modified layer is a promising strategy to enhance inverted PSCs performance.« less

Adhesion and migration of CHO cells on micropatterned single layer graphene

NASA Astrophysics Data System (ADS)

Keshavan, S.; Oropesa-Nuñez, R.; Diaspro, A.; Canale, C.; Dante, S.

2017-06-01

Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

Scanning probe microscopy in mineralogical studies: about origin of the observed roughness of natural silica-rich glasses

NASA Astrophysics Data System (ADS)

Golubev, Ye A.; Isaenko, S. I.

2017-10-01

We have studied different mineralogical objects: natural glasses of impact (tektites, impactites) and volcanic (obsidians) origin, using atomic force microscopy, X-ray microanalysis, infrared and Raman spectroscopy. The spectroscopy showed the difference in the structure and chemical composition of the glasses of different origin. The analysis of the dependence of nanoscale heterogeneity of the glasses, revealed by the atomic force microscopy, on their structural and chemical features was carried out.

Atomic Force Microscope for Imaging and Spectroscopy

NASA Technical Reports Server (NTRS)

Pike, W. T.; Hecht, M. H.; Anderson, M. S.; Akiyama, T.; Gautsch, S.; deRooij, N. F.; Staufer, U.; Niedermann, Ph.; Howald, L.; Mueller, D.

2000-01-01

We have developed, built, and tested an atomic force microscope (AFM) for extraterrestrial applications incorporating a micromachined tip array to allow for probe replacement. It is part of a microscopy station originally intended for NASA's 2001 Mars lander to identify the size, distribution, and shape of Martian dust and soil particles. As well as imaging topographically down to nanometer resolution, this instrument can be used to reveal chemical information and perform infrared and Raman spectroscopy at unprecedented resolution.

Vacuum arc plasma deposition of thin titanium dioxide films on silicone elastomer as a functional coating for medical applications.

PubMed

Boudot, Cécile; Kühn, Marvin; Kühn-Kauffeldt, Marina; Schein, Jochen

2017-05-01

Silicone elastomer is a promising material for medical applications and is widely used for implants with blood and tissue contact. However, its strong hydrophobicity limits adhesion of tissue cells to silicone surfaces, which can impair the healing process. To improve the biological properties of silicone, a triggerless pulsed vacuum cathodic arc plasma deposition technique was applied to deposit titanium dioxide (TiO 2 ) films onto the surface. Scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and contact angle measurements were used for coating characterization. Deposited films were about 150nm thick and exhibited good adhesion to the underlying silicone substrate. Surface wettability and roughness both increased after deposition of the TiO 2 layer. In addition, cell-biological investigations demonstrated that the in-vitro cytocompatibility of TiO 2 -coated samples was greatly improved without impacting silicone's nontoxicity. For validation of use in medical devices, further investigations were conducted and demonstrated stability of surface properties in an aqueous environment for a period of 68days and the coating's resistance to several sterilization methods. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of power on growth of nanocrystalline silicon films deposited by VHF PECVD technique for solar cell applications

NASA Astrophysics Data System (ADS)

Juneja, Sucheta; Verma, Payal; Savelyev, Dmitry A.; Khonina, Svetlana N.; Sudhakar, S.; Kumar, Sushil

2016-04-01

An investigation of the effect of power on the deposition of nanocrystalline silicon thin films were carried out using a gaseous mixture of silane and hydrogen in the 60MHz assisted VHF plasma enhanced chemical vapor deposition (PECVD) technique. The power was varied from 10 to 50 watt maintaining all other parameters constant. Corresponding layer properties w.r.t. material microstructure, optical, hydrogen content and electrical transport are studied in detail. The structural properties have been studied by Raman spectroscopy and x-ray diffraction (XRD). The presence of nano-sized crystals and their morphology have been investigated using atomic force microscopy (AFM). The role of bonded hydrogen content in the films have been studied from the results of Fourier transform infrared spectroscopy. It was observed from the results that with increase in power, crystalline volume fraction increases and crystallite size changes from 4 to 9 nm. The optical band gap varies from 1.7 to 2.1eV due to quantum confinement effect and which further can be explained with reduced hydrogen content. These striking features of nc-Si films can be used to fabricate stable thin film solar cells.

Structure and Dynamics Investigations of Sr/Ca-Doped LaPO 4 Proton Conductors

DOE Office of Scientific and Technical Information (OSTI.GOV)

al-Wahish, Amal; al-Binni, U.; Tetard, L.

Proton conductors loom out of the pool of candidate materials with great potential to boost hydrogen alternatives to fossil-based resources for energy. Acceptor doped lanthanum orthophosphates are considered for solid oxide fuel cells (SOFCs) for their potential stability and conductivity at high temperature. By exploring the crystal and defect structure of x% Sr/Ca-doped LaPO 4 with different nominal Sr/Ca concentrations (x = 0 – 10) with Neutron powder diffraction (NPD) and X-ray powder diffraction (XRD), we confirm that Sr/Ca-doped LaPO 4 can exist as self-supported structures at high temperatures during solid oxide fuel cell operation. Thermal stability, surface topography, sizemore » distribution are also studied to better understand the proton conductivity for dry and wet compounds obtained at sintering temperatures ranging from 1200 to 1400 °C using a combination of scanning electron microscopy (SEM), Atomic Force Microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS). In conclusion, the results suggest that Sr doped samples exhibit the highest proton conductivity of our samples and illustrate the impact of material design and versatile characterization schemes on the development of proton conductors with superior functionality.« less

Direct Observation of Markovian Behavior of the Mechanical Unfolding of Individual Proteins

PubMed Central

Cao, Yi; Kuske, Rachel; Li, Hongbin

2008-01-01

Single-molecule force-clamp spectroscopy is a valuable tool to analyze unfolding kinetics of proteins. Previous force-clamp spectroscopy experiments have demonstrated that the mechanical unfolding of ubiquitin deviates from the generally assumed Markovian behavior and involves the features of glassy dynamics. Here we use single molecule force-clamp spectroscopy to study the unfolding kinetics of a computationally designed fast-folding mutant of the small protein GB1, which shares a similar β-grasp fold as ubiquitin. By treating the mechanical unfolding of polyproteins as the superposition of multiple identical Poisson processes, we developed a simple stochastic analysis approach to analyze the dwell time distribution of individual unfolding events in polyprotein unfolding trajectories. Our results unambiguously demonstrate that the mechanical unfolding of NuG2 fulfills all criteria of a memoryless Markovian process. This result, in contrast with the complex mechanical unfolding behaviors observed for ubiquitin, serves as a direct experimental demonstration of the Markovian behavior for the mechanical unfolding of a protein and reveals the complexity of the unfolding dynamics among structurally similar proteins. Furthermore, we extended our method into a robust and efficient pseudo-dwell-time analysis method, which allows one to make full use of all the unfolding events obtained in force-clamp experiments without categorizing the unfolding events. This method enabled us to measure the key parameters characterizing the mechanical unfolding energy landscape of NuG2 with improved precision. We anticipate that the methods demonstrated here will find broad applications in single-molecule force-clamp spectroscopy studies for a wide range of proteins. PMID:18375518

PREFACE: Cell-substrate interactions Cell-substrate interactions

NASA Astrophysics Data System (ADS)

Gardel, Margaret; Schwarz, Ulrich

2010-05-01

One of the most striking achievements of evolution is the ability to build cellular systems that are both robust and dynamic. Taken by themselves, both properties are obvious requirements: robustness reflects the fact that cells are there to survive, and dynamics is required to adapt to changing environments. However, it is by no means trivial to understand how these two requirements can be implemented simultaneously in a physical system. The long and difficult quest to build adaptive materials is testimony to the inherent difficulty of this goal. Here materials science can learn a lot from nature, because cellular systems show that robustness and dynamics can be achieved in a synergetic fashion. For example, the capabilities of tissues to repair and regenerate are still unsurpassed in the world of synthetic materials. One of the most important aspects of the way biological cells adapt to their environment is their adhesive interaction with the substrate. Numerous aspects of the physiology of metazoan cells, including survival, proliferation, differentiation and migration, require the formation of adhesions to the cell substrate, typically an extracellular matrix protein. Adhesions guide these diverse processes both by mediating force transmission from the cell to the substrate and by controlling biochemical signaling pathways. While the study of cell-substrate adhesions is a mature field in cell biology, a quantitative biophysical understanding of how the interactions of the individual molecular components give rise to the rich dynamics and mechanical behaviors observed for cell-substrate adhesions has started to emerge only over the last decade or so. The recent growth of research activities on cell-substrate interactions was strongly driven by the introduction of new physical techniques for surface engineering into traditional cell biological work with cell culture. For example, microcontact printing of adhesive patterns was used to show that cell fate depends not on the amount of ligand for adhesion receptors, but on its spatial distribution [1]. New protocols for the preparation of soft elastic substrates were essential to show that adhesion structures and cytoskeleton of adherent cells strongly adapt to substrate stiffness [2], with dramatic effects for cellular decision making. For example, it has been shown recently that differentiation of mesenchymal stem cells is strongly influenced by substrate stiffness [3]. Thus, physical factors appear to be equally important as biochemical ones in determining the cellular response to its substrate [4]. The introduction of novel physical techniques not only opened up completely new perspectives regarding biological function, it also introduced a new quantitative element into this field. For example, the availability of soft elastic substrates with controlled stiffness allows us to reconstruct cellular traction forces and to correlate them with other cellular features. This development enables modeling approaches to work in close contact with experimental data, thus opening up the perspective that the field of cell-substrate interactions will become a quantitative and predictive science in the future. Because physical research into cell-substrate interactions has become one of the fastest growing research areas in cellular biophysics and materials science, we believe that it is very timely that this special issue gathers some of the on-going research effort in this field. In contrast to the non-living world, cellular systems usually interact with their environment through specific adhesion, mainly based on adhesion receptors from the integrin family. During recent years, force spectroscopy has emerged as one of the main methods to study the physics of specific adhesion. In this special issue, single cell force spectroscopy is used by Boettiger and Wehrle-Haller to characterize the strength of cell-matrix adhesion and how it is modulated by the glycocalyx [5], while Chirasatitsin and Engler use force spectroscopy mapping to characterize the spatial distribution of adhesive sites on the substrate [6]. Scrimgeour et al describe a new method to adhesively pattern self-assembled monolayers for cell adhesion by a simple photobleaching setup [7] and Stricker et al demonstrate how elastic substrates can be combined with microcontact printing to improve the reconstruction of traction forces [8]. The work by Metzner et al shows that meaningful results on the cell-substrate interactions can be extracted also from experiments in which cells interact with biofunctionalized beads [9]. If cells start to adhere to a substrate, the main rate-limiting step is establishment of close contact between the plasma membrane and the substrate. This process can be followed with high spatial and temporal resolution with reflection interference microscopy, as demonstrated by Ryzhkov et al for mouse embryonic fibroblasts [10] and by Cretel et al for T lymphocytes [11]. Once mature adhesion has been achieved, the integrin-based focal adhesions providing anchorage to the substrate are strongly connected to the actin cytoskeleton, the main determinant of cell shape and structure. Heil and Spatz use microfabricated pillars to perturb the mechanical balance and quantitatively characterize the fast response of the focal adhesions [12]. A similar approach is used by Kirchenbüchler et al, who use deformation of an elastic substrate to demonstrate that the weak link in the mechanical system of substrate, adhesions and actin cytoskeleton is most likely located at the adhesion-cytoskeleton interface [13]. Rather than using external perturbations, Zemel et al quantify and model how cells spontaneously polarize their cytoskeleton in response to the physical properties of the substrate [14]. Quantitative analysis of cellular data has become standard in the field of cell-substrate interactions. Moreover, theoretical models for cell-substrate interactions help us to identify and understand the mechanisms underlying the observed phenomena in these complex systems. Recently, a large effort has been invested into understanding how force transmitted by the actin cytoskeleton changes the state of focal adhesions. In the contribution by Biton and Safran, this issue is addressed for the case that force arises from shear flow over an adhering cell [15]. Another important source for force on focal adhesions is actin retrograde flow, which has been demonstrated before to show variable coupling to the underlying layer of adhesion receptors. Two contributions discuss how stochastic bond dynamics at the cell-substrate interface is modulated by physical factors. The model by Sabass and Schwarz suggests that dissipation in the actin cytoskeleton stabilizes bond dynamics [16] and the model by Li et al suggests that catch bonding and multiple layers are important elements of the way focal adhesions function [17]. If interacting with an elastic environment, the combined system of focal adhesions and actin cytoskeleton can be used by cells to sense its rigidity and to make decisions on its response. Moshayedi et al show that great care has to be taken when preparing soft elastic substrates for cell culture studies and then use their protocols to quantitatively evaluate the mechanosensitive response of astrocytes from the brain [18]. The cellular system used by Lee et al is pericytes from the microvasculature, for which the authors show that they exert sufficient forces to stimulate vascular endothelial cells [19]. Buxboim et al use the technology of soft elastic substrates to measure how far mesenchymal stem cells can mechanically sense into their substrate [20]. The mechanical activity of cells observed in two-dimensional cell culture has significant consequences for both physiological and disease-related situations, including cell migration, tissue maintenance and tumor growth. Jannat et al show that chemotaxis of neutrophils, that is the first line of the immune system, is strongly modulated by mechanosensing on substrates of varying stiffness [21]. Mogilner and Rubinstein present a theoretical systems analysis for the shape of rapidly migrating keratocytes [22]. Saez et al show, with microfabricated pillar assays, how force is distributed within a layer of epithelial cells [23]. For three-dimensional tissue models, new techniques have to be developed to characterize the complex mechanics of hydrogels. Levental et al [24] and Kotlarchyk et al [25] approach this challenge with mechanical and optical methods, respectively. Narayanan et al combine experiments and continuum models to explore how chemo-mechanical interactions influence tumor growth [26]. References [1] Chen C S, Mrksich M, Huang S, Whitesides G M and Ingber D E 1997 Geometric control of cell life and death Science 276 1425 [2] Pelham R J Jr and Wang Y-L 1997 Cell locomotion and focal adhesions are regulated by substrate flexibility Proc. Natl. Acad. Sci. USA 94 13661 [3] Engler A J, Sen S, Sweeney H L and Discher D E 2006 Matrix elasticity directs stem cell lineage specification Cell 126 677-89 [4] Geiger B, Spatz J P and Bershadsky A D 2009 Environmental sensing through focal adhesions Nat. Rev. Mol. Cell Biol. 10 21 [5] Boettiger D and Wehrle-Haller B 2010 Integrin and glycocalyx mediated contributions to cell adhesion identified by single cell force spectroscopy J. Phys.: Condens. Matter 22 194101 [6] Chirasatitsin S and Engler A J 2010 Detecting cell-adhesive sites in extracellular matrix using force spectroscopy mapping J. Phys.: Condens. Matter 22 194102 [7] Scrimgeour J, Kodali V K, Kovari D T and Curtis J E 2010 Photobleaching-activated micropatterning on self-assembled monolayers J. Phys.: Condens. Matter 22 194103 [8] Stricker J, Sabass B, Schwarz U S and Gardel M L 2010 Optimization of traction force microscopy for micron-sized focal adhesions J. Phys.: Condens. Matter 22 194104 [9] Metzner C, Raupach C, Mierke C T and Fabry B 2010 Fluctuations of cytoskeleton-bound microbeads—the effect of bead-receptor binding dynamics J. Phys.: Condens. Matter 22 194105 [10] Ryzhkov P, Prass M, Gummich M, Kühn J-S, Oettmeier C and Döbereiner H-G 2010 Adhesion patterns in early cell spreading J. Phys.: Condens. Matter 22 194106 [11] Cretel E, Touchard D, Benoliel A M, Bongrand P and Pierres A 2010 Early contacts between T lymphocytes and activating surfaces J. Phys.: Condens. Matter 22 194107 [12] Heil P and Spatz J P 2010 Lateral shear forces applied to cells with single elastic micropillars to influence focal adhesion dynamics J. Phys.: Condens. Matter 22 194108 [13] Kirchenbüchler D, Born S, Kirchgeßner N, Houben S, Hoffmann B and Merkel R 2010 Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins J. Phys.: Condens. Matter 22 194109 [14] Zemel A, Rehfeldt F, Brown A E X, Discher D E and Safran S A 2010 Cell shape, spreading symmetry, and the polarization of stress-fibers in cells J. Phys.: Condens. Matter 22 194110 [15] Biton Y Y and Safran S A 2010 Theory of the mechanical response of focal adhesions to shear flow J. Phys.: Condens. Matter 22 194111 [16] Sabass B and Schwarz U S 2010 Modeling cytoskeletal flow over adhesion sites: competition between stochastic bond dynamics and intracellular relaxation J. Phys.: Condens. Matter 22 194112 [17] Li Y, Bhimalapuram P and Dinner A R 2010 Model for how retrograde actin flow regulates adhesion traction stresses J. Phys.: Condens. Matter 22 194113 [18] Moshayedi P, da F Costa L, Christ A, Lacour S P, Fawcett J, Guck J and Franze K 2010 Mechanosensitivity of astrocytes on optimized polyacrylamide gels analyzed by quantitative morphometry J. Phys.: Condens. Matter 22 194114 [19] Lee S, Zeiger A, Maloney J M, Kotecki M, Van Vliet K J and Herman I M 2010 Pericyte contraction at the cell-material interface can modulate the microvascular niche J. Phys.: Condens. Matter 22 194115 [20] Buxboim A, Rajagopal K, Brown A E X and Discher D E 2010 How deeply cells feel: methods for thin gels J. Phys.: Condens. Matter 22 194116 [21] Jannat R A, Robbins G P, Ricart B G, Dembo M and Hammer D A 2010 Neutrophil adhesion and chemotaxis depend on substrate mechanics J. Phys.: Condens. Matter 22 194117 [22] Mogilner A and Rubinstein B 2010 Actin disassembly 'clock' and membrane tension determine cell shape and turning: a mathematical method J. Phys.: Condens. Matter 22 194118 [23] Saez A, Anon E, Ghibaudo M, du Roure O, Di Meglio J-M, Hersen P, Silberzan P, Buguin A, Ladoux B 2010 Traction forces exerted by epithelial cell sheets J. Phys.: Condens. Matter 22 194119 [24] Levental I, Levental K R, Klein E A, Assoian R, Miller R T, Wells R G and Janmey P A 2010 A simple indentation device for measuring micrometer-scale tissue stiffness J. Phys.: Condens. Matter 22 194120 [25] Kotlarchyk M A, Botvinick E L and Putnam A J 2010 Characterization of hydrogel microstructure using laser tweezers particle tracking and confocal reflection imaging J. Phys.: Condens. Matter 22 194121 [26] Narayanan H, Verner S N, Mills K L, Kemkemer R and Garikipati K 2010 In silico estimates of the free energy rates in growing tumor spheroids J. Phys.: Condens. Matter 22 194122

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

PubMed

Paul, Biplab C; El-Ganiny, Amira M; Abbas, Mariam; Kaminskyj, Susan G W; Dahms, Tanya E S

2011-05-01

The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.

Molecular mechanism of extreme mechanostability in a pathogen adhesin.

PubMed

Milles, Lukas F; Schulten, Klaus; Gaub, Hermann E; Bernardi, Rafael C

2018-03-30

High resilience to mechanical stress is key when pathogens adhere to their target and initiate infection. Using atomic force microscopy-based single-molecule force spectroscopy, we explored the mechanical stability of the prototypical staphylococcal adhesin SdrG, which targets a short peptide from human fibrinogen β. Steered molecular dynamics simulations revealed, and single-molecule force spectroscopy experiments confirmed, the mechanism by which this complex withstands forces of over 2 nanonewtons, a regime previously associated with the strength of a covalent bond. The target peptide, confined in a screwlike manner in the binding pocket of SdrG, distributes forces mainly toward the peptide backbone through an intricate hydrogen bond network. Thus, these adhesins can attach to their target with exceptionally resilient mechanostability, virtually independent of peptide side chains. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Exploring the folding pattern of a polymer chain in a single crystal by combining single-molecule force spectroscopy and steered molecular dynamics simulations.

PubMed

Song, Yu; Feng, Wei; Liu, Kai; Yang, Peng; Zhang, Wenke; Zhang, Xi

2013-03-26

Understanding the folding pattern of a single polymer chain within its single crystal will shed light on the mechanism of crystallization. Here, we use the combined techniques of atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations to study the folding pattern of a polyethylene oxide (PEO) chain in its single crystal. Our results show that the folding pattern of a PEO chain in the crystal formed in dilute solution follows the adjacent re-entry folding model. While in the crystal obtained from the melt, the nonadjacent folding with large and irregular loops contributes to big force fluctuations in the force-extension curves. The method established here can offer a novel strategy to directly unravel the chain-folding pattern of polymer single crystals at single-molecule level.

A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force

NASA Astrophysics Data System (ADS)

Bano, Fouzia; Banerji, Suneale; Howarth, Mark; Jackson, David G.; Richter, Ralf P.

2016-09-01

Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG·protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG·protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices.

Perspective: Mechanochemistry of biological and synthetic molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makarov, Dmitrii E., E-mail: makarov@cm.utexas.edu

Coupling of mechanical forces and chemical transformations is central to the biophysics of molecular machines, polymer chemistry, fracture mechanics, tribology, and other disciplines. As a consequence, the same physical principles and theoretical models should be applicable in all of those fields; in fact, similar models have been invoked (and often repeatedly reinvented) to describe, for example, cell adhesion, dry and wet friction, propagation of cracks, and action of molecular motors. This perspective offers a unified view of these phenomena, described in terms of chemical kinetics with rates of elementary steps that are force dependent. The central question is then tomore » describe how the rate of a chemical transformation (and its other measurable properties such as the transition path) depends on the applied force. I will describe physical models used to answer this question and compare them with experimental measurements, which employ single-molecule force spectroscopy and which become increasingly common. Multidimensionality of the underlying molecular energy landscapes and the ensuing frequent misalignment between chemical and mechanical coordinates result in a number of distinct scenarios, each showing a nontrivial force dependence of the reaction rate. I will discuss these scenarios, their commonness (or its lack), and the prospects for their experimental validation. Finally, I will discuss open issues in the field.« less

Probing and mapping electrode surfaces in solid oxide fuel cells.

PubMed

Blinn, Kevin S; Li, Xiaxi; Liu, Mingfei; Bottomley, Lawrence A; Liu, Meilin

2012-09-20

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen (1-7). The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion(2). Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation(8-12). It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition(8, 10, 13, 14) ("coking") and sulfur poisoning(11, 15) and the manner in which surface modifications stave off this degradation(16). The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated(17-22). In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H2S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

PubMed Central

Blinn, Kevin S.; Li, Xiaxi; Liu, Mingfei; Bottomley, Lawrence A.; Liu, Meilin

2012-01-01

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen 1-7. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion2. Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation8-12. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition8, 10, 13, 14 ("coking") and sulfur poisoning11, 15 and the manner in which surface modifications stave off this degradation16. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated17-22. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H2S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode. PMID:23023264

Quartz tuning fork-based frequency modulation atomic force spectroscopy and microscopy with all digital phase-locked loop

NASA Astrophysics Data System (ADS)

An, Sangmin; Hong, Mun-heon; Kim, Jongwoo; Kwon, Soyoung; Lee, Kunyoung; Lee, Manhee; Jhe, Wonho

2012-11-01

We present a platform for the quartz tuning fork (QTF)-based, frequency modulation atomic force microscopy (FM-AFM) system for quantitative study of the mechanical or topographical properties of nanoscale materials, such as the nano-sized water bridge formed between the quartz tip (˜100 nm curvature) and the mica substrate. A thermally stable, all digital phase-locked loop is used to detect the small frequency shift of the QTF signal resulting from the nanomaterial-mediated interactions. The proposed and demonstrated novel FM-AFM technique provides high experimental sensitivity in the measurement of the viscoelastic forces associated with the confined nano-water meniscus, short response time, and insensitivity to amplitude noise, which are essential for precision dynamic force spectroscopy and microscopy.

Quartz tuning fork-based frequency modulation atomic force spectroscopy and microscopy with all digital phase-locked loop.

PubMed

An, Sangmin; Hong, Mun-heon; Kim, Jongwoo; Kwon, Soyoung; Lee, Kunyoung; Lee, Manhee; Jhe, Wonho

2012-11-01

We present a platform for the quartz tuning fork (QTF)-based, frequency modulation atomic force microscopy (FM-AFM) system for quantitative study of the mechanical or topographical properties of nanoscale materials, such as the nano-sized water bridge formed between the quartz tip (~100 nm curvature) and the mica substrate. A thermally stable, all digital phase-locked loop is used to detect the small frequency shift of the QTF signal resulting from the nanomaterial-mediated interactions. The proposed and demonstrated novel FM-AFM technique provides high experimental sensitivity in the measurement of the viscoelastic forces associated with the confined nano-water meniscus, short response time, and insensitivity to amplitude noise, which are essential for precision dynamic force spectroscopy and microscopy.

Synthesis of strongly fluorescent molybdenum disulfide nanosheets for cell-targeted labeling.

PubMed

Wang, Nan; Wei, Fang; Qi, Yuhang; Li, Hongxiang; Lu, Xin; Zhao, Guoqiang; Xu, Qun

2014-11-26

MoS2 nanosheets with polydispersity of the lateral dimensions from natural mineral molybdenite have been prepared in the emulsions microenvironment built by the water/surfactant/CO2 system. The size, thickness, and atomic structure are characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), and laser-scattering particle size analysis. Meanwhile, by the analysis of photoluminescence spectroscopy and microscope, the MoS2 nanosheets with smaller lateral dimensions exhibit extraordinary photoluminescence properties different from those with relatively larger lateral dimensions. The discovery of the excitation dependent photoluminescence for MoS2 nanosheets makes them potentially of interests for the applications in optoelectronics and biology. Moreover, we demonstrate that the fabricated MoS2 nanosheets can be a nontoxic fluorescent label for cell-targeted labeling application.

Reversible Stabilization of Vesicles: Redox-Responsive Polymer Nanocontainers for Intracellular Delivery.

PubMed

de Vries, Wilke C; Grill, David; Tesch, Matthias; Ricker, Andrea; Nüsse, Harald; Klingauf, Jürgen; Studer, Armido; Gerke, Volker; Ravoo, Bart Jan

2017-08-01

We present the self-assembly of redox-responsive polymer nanocontainers comprising a cyclodextrin vesicle core and a thin reductively cleavable polymer shell anchored via host-guest recognition on the vesicle surface. The nanocontainers are of uniform size, show high stability, and selectively respond to a mild reductive trigger as revealed by dynamic light scattering, transmission electron microscopy, atomic force microscopy, a quantitative thiol assay, and fluorescence spectroscopy. Live cell imaging experiments demonstrate a specific redox-responsive release and cytoplasmic delivery of encapsulated hydrophilic payloads, such as the pH-probe pyranine, and the fungal toxin phalloidin. Our results show the high potential of these stimulus-responsive nanocontainers for cell biological applications requiring a controlled delivery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improving single molecule force spectroscopy through automated real-time data collection and quantification of experimental conditions

PubMed Central

Scholl, Zackary N.; Marszalek, Piotr E.

2013-01-01

The benefits of single molecule force spectroscopy (SMFS) clearly outweigh the challenges which include small sample sizes, tedious data collection and introduction of human bias during the subjective data selection. These difficulties can be partially eliminated through automation of the experimental data collection process for atomic force microscopy (AFM). Automation can be accomplished using an algorithm that triages usable force-extension recordings quickly with positive and negative selection. We implemented an algorithm based on the windowed fast Fourier transform of force-extension traces that identifies peaks using force-extension regimes to correctly identify usable recordings from proteins composed of repeated domains. This algorithm excels as a real-time diagnostic because it involves <30 ms computational time, has high sensitivity and specificity, and efficiently detects weak unfolding events. We used the statistics provided by the automated procedure to clearly demonstrate the properties of molecular adhesion and how these properties change with differences in the cantilever tip and protein functional groups and protein age. PMID:24001740

Stimulated Raman spectroscopy and nanoscopy of molecules using near field photon induced forces without resonant electronic enhancement gain

NASA Astrophysics Data System (ADS)

Tamma, Venkata Ananth; Huang, Fei; Nowak, Derek; Kumar Wickramasinghe, H.

2016-06-01

We report on stimulated Raman spectroscopy and nanoscopy of molecules, excited without resonant electronic enhancement gain, and recorded using near field photon induced forces. Photon-induced interaction forces between the sharp metal coated silicon tip of an Atomic Force Microscope (AFM) and a sample resulting from stimulated Raman excitation were detected. We controlled the tip to sample spacing using the higher order flexural eigenmodes of the AFM cantilever, enabling the tip to come very close to the sample. As a result, the detection sensitivity was increased compared with previous work on Raman force microscopy. Raman vibrational spectra of azobenzene thiol and l-phenylalanine were measured and found to agree well with published results. Near-field force detection eliminates the need for far-field optical spectrometer detection. Recorded images show spatial resolution far below the optical diffraction limit. Further optimization and use of ultrafast pulsed lasers could push the detection sensitivity towards the single molecule limit.

Stimulated Raman spectroscopy and nanoscopy of molecules using near field photon induced forces without resonant electronic enhancement gain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tamma, Venkata Ananth; Huang, Fei; Kumar Wickramasinghe, H., E-mail: hkwick@uci.edu

We report on stimulated Raman spectroscopy and nanoscopy of molecules, excited without resonant electronic enhancement gain, and recorded using near field photon induced forces. Photon-induced interaction forces between the sharp metal coated silicon tip of an Atomic Force Microscope (AFM) and a sample resulting from stimulated Raman excitation were detected. We controlled the tip to sample spacing using the higher order flexural eigenmodes of the AFM cantilever, enabling the tip to come very close to the sample. As a result, the detection sensitivity was increased compared with previous work on Raman force microscopy. Raman vibrational spectra of azobenzene thiol andmore » l-phenylalanine were measured and found to agree well with published results. Near-field force detection eliminates the need for far-field optical spectrometer detection. Recorded images show spatial resolution far below the optical diffraction limit. Further optimization and use of ultrafast pulsed lasers could push the detection sensitivity towards the single molecule limit.« less

Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

PubMed Central

2013-01-01

Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. PMID:24059222

Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

NASA Astrophysics Data System (ADS)

Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

2013-09-01

Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a `green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

Nanofibers of Human Tropoelastin-inspired peptides: Structural characterization and biological properties.

PubMed

Secchi, Valeria; Franchi, Stefano; Fioramonti, Marco; Polzonetti, Giovanni; Iucci, Giovanna; Bochicchio, Brigida; Battocchio, Chiara

2017-08-01

Regenerative medicine is taking great advantage from the use of biomaterials in the treatments of a wide range of diseases and injuries. Among other biomaterials, self-assembling peptides are appealing systems due to their ability to spontaneously form nanostructured hydrogels that can be directly injected into lesions. Indeed, self-assembling peptide scaffolds are expected to behave as biomimetic matrices able to surround cells, to promote specific interactions, and to control and modify cell behavior by mimicking the native environment as well. We selected three pentadecapeptides inspired by Human Tropoelastin, a natural protein of the extracellular matrix, expected to show high biocompatibility. Moreover, the here proposed self-assembling peptides (SAPs) are able to spontaneously aggregate in nanofibers in biological environment, as revealed by AFM (Atomic Force Microscopy). Peptides were characterized by XPS (X-ray Photoelectron Spectroscopy) and IRRAS (Infrared Reflection Absorption Spectroscopy) both as lyophilized (not aggregated) and as aggregated (nanofibers) samples in order to investigate some potential differences in their chemical composition and intermolecular interactions, and to analyze the surface and interface of nanofibers. Finally, an accurate investigation of the biological properties of the SAPs and of their interaction with cells was performed by culturing for the first time human Mesenchymal Stem Cells (hMSCs) in presence of SAPs. The final aim of this work was to assess if Human Tropoelastin-inspired nanostructured fibers could exert a cytotoxic effect and to evaluate their biocompatibility, cellular adhesion and proliferation. Copyright © 2017 Elsevier B.V. All rights reserved.

Improved Carrier Transport in Perovskite Solar Cells Probed by Femtosecond Transient Absorption Spectroscopy.

PubMed

Serpetzoglou, Efthymis; Konidakis, Ioannis; Kakavelakis, George; Maksudov, Temur; Kymakis, Emmanuel; Stratakis, Emmanuel

2017-12-20

CH 3 NH 3 PbI 3 perovskite thin films have been deposited on glass/indium tin oxide/hole transport layer (HTL) substrates, utilizing two different materials as the HTLs. In the first configuration, the super hydrophilic polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), known as PEDOT:PSS, was employed as the HTL material, whereas in the second case, the nonwetting poly(triarylamine) semiconductor polymer, known as PTAA, was used. It was found that when PTAA is used as the HTL material, the averaged power conversion efficiency (PCE) of the perovskite solar cells (PSCs) remarkably increases from 12.60 to 15.67%. To explore the mechanism behind this enhancement, the aforementioned perovskite/HTL arrangements were investigated by time-resolved transient absorption spectroscopy (TAS) performed under inert conditions. By means of TAS, the charge transfer, carrier trapping, and hole injection dynamics from the photoexcited perovskite layers to the HTL can be directly monitored via the characteristic bleaching profile of the perovskite at ∼750 nm. TAS studies revealed faster relaxation times and decay dynamics when the PTAA polymer is employed, which potentially account for the enhanced PCE observed. The TAS results are correlated with the structure and crystalline quality of the corresponding perovskite films, investigated by scanning electron microscopy, X-ray diffraction, atomic force microscopy, micro-photoluminescence, and transmittance spectroscopy. It is concluded that TAS is a benchmark technique for the understanding of the carrier transport mechanisms in PSCs and constitutes a figure-of-merit tool toward their efficiency improvement.

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies.

PubMed

Kumorek, Marta; Kubies, Dana; Filová, Elena; Houska, Milan; Kasoju, Naresh; Mázl Chánová, Eliška; Matějka, Roman; Krýslová, Markéta; Bačáková, Lucie; Rypáček, František

2015-01-01

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm(2). The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm(2)) than on surfaces with a higher concentration of FGF-2 (120 ng/cm(2)).

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies

PubMed Central

Kumorek, Marta; Kubies, Dana; Filová, Elena; Houska, Milan; Kasoju, Naresh; Mázl Chánová, Eliška; Matějka, Roman; Krýslová, Markéta; Bačáková, Lucie; Rypáček, František

2015-01-01

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm2. The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm2) than on surfaces with a higher concentration of FGF-2 (120 ng/cm2). PMID:25945799

Force related hemodynamic responses during execution and imagery of a hand grip task: A functional near infrared spectroscopy study.

PubMed

Wriessnegger, Selina C; Kirchmeyr, Daniela; Bauernfeind, Günther; Müller-Putz, Gernot R

2017-10-01

We examined force related hemodynamic changes during the performance of a motor execution (ME) and motor imagery (MI) task by means of multichannel functional near infrared spectroscopy (fNIRS). The hemodynamic responses of fourteen healthy participants were measured while they performed a hand grip execution or imagery task with low and high grip forces. We found an overall higher increase of [oxy-Hb] concentration changes during ME for both grip forces but with a delayed peak maximum for the lower grip force. During the MI task with lower grip force, the [oxy-Hb] level increases are stronger compared to the MI with higher grip force. The facilitation in performing MI with higher grip strength might thus indicate less inhibition of the actual motor act which could also explain the later increase onset of [oxy-Hb] in the ME task with the lower grip force. Our results suggest that execution and imagery of a hand grip task with high and low grip forces, leads to different cortical activation patterns. Since impaired control of grip forces during object manipulation in particular is one aspect of fine motor control deficits after stroke, our study will contribute to future rehabilitation programs enhancing patient's grip force control. Copyright © 2017 Elsevier Inc. All rights reserved.

Bottom-up electrochemical preparation of solid-state carbon nanodots directly from nitriles/ionic liquids using carbon-free electrodes and the applications in specific ferric ion detection and cell imaging

NASA Astrophysics Data System (ADS)

Niu, Fushuang; Xu, Yuanhong; Liu, Mengli; Sun, Jing; Guo, Pengran; Liu, Jingquan

2016-03-01

Carbon nanodots (C-dots), a new type of potential alternative to conventional semiconductor quantum dots, have attracted numerous attentions in various applications including bio-chemical sensing, cell imaging, etc., due to their chemical inertness, low toxicity and flexible functionalization. Various methods including electrochemical (EC) methods have been reported for the synthesis of C-dots. However, complex procedures and/or carbon source-containing electrodes are often required. Herein, solid-state C-dots were simply prepared by bottom-up EC carbonization of nitriles (e.g. acetonitrile) in the presence of an ionic liquid [e.g. 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6)], using carbon-free electrodes. Due to the positive charges of BMIM+ on the C-dots, the final products presented in a precipitate form on the cathode, and the unreacted nitriles and BMIMPF6 can be easily removed by simple vacuum filtration. The as-prepared solid-state C-dots can be well dispersed in an aqueous medium with excellent photoluminescence properties. The average size of the C-dots was found to be 3.02 +/- 0.12 nm as evidenced by transmission electron microscopy. Other techniques such as UV-vis spectroscopy, fluorescence spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy were applied for the characterization of the C-dots and to analyze the possible generation mechanism. These C-dots have been successfully applied in efficient cell imaging and specific ferric ion detection.Carbon nanodots (C-dots), a new type of potential alternative to conventional semiconductor quantum dots, have attracted numerous attentions in various applications including bio-chemical sensing, cell imaging, etc., due to their chemical inertness, low toxicity and flexible functionalization. Various methods including electrochemical (EC) methods have been reported for the synthesis of C-dots. However, complex procedures and/or carbon source-containing electrodes are often required. Herein, solid-state C-dots were simply prepared by bottom-up EC carbonization of nitriles (e.g. acetonitrile) in the presence of an ionic liquid [e.g. 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6)], using carbon-free electrodes. Due to the positive charges of BMIM+ on the C-dots, the final products presented in a precipitate form on the cathode, and the unreacted nitriles and BMIMPF6 can be easily removed by simple vacuum filtration. The as-prepared solid-state C-dots can be well dispersed in an aqueous medium with excellent photoluminescence properties. The average size of the C-dots was found to be 3.02 +/- 0.12 nm as evidenced by transmission electron microscopy. Other techniques such as UV-vis spectroscopy, fluorescence spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy were applied for the characterization of the C-dots and to analyze the possible generation mechanism. These C-dots have been successfully applied in efficient cell imaging and specific ferric ion detection. Electronic supplementary information (ESI) available: Fig. S1. TEM image of the products generated via an electrochemical method using pure BMIMBF4 aqueous solution as the electrolyte; Fig. S2. TEM and HRTEM (inset) images of the water-dispersed solution of C-dots generated from the EC process using electrolytes with a BMIMPF6/3-methylaminopropionitrile volume ratio of 1 : 9 Fig. S3. The effect of pH on the fluorescence intensity (I) of the C-dots; experimental details for detection of Fe3+ in tap water; Fig. S4. Calibration curve for detection of Fe3+ in tap water using the standard addition method. See DOI: 10.1039/c6nr00023a

The application of atomic force microscopy in mineral flotation.

PubMed

Xing, Yaowen; Xu, Mengdi; Gui, Xiahui; Cao, Yijun; Babel, Bent; Rudolph, Martin; Weber, Stefan; Kappl, Michael; Butt, Hans-Jürgen

2018-06-01

During the past years, atomic force microscopy (AFM) has matured to an indispensable tool to characterize nanomaterials in colloid and interface science. For imaging, a sharp probe mounted near to the end of a cantilever scans over the sample surface providing a high resolution three-dimensional topographic image. In addition, the AFM tip can be used as a force sensor to detect local properties like adhesion, stiffness, charge etc. After the invention of the colloidal probe technique it has also become a major method to measure surface forces. In this review, we highlight the advances in the application of AFM in the field of mineral flotation, such as mineral morphology imaging, water at mineral surface, reagent adsorption, inter-particle force, and bubble-particle interaction. In the coming years, the complementary characterization of chemical composition such as using infrared spectroscopy and Raman spectroscopy for AFM topography imaging and the synchronous measurement of the force and distance involving deformable bubble as a force sensor will further assist the fundamental understanding of flotation mechanism. Copyright © 2018 Elsevier B.V. All rights reserved.

Galleria mellonella apolipophorin III - an apolipoprotein with anti-Legionella pneumophila activity.

PubMed

Zdybicka-Barabas, Agnieszka; Palusińska-Szysz, Marta; Gruszecki, Wiesław I; Mak, Paweł; Cytryńska, Małgorzata

2014-10-01

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III. Copyright © 2014 Elsevier B.V. All rights reserved.

Positron Annihilation Ratio Spectroscopy Study of Electric Fields Applied to Positronium at Material Interfaces

DTIC Science & Technology

2011-03-01

from 142 ns to a few ns [3:3]. Through the application of positron annihilation lifetime spectroscopy (PALS) on a material, the o-Ps lifetime can be...Force Base, Ohio APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED. POSITRON ANNIHILATION RATIO SPECTROSCOPY STUDY OF ELECTRIC FIELDS APPLIED TO...protection in the United States. AFIT/GNE/ENP/11-M19 POSITRON ANNIHILATION RATIO SPECTROSCOPY STUDY OF ELECTRIC FIELDS APPLIED TO POSITRONIUM AT

Fourier Transform Infrared (FTIR) Spectroscopy, Ultraviolet Resonance Raman (UVRR) Spectroscopy, and Atomic Force Microscopy (AFM) for Study of the Kinetics of Formation and Structural Characterization of Tau Fibrils.

PubMed

Ramachandran, Gayathri

2017-01-01

Kinetic studies of tau fibril formation in vitro most commonly employ spectroscopic probes such as thioflavinT fluorescence and laser light scattering or negative stain transmission electron microscopy. Here, I describe the use of Fourier transform infrared (FTIR) spectroscopy, ultraviolet resonance Raman (UVRR) spectroscopy, and atomic force microscopy (AFM) as complementary probes for studies of tau aggregation. The sensitivity of vibrational spectroscopic techniques (FTIR and UVRR) to secondary structure content allows for measurement of conformational changes that occur when the intrinsically disordered protein tau transforms into cross-β-core containing fibrils. AFM imaging serves as a gentle probe of structures populated over the time course of tau fibrillization. Together, these assays help further elucidate the structural and mechanistic complexity inherent in tau fibril formation.

Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase

PubMed Central

Baumann, Fabian; Bauer, Magnus Sebastian; Rees, Martin; Alexandrovich, Alexander; Gautel, Mathias; Pippig, Diana Angela; Gaub, Hermann Eduard

2017-01-01

Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region. DOI: http://dx.doi.org/10.7554/eLife.26473.001 PMID:28696205

Electromagnetic torque tweezers: a versatile approach for measurement of single-molecule twist and torque.

PubMed

Janssen, Xander J A; Lipfert, Jan; Jager, Tessa; Daudey, Renier; Beekman, Jaap; Dekker, Nynke H

2012-07-11

The well-established single-molecule force-spectroscopy techniques have recently been complemented by methods that can measure torque and twist directly, notably magnetic torque tweezers and the optical torque wrench. A limitation of the current torque measurement schemes is the intrinsic coupling between the force and torque degrees of freedom. Here we present electromagnetic torque tweezers (eMTT) that combine permanent and electromagnets to enable independent control of the force and torsional trap stiffness for sensitive measurements of single molecule torque and twist. Using the eMTT, we demonstrate sensitive torque measurements on tethered DNA molecules from simple tracking of the beads' (x,y)-position, obviating the need for any angular tracking algorithms or markers. Employing the eMTT for high-resolution torque measurements, we experimentally confirm the theoretically predicted torque overshoot at the DNA buckling transition in high salt conditions. We envision that the flexibility and control afforded by the eMTT will enable a range of new torque and twist measurement schemes from single-molecules to living cells.

Structural, optical, electrochemical and photovoltaic studies of spider web like Silver Indium Diselenide Quantum dots synthesized by ligand mediated colloidal sol-gel approach

NASA Astrophysics Data System (ADS)

Adhikari, Tham; Pathak, Dinesh; Wagner, Tomas; Jambor, Roman; Jabeen, Uzma; Aamir, Muhammad; Nunzi, Jean-Michel

2017-11-01

Silver indium diselenide quantum dots were successively synthesized by colloidal sol-gel method by chelating with organic ligand oleylamine (OLA). The particle size was studied by transmission electron microscopy (TEM) and the size was found about 10 nm. X-ray diffraction (XRD) was used to study crystalline structure of the nanocrystals. The grain size and morphology were further studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The elemental composition was studied by X-ray photon electron spectroscopy (XPS) and energy dispersive x-ray spectroscopy (EDAX). The capping property of OLA in nanocrystal was also demonstrated by Fourier Transform Infrared spectroscopy (FTIR). The band gap was calculated from both cyclic voltammetry and optical absorption and suggest quantum confinement. The solution processed bilayer thin film solar cells were fabricated with n-type Zinc oxide using doctor blading/spin coating method and their photovoltaic performance was studied. The best device sintered at 450 °C showed an efficiency 0.75% with current density of 4.54 mAcm-2, open-circuit voltage 0.44 V and fill factor 39.4%.

Protein-Bound Polysaccharide from Corbicula fluminea Inhibits Cell Growth in MCF-7 and MDA-MB-231 Human Breast Cancer Cells.

PubMed

Liao, Ningbo; Zhong, Jianjun; Zhang, Ronghua; Ye, Xingqian; Zhang, Yanjun; Wang, Wenjun; Wang, Yuexia; Chen, Shiguo; Liu, Donghong; Liu, Ruihai

2016-01-01

A novel protein-bound polysaccharide, CFPS-1, isolated from Corbicula fluminea, is composed predominantly of mannose (Man) and glucose (Glc) in a molar ratio of 3.1:12.7. The polysaccharide, with an average molecular weight of about 283 kDa, also contains 10.8% protein. Atomic force microscopy, high-performance liquid chromatography, Fourier transform infrared spectroscopy, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy analyses revealed that CFPS-1 has a backbone of 1,6-linked and 1,4,6-linked-α-D-Glc, which is terminated with a 1-linked-α-D-Man residue at the O-4 position of 1,4,6-linked-α-D-Glc, in a molar ratio of 3:1:1. Preliminary in vitro bioactivity tests revealed that CFPS-1 effectively and dose-dependently inhibits human breast cancer MCF-7 and MDA-MB-231 cell growth, with an IC50 of 243 ± 6.79 and 1142 ± 14.84 μg/mL, respectively. In MCF-7, CFPS-1 produced a significant up-regulation of p53, p21, Bax and cleaved caspase-7 and down-regulation of Cdk4, cyclin D1, Bcl-2 and caspase-7. These effects resulted in cell cycle blockade at the S-phase and apoptosis induction. In contrast, in MDA-MB-231, with limited degree of change in cell cycle distribution, CFPS-1 increases the proportion of cells in apoptotic sub-G1 phase executed by down-regulation of Bcl-2 and caspase-7 and up-regulation of Bax and cleaved caspase-7. This study extends our understanding of the anticancer mechanism of C. fluminea protein-bound polysaccharide.

Mapping mechanical force propagation through biomolecular complexes

DOE PAGES

Schoeler, Constantin; Bernardi, Rafael C.; Malinowska, Klara H.; ...

2015-08-11

In this paper, we employ single-molecule force spectroscopy with an atomic force microscope (AFM) and steered molecular dynamics (SMD) simulations to reveal force propagation pathways through a mechanically ultrastable multidomain cellulosome protein complex. We demonstrate a new combination of network-based correlation analysis supported by AFM directional pulling experiments, which allowed us to visualize stiff paths through the protein complex along which force is transmitted. Finally, the results implicate specific force-propagation routes nonparallel to the pulling axis that are advantageous for achieving high dissociation forces.

Photoinduced force microscopy: A technique for hyperspectral nanochemical mapping

NASA Astrophysics Data System (ADS)

Murdick, Ryan A.; Morrison, William; Nowak, Derek; Albrecht, Thomas R.; Jahng, Junghoon; Park, Sung

2017-08-01

Advances in nanotechnology have intensified the need for tools that can characterize newly synthesized nanomaterials. A variety of techniques has recently been shown which combines atomic force microscopy (AFM) with optical illumination including tip-enhanced Raman spectroscopy (TERS), scattering-type scanning near-field optical microscopy (sSNOM), and photothermal induced resonance microscopy (PTIR). To varying degrees, these existing techniques enable optical spectroscopy with the nanoscale spatial resolution inherent to AFM, thereby providing nanochemical interrogation of a specimen. Here we discuss photoinduced force microscopy (PiFM), a recently developed technique for nanoscale optical spectroscopy that exploits image forces acting between an AFM tip and sample to detect wavelength-dependent polarization within the sample to generate absorption spectra. This approach enables ∼10 nm spatial resolution with spectra that show correlation with macroscopic optical absorption spectra. Unlike other techniques, PiFM achieves this high resolution with virtually no constraints on sample or substrate properties. The applicability of PiFM to a variety of archetypal systems is reported here, highlighting the potential of PiFM as a useful tool for a wide variety of industrial and academic investigations, including semiconducting nanoparticles, nanocellulose, block copolymers, and low dimensional systems, as well as chemical and morphological mixing at interfaces.

Lateral dimension-dependent antibacterial activity of graphene oxide sheets.

PubMed

Liu, Shaobin; Hu, Ming; Zeng, Tingying Helen; Wu, Ran; Jiang, Rongrong; Wei, Jun; Wang, Liang; Kong, Jing; Chen, Yuan

2012-08-21

Graphene oxide (GO) is a promising precursor to produce graphene-family nanomaterials for various applications. Their potential health and environmental impacts need a good understanding of their cellular interactions. Many factors may influence their biological interactions with cells, and the lateral dimension of GO sheets is one of the most relevant material properties. In this study, a model bacterium, Escherichia coli ( E. coli ), was used to evaluate the antibacterial activity of well-dispersed GO sheets, whose lateral size differs by more than 100 times. Our results show that the antibacterial activity of GO sheets toward E. coli cells is lateral size dependent. Larger GO sheets show stronger antibacterial activity than do smaller ones, and they have different time- and concentration-dependent antibacterial activities. Large GO sheets lead to most cell loss after 1 h incubation, and their concentration strongly influences antibacterial activity at relative low concentration (<10 μg/mL). In contrast, when incubating with small GO sheets up to 4 h, the inactivation rate of E. coli cells continues increasing. The increase of small GO sheet concentration also results in persistent increases in their antibacterial activity. In this study, GO sheets with different lateral sizes are all well dispersed, and their oxidation capacity toward glutathione is similar, consistent with X-ray photoelectron spectroscopy and ultraviolet-visible absorption spectroscopy results. This suggests the lateral size-dependent antibacterial activity of GO sheets is caused by neither their aggregation states, nor oxidation capacity. Atomic force microscope analysis of GO sheets and cells shows that GO sheets interact strongly with cells. Large GO sheets more easily cover cells, and cells cannot proliferate once fully covered, resulting in the cell viability loss observed in the followed colony counting test. In contrast, small GO sheets adhere to the bacterial surfaces, which cannot effectively isolate cells from environment. This study highlights the importance of tailoring the lateral dimension of GO sheets to optimize the application potential with minimal risks for environmental health and safety.

As-prepared MoS2 quantum dot as a facile fluorescent probe for long-term tracing of live cells

NASA Astrophysics Data System (ADS)

Zhou, Kai; Zhang, Yue; Xia, Zhining; Wei, Weili

2016-07-01

Recently, the newly emerged two-dimensional nanomaterials, layered transition metal dichalcogenide (e.g. MoS2) nanosheets, have drawn tremendous attentions due to their extraordinary electronic and optical properties, and MoS2 quantum dots (MoS2 QDs) with lateral sizes less than 10 nm have been found to be highly luminescent. In the present study, a facile approach for large-scale preparation of MoS2 QDs by Na intercalation reaction without using any toxic organic reagents is proposed. MoS2 QDs were carefully characterized by various techniques including transmission electron microscopy, atomic force microscopy, dynamic light scattering, spectroscopy, in vitro cytotoxicology, and capillary electrophoresis. The as-prepared MoS2 QDs were strongly fluorescent, highly photo-stable, low in cytotoxicity, and readily reactive to thiols. These inherent properties of MoS2 QDs make them excellent fluorescent probes for long-term live cell tracing. The results of live cells imaging indicated that MoS2 QD stained cells remained highly fluorescent after long-term culture, and could be easily traced from other co-cultured cell lines.

Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling

NASA Astrophysics Data System (ADS)

Li, Qian; Huth, Steven; Adam, Dieter; Selhuber-Unkel, Christine

2016-07-01

Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery.

Using Brillouin microspectroscopy to characterize adipocytes' response to lipid droplet accumulation

NASA Astrophysics Data System (ADS)

Troyanova-Wood, Maria; Coker, Zachary; Traverso, Andrew; Yakovlev, Vladislav V.

2017-02-01

Obesity and overweight are accompanied by an enlargement of adipocytes, which is commonly related to the increasing number or size of lipid droplets within the cells. Some studies have shown that the accumulation of lipid droplets within adipocytes results in their increased stiffness. Recently, Brillouin microspectroscopy has been introduced as a nondestructive method of imaging the elasticity of cells. Unlike other imaging modalities, it is capable of assessing the elastic properties on both tissue- and cell levels. In this study, Brillouin spectroscopy was used to measure the elasticity changes in response to accumulation of lipid droplets within adipocyte during adipogenesis. The cell line used in the study is 3T3-L1, with chemically-induced differentiation from pre-adipocytes to mature adipocytes. The Brillouin shift measurements of the cells before and after differentiation indicate that the stiffness of adipocytes increases due to accumulation of lipid droplets. The results are in agreement with previous atomic force microscopy (AFM) nanoindentation studies. Brillouin microspectroscopy is a technique suitable for measuring the changes of elasticity of adipocytes in response to lipid droplet accumulation.

Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface*

PubMed Central

Heim, Kyle P.; Sullan, Ruby May A.; Crowley, Paula J.; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F.; Brady, L. Jeannine

2015-01-01

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. PMID:25666624

Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface.

PubMed

Heim, Kyle P; Sullan, Ruby May A; Crowley, Paula J; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F; Brady, L Jeannine

2015-04-03

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

HDL particles incorporate into lipid bilayers - a combined AFM and single molecule fluorescence microscopy study.

PubMed

Plochberger, Birgit; Röhrl, Clemens; Preiner, Johannes; Rankl, Christian; Brameshuber, Mario; Madl, Josef; Bittman, Robert; Ros, Robert; Sezgin, Erdinc; Eggeling, Christian; Hinterdorfer, Peter; Stangl, Herbert; Schütz, Gerhard J

2017-11-21

The process, how lipids are removed from the circulation and transferred from high density lipoprotein (HDL) - a main carrier of cholesterol in the blood stream - to cells, is highly complex. HDL particles are captured from the blood stream by the scavenger receptor, class B, type I (SR-BI), the so-called HDL receptor. The details in subsequent lipid-transfer process, however, have not yet been completely understood. The transfer has been proposed to occur directly at the cell surface across an unstirred water layer, via a hydrophobic channel in the receptor, or after HDL endocytosis. The role of the target lipid membrane for the transfer process, however, has largely been overlooked. Here, we studied at the single molecule level how HDL particles interact with synthetic lipid membranes. Using (high-speed) atomic force microscopy and fluorescence correlation spectroscopy (FCS) we found out that, upon contact with the membrane, HDL becomes integrated into the lipid bilayer. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of fluorescently labelled amphiphilic lipid probe from HDL particles to the lipid bilayer upon contact.

Combining Portable Raman Probes with Nanotubes for Theranostic Applications

PubMed Central

Bhirde, Ashwinkumar A.; Liu, Gang; Jin, Albert; Iglesias-Bartolome, Ramiro; Sousa, Alioscka A.; Leapman, Richard D.; Gutkind, J. Silvio; Lee, Seulki; Chen, Xiaoyuan

2011-01-01

Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT) characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) were dispersed aqueously using a lipid-polymer (LP) coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC)-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells using a simple handheld Raman instrument. This approach could potentially be used to treat various diseases, including cancer. PMID:21769298

Combining portable Raman probes with nanotubes for theranostic applications.

PubMed

Bhirde, Ashwinkumar A; Liu, Gang; Jin, Albert; Iglesias-Bartolome, Ramiro; Sousa, Alioscka A; Leapman, Richard D; Gutkind, J Silvio; Lee, Seulki; Chen, Xiaoyuan

2011-01-01

Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT) characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) were dispersed aqueously using a lipid-polymer (LP) coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC)-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells using a simple handheld Raman instrument. This approach could potentially be used to treat various diseases, including cancer.

Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells

PubMed Central

Zhang, Guo-Liang; Huang, Yi-Fan; Gurunathan, Sangiliyandi

2017-01-01

Background Zinc oxide nanoparticles (ZnO NPs) are frequently used in industrial products such as paint, surface coating, and cosmetics, and recently, they have been explored in biologic and biomedical applications. Therefore, this study was undertaken to investigate the effect of ZnO NPs on cytotoxicity, apoptosis, and autophagy in human ovarian cancer cells (SKOV3). Methods ZnO NPs with a crystalline size of 20 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays. Results Exposure of cells to ZnO NPs resulted in a dose-dependent loss of cell viability, and the characteristic apoptotic features such as rounding and loss of adherence, enhanced reactive oxygen species generation, and loss of mitochondrial membrane potential were observed in the ZnO NP-treated cells. Furthermore, the cells treated with ZnO NPs showed significant double-strand DNA breaks, which are gained evidences from significant number of γ-H2AX and Rad51 expressed cells. ZnO NP-treated cells showed upregulation of p53 and LC3, indicating that ZnO NPs are able to upregulate apoptosis and autophagy. Finally, the Western blot analysis revealed upregulation of Bax, caspase-9, Rad51, γ-H2AX, p53, and LC3 and downregulation of Bcl-2. Conclusion The study findings demonstrated that the ZnO NPs are able to induce significant cytotoxicity, apoptosis, and autophagy in human ovarian cells through reactive oxygen species generation and oxidative stress. Therefore, this study suggests that ZnO NPs are suitable and inherent anticancer agents due to their several favorable characteristic features including favorable band gap, electrostatic charge, surface chemistry, and potentiation of redox cycling cascades. PMID:28919752

Understanding the role of clay minerals in the chromium(VI) bioremoval by Pseudomonas aeruginosa CCTCC AB93066 under growth condition: microscopic, spectroscopic and kinetic analysis.

PubMed

Kang, Chunxi; Wu, Pingxiao; Li, Yuewu; Ruan, Bo; Li, Liping; Tran, Lytuong; Zhu, Nengwu; Dang, Zhi

2015-11-01

Laboratory batch experiments were conducted to investigate the role of clay minerals, e.g., kaolinite and vermiculite, in microbial Cr(VI) reduction by Pseudomonas aeruginosa under growth condition in glucose-amended mediums as a method for treating Cr(VI)-contaminated subsurface environment such as soil. Our results indicated that glucose could acted as an essential electron donor, and clay minerals significantly enhanced microbial Cr(VI) reduction rates by improving the consumption rate of glucose and stimulating the growth and propagation of P. aeruginosa. Cr(VI) bioreduction by both free cells and clay minerals-amended cells followed the pseudo-first-order kinetic model, with the latter one fitting better. The mass balance analyses and X-ray photoelectron spectroscopy analysis found that Cr(VI) was reduced to Cr(III) and the adsorption of total chromium on clay minerals-bacteria complex was small, implying that Cr(VI) bioremoval was not mainly due to the adsorption of Cr(VI) onto cells or clay minerals or clay minerals-cells complex but mainly due to the Cr(VI) reduction capacity of P. aeruginosa under the experimental conditions studied (e.g., pH 7). Atomic force microscopy revealed that the addition of clay minerals (e.g. vermiculite) decreased the surface roughness of Cr(VI)-laden cells and changed the cell morphology and dimension. Fourier transform infrared spectroscopy revealed that organic matters such as aliphatic species and/or proteins played an important role in the combination of cells and clay minerals. Scanning electron microscopy confirmed the attachment of cells on the surface of clay minerals, indicating that clay minerals could provide a microenvironment to protect cells from Cr(VI) toxicity and serve as growth-supporting materials. These findings manifested the underlying influence of clay minerals on microbial reduction of Cr(VI) and gave an understanding of the interaction between pollutants, the environment and the biota.

Nanostructuring on zinc phthalocyanine thin films for single-junction organic solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaudhary, Dhirendra K.; Kumar, Lokendra, E-mail: lokendrakr@allduniv.ac.in

2016-05-23

Vertically aligned and random oriented crystalline molecular nanorods of organic semiconducting Zinc Phthalocyanine (ZnPc) have been grown on ITO coated glass substrate using solvent volatilization method. Interesting changes in surface morphology were observed under different solvent treatment. Vertically aligned nanorods of ZnPc thin film were observed in the films treated with acetone, where as the random oriented nanorods were observed in the films treated with chloroform. The X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM) have been used for characterization of nanostructures. The optical properties of the nanorods have been investigated by UV-Vis. absorption spectroscopy.

Raman spectroscopy and atomic force microscopy study of interfacial polytypism in GaP/Ge(111) heterostructures

NASA Astrophysics Data System (ADS)

Aggarwal, R.; Ingale, Alka A.; Dixit, V. K.

2018-01-01

Effects of lattice and polar/nonpolar mismatch between the GaP layer and Ge(111) substrate are investigated by spatially resolved Raman spectroscopy. The red shifted transverse optical (TO) and longitudinal optical (LO) phonons due to residual strain, along with asymmetry to TO phonon ∼358 cm-1 are observed in GaP/Ge(111). The peak intensity variation of mode ∼358 cm-1 with respect to TO phonon across the crystallographic morphed surface of GaP micro structures is associated with the topographical variations using atomic force microscopy mapping and Raman spectroscopy performed on both in plane and cross-sectional surface. Co-existence of GaP allotropes, i.e. wurtzite phase near heterojunction interface and dominant zinc-blende phase near surface is established using the spatially resolved polarized Raman spectroscopy from the cross sectional surface of heterostructures. This consistently explains effect of surface morphology on Raman spectroscopy from GaP(111). The study shows the way to identify crystalline phases in other advanced semiconductor heterostructures without any specific sample preparation.

Hydrophobic Hydration of Stimulus-Responsive Polyproteins Measured by Single Molecule Force Spectroscopy

NASA Astrophysics Data System (ADS)

Zauscher, Stefan

2007-03-01

We present a new procedure to reduce and analyze force-extension data obtained by single molecule force spectroscopy (SMFS). This approach allows, for the first time, to infer effects of solvent quality and minor changes in molecular architecture on molecular-elasticity of individual (bio)macromolecules. Specifically, we show how changes in the effective Kuhn segment length can be used to interpret the hydrophobic hydration behavior of elastin-like polypeptides (ELPs).Our results are intriguing as they suggest that SMFS in combination with our analysis procedure can be used to study the subtleties of polypeptide-water interactions on the single molecule level. We also report on the force-induced cis-trans isomerization of prolines, which are repeated every fifth residue in the main chain of ELPs. We present evidence for this mechanism by Monte Carlo simulations of the force-extension curves using an elastically coupled two-state system. Our results suggest that SMFS could be used to assay proline cis-trans isomerization in proteins and may thus have significant potential diagnostic utility.

Mechanical Properties of Single Collagen Fibrils Revealed by Force Spectroscopy

NASA Astrophysics Data System (ADS)

Graham, John; Phillips, Charlotte; Grandbois, Michel

2004-03-01

In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin and tendon. Here we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy-based force spectroscopy. The elongation profiles indicate that in vitro assembled heterotrimeric type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurs within the fibrils in the 1.5 -- 4.5 nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in cellular motion in the context of tissue growth and morphogenesis. In contrast, homotrimeric collagen fibrils corresponding to osteogenesis imperfecta pathology exhibit a marked difference in their elasticity profile.

Probing Long-Range Neutrino-Mediated Forces with Atomic and Nuclear Spectroscopy.

PubMed

Stadnik, Yevgeny V

2018-06-01

The exchange of a pair of low-mass neutrinos between electrons, protons, and neutrons produces a "long-range" 1/r^{5} potential, which can be sought for in phenomena originating on the atomic and subatomic length scales. We calculate the effects of neutrino-pair exchange on transition and binding energies in atoms and nuclei. In the case of atomic s-wave states, there is a large enhancement of the induced energy shifts due to the lack of a centrifugal barrier and the highly singular nature of the neutrino-mediated potential. We derive limits on neutrino-mediated forces from measurements of the deuteron binding energy and transition energies in positronium, muonium, hydrogen, and deuterium, as well as isotope-shift measurements in calcium ions. Our limits improve on existing constraints on neutrino-mediated forces from experiments that search for new macroscopic forces by 18 orders of magnitude. Future spectroscopy experiments have the potential to probe long-range forces mediated by the exchange of pairs of standard-model neutrinos and other weakly charged particles.

Probing Long-Range Neutrino-Mediated Forces with Atomic and Nuclear Spectroscopy

NASA Astrophysics Data System (ADS)

Stadnik, Yevgeny V.

2018-06-01

The exchange of a pair of low-mass neutrinos between electrons, protons, and neutrons produces a "long-range" 1 /r5 potential, which can be sought for in phenomena originating on the atomic and subatomic length scales. We calculate the effects of neutrino-pair exchange on transition and binding energies in atoms and nuclei. In the case of atomic s -wave states, there is a large enhancement of the induced energy shifts due to the lack of a centrifugal barrier and the highly singular nature of the neutrino-mediated potential. We derive limits on neutrino-mediated forces from measurements of the deuteron binding energy and transition energies in positronium, muonium, hydrogen, and deuterium, as well as isotope-shift measurements in calcium ions. Our limits improve on existing constraints on neutrino-mediated forces from experiments that search for new macroscopic forces by 18 orders of magnitude. Future spectroscopy experiments have the potential to probe long-range forces mediated by the exchange of pairs of standard-model neutrinos and other weakly charged particles.

Force spectroscopy of multivalent binding of riboflavin-conjugated dendrimers to riboflavin binding protein.

PubMed

Leistra, Abigail N; Han, Jong Hyun; Tang, Shengzhuang; Orr, Bradford G; Banaszak Holl, Mark M; Choi, Seok Ki; Sinniah, Kumar

2015-05-07

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates.

Lactoferrin-Immobilized Surfaces onto Functionalized PLA Assisted by the Gamma-Rays and Nitrogen Plasma to Create Materials with Multifunctional Properties.

PubMed

Stoleru, Elena; Zaharescu, Traian; Hitruc, Elena Gabriela; Vesel, Alenka; Ioanid, Emil G; Coroaba, Adina; Safrany, Agnes; Pricope, Gina; Lungu, Maria; Schick, Christoph; Vasile, Cornelia

2016-11-23

Both cold nitrogen radiofrequency plasma and gamma irradiation have been applied to activate and functionalize the polylactic acid (PLA) surface and the subsequent lactoferrin immobilization. Modified films were comparatively characterized with respect to the procedure of activation and also with unmodified sample by water contact angle measurements, mass loss, X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM), and chemiluminescence measurements. All modified samples exhibit enhanced surface properties mainly those concerning biocompatibility, antimicrobial, and antioxidant properties, and furthermore, they are biodegradable and environmentally friendly. Lactoferrin deposited layer by covalent coupling using carbodiimide chemistry showed a good stability. It was found that the lactoferrin-modified PLA materials present significantly increased oxidative stability. Gamma-irradiated samples and lactoferrin-functionalized samples show higher antioxidant, antimicrobial, and cell proliferation activity than plasma-activated and lactoferrin-functionalized ones. The multifunctional materials thus obtained could find application as biomaterials or as bioactive packaging films.

Research on the differences between 2-(2-Chlorophenyl)benzimidazole and 2-(4-Chlorophenyl)benzimidazole based on terahertz time domain spectroscopy

NASA Astrophysics Data System (ADS)

Song, Maojiang; Yang, Fei; Liu, Liping; Shen, Li; Hu, Pengfei; Zhang, Li; Su, Caixia

2018-05-01

Due to wide variety of biological and pharmacological activities of benzimidazole derivatives, the differences between 2-(2-Chlorophenyl)benzimidazole and 2-(4-Chlorophenyl) benzimidazole were researched by employing terahertz time-domain spectroscopy and density functional theory systematically. Although the only difference between their molecular configurations is the arrangement of chlorine atom on chlorophenyl ring, there are distinctive differences in their fingerprint spectra in the range of 0.2-2.5 THz, such as amount, amplitude, and frequency position of absorption peaks. The validity of these results was confirmed by the theoretical results simulated by using density functional theory. The possible reasons of these differences originate from the different van der Waals forces and the different dihedral angles of the molecules within crystal cell. These results indicate the importance of this spectral range as a conformational fingerprint region where even minor changes in the molecular configuration lead to major differences in its THz absorption.

CdSe magic-sized quantum dots incorporated in biomembrane models at the air-water interface composed of components of tumorigenic and non-tumorigenic cells.

PubMed

Goto, Thiago E; Lopes, Carla C; Nader, Helena B; Silva, Anielle C A; Dantas, Noelio O; Siqueira, José R; Caseli, Luciano

2016-07-01

Cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) are semiconductor nanocrystals with stable luminescence that are feasible for biomedical applications, especially for in vivo and in vitro imaging of tumor cells. In this work, we investigated the specific interaction of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and Langmuir-Blodgett (LB) films of lipids as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers constituted either of selected lipids or of tumorigenic and non-tumorigenic cell extracts. The films were transferred to solid supports to obtain microscopic images, providing information on their morphology. Similarity between films with different compositions representing cell membranes, with or without the quantum dots, was evaluated by atomic force microscopy (AFM) and confocal microscopy. This study demonstrates that the affinity of quantum dots for models representing cancer cells permits the use of these systems as devices for cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Quadrature transmit coil for breast imaging at 7 tesla using forced current excitation for improved homogeneity.

PubMed

McDougall, Mary Preston; Cheshkov, Sergey; Rispoli, Joseph; Malloy, Craig; Dimitrov, Ivan; Wright, Steven M

2014-11-01

To demonstrate the use of forced current excitation (FCE) to create homogeneous excitation of the breast at 7 tesla, insensitive to the effects of asymmetries in the electrical environment. FCE was implemented on two breast coils: one for quadrature (1) H imaging and one for proton-decoupled (13) C spectroscopy. Both were a Helmholtz-saddle combination, with the saddle tuned to 298 MHz for imaging and 75 MHz for spectroscopy. Bench measurements were acquired to demonstrate the ability to force equal currents on elements in the presence of asymmetric loading to improve homogeneity. Modeling and temperature measurements were conducted per safety protocol. B1 mapping, imaging, and proton-decoupled (13) C spectroscopy were demonstrated in vivo. Using FCE to ensure balanced currents on elements enabled straightforward tuning and maintaining of isolation between quadrature elements of the coil. Modeling and bench measurements confirmed homogeneity of the field, which resulted in images with excellent fat suppression and in broadband proton-decoupled carbon-13 spectra. FCE is a straightforward approach to ensure equal currents on multiple coil elements and a homogeneous excitation field, insensitive to the effects of asymmetries in the electrical environment. This enabled effective breast imaging and proton-decoupled carbon-13 spectroscopy at 7T. © 2014 Wiley Periodicals, Inc.

Synthesis And Single Molecule Force Spectroscopy Of Poly(hydroxyethyl methacrylate-g-ethylene glycol)

NASA Astrophysics Data System (ADS)

Zhang, Dong; Ortiz, Christine

2003-03-01

With the advent of nanotechnology, miniaturized devices will soon need nanoscale springs with well-controlled nanomechanical properties such as shock absorbers, or to control the adhesive interactions between two components. In order to understand, manipulate, and control single macromolecule nanomechanical properties, mono(thiol)-terminated poly(hydroxyethyl methacrylate-g-ethylene glycol) has been synthesized via atom transfer radical polymerization. End-functionalization, chemical structure, molecular weight, side-chain graft density, radius of gyration, and polydispersity were characterized by 1H nuclear magnetic resonance, static light scattering, and gel permeation chromatography. The polymer chains were attached to Au-coated Si wafers via chemisorption to prepare well-separated "mushrooms", as verified by atomic force microscopy. Single molecule force spectroscopy was then used to measure the extensional elastic properties, i.e. force (nN) versus end-to-end separation distance (nm), of the individual chains by tethering to a Si3N4 probe tip via nonspecific, physisorption interactions.

WEATHERING DEGRADATION OF A POLYURETHANE COATING. (R828081E01)

EPA Science Inventory

The degradation of polyurethane topcoat over a chromate pigmented epoxy primer was examined by atomic force microscopy (AFM), scanning electronic microscopy (SEM), X-ray photo-electron spectroscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR) after the coated pane...

Mid-infrared spectroscopy in skin cancer cell type identification

NASA Astrophysics Data System (ADS)

Kastl, Lena; Kemper, Björn; Lloyd, Gavin R.; Nallala, Jayakrupakar; Stone, Nick; Naranjo, Valery; Penaranda, Francisco; Schnekenburger, Jürgen

2017-07-01

Mid infrared spectroscopy samples were developed for the analysis of skin tumor cell types and three dimensional tissue phantoms towards the application of midIR spectroscopy for fast and reliable skin cancer diagnostics.

Influence of Cu-Ti thin film surface properties on antimicrobial activity and viability of living cells.

PubMed

Wojcieszak, Damian; Kaczmarek, Danuta; Antosiak, Aleksandra; Mazur, Michal; Rybak, Zbigniew; Rusak, Agnieszka; Osekowska, Malgorzata; Poniedzialek, Agata; Gamian, Andrzej; Szponar, Bogumila

2015-11-01

The paper describes properties of thin-film coatings based on copper and titanium. Thin films were prepared by co-sputtering of Cu and Ti targets in argon plasma. Deposited coatings consist of 90at.% of Cu and 10at.% of Ti. Characterization of the film was made on the basis of investigations of microstructure and physicochemical properties of the surface. Methods such as scanning electron microscopy, x-ray microanalysis, x-ray diffraction, x-ray photoelectron spectroscopy, atomic force microscopy, optical profilometry and wettability measurements were used to assess the properties of deposited thin films. An impact of Cu-Ti coating on the growth of selected bacteria and viability of the living cells (line L929, NCTC clone 929) was described in relation to the structure, surface state and wettability of the film. It was found that as-deposited films were amorphous. However, in such surroundings the nanocrystalline grains of 10-15nm and 25-35nm size were present. High surface active area with a roughness of 8.9nm, had an effect on receiving relatively high water contact angle value (74.1°). Such wettability may promote cell adhesion and result in an increase of the probability of copper ion transfer from the film surface into the cell. Thin films revealed bactericidal and fungicidal effects even in short term-contact. High activity of prepared films was directly related to high amount (ca. 51 %) of copper ions at 1+ state as x-ray photoelectron spectroscopy results have shown. Copyright © 2015 Elsevier B.V. All rights reserved.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies.

PubMed

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia ( pml ) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

Sandwiching spherical 1,2-dioleoyltrimethylammoniumpropane liposome in gold nanoparticle on solid transducer for electrochemical ultrasensitive DNA detection and transfection.

PubMed

Shankara Narayanan, Jeyaraman; Bhuvana, Mohanlal; Dharuman, Venkataraman

2014-08-15

Cationic N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium propane (DOTAP) liposome is spherically sandwiched in gold nanoparticle (abbreviated as sDOTAP-AuNP) onto a gold electrode surface. The sDOTAP-AuNP is applied for electrochemical label free DNA sensing and Escherichia coli cell transfection for the first time. Complementary target (named as hybridized), non-complementary target (un-hybridized) and single base mismatch target (named as SMM) hybridized surfaces are discriminated sensitively and selectively in presence of [Fe(CN)6](3-/4-). Double strand specific intercalator methylene blue in combination with [Fe(CN)6](3-) is used to enhance target detection limit down to femtomolar concentration. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) techniques are used for characterizing DNA sensing. High Resolution Transmission Electron Microscopy (HRTEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) techniques are used to confirm the spherical nature of the sDOTAP-AuNP-DNA composite in solution and on the solid surface. DNA on the sDOTAP-ssDNA is transferred by potential stripping method (+0.2V (Ag/AgCl)) into buffer solution containing E. coli cells. The transfection is confirmed by the contrast images for the transfected and non-transfected cell from Confocal Laser Scanning Microscopy (CLSM). The results demonstrate effectiveness of the electrochemical DNA transfection method developed and could be applied for other cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Bovine α-lactalbumin functionalized graphene oxide nano-sheet exhibits enhanced biocompatibility: A rational strategy for graphene-based targeted cancer therapy.

PubMed

Mahanta, Sailendra; Paul, Subhankar

2015-10-01

Graphene oxide nanosheet (GOns) with sharp edges was synthesized using controlled pyrolysis of citric acid. Scanning electron, as well as atomic force microscopy of the sample confirmed the formation of multilayered GOns with an average sheet length of 150 nm. X-ray diffraction pattern and Raman spectra also confirmed the formation of GOns. Furthermore, GOns was successfully functionalized (FGOns) by cross-linking with a small protein bovine α-lactalbumin (BLA). The crosslinking of protein with GOns in FGOns was confirmed by infrared spectroscopy, and the conformational change of BLA was observed by fluorescence, as well as circular dichroism spectroscopy. When applied to human erythrocytes, GOns demonstrated profound hemolysis; however, such hemolytic effect was drastically reduced by FGOns. To evaluate the potential biomedical application of FGOns, the cytotoxicity of the sample was also assessed. The administration of both GOns and FGOns in breast cancer cells MCF-7 and MDAMB-231 demonstrated more than 88% cell death within 24 h and such cytotoxicity against cancer cells was caused due to the generation of reactive oxygen species (ROS), as revealed from the N-acetyl-L-cysteine (NAC, a ROS-inhibitor)-based assay. FGOns demonstrated excellent biocompatibility against normal cells such as HaCaT and 3T3 compared to GOns that demonstrated dose-dependent toxicity. Moreover, FGOns demonstrated more efficient cellular uptake than GOns by cancer cells. Therefore, our present study demonstrated that the functionalization of GOns using small protein could improve its biocompatibility multifold and such strategy might represent wide opportunity to use GO like nanomaterial safely in various biomedical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Improving Osteoblast Response In Vitro by a Nanostructured Thin Film with Titanium Carbide and Titanium Oxides Clustered around Graphitic Carbon.

PubMed

Longo, Giovanni; Ioannidu, Caterina Alexandra; Scotto d'Abusco, Anna; Superti, Fabiana; Misiano, Carlo; Zanoni, Robertino; Politi, Laura; Mazzola, Luca; Iosi, Francesca; Mura, Francesco; Scandurra, Roberto

2016-01-01

Recently, we introduced a new deposition method, based on Ion Plating Plasma Assisted technology, to coat titanium implants with a thin but hard nanostructured layer composed of titanium carbide and titanium oxides, clustered around graphitic carbon. The nanostructured layer has a double effect: protects the bulk titanium against the harsh conditions of biological tissues and in the same time has a stimulating action on osteoblasts. The aim of this work is to describe the biological effects of this layer on osteoblasts cultured in vitro. We demonstrate that the nanostructured layer causes an overexpression of many early genes correlated to proteins involved in bone turnover and an increase in the number of surface receptors for α3β1 integrin, talin, paxillin. Analyses at single-cell level, by scanning electron microscopy, atomic force microscopy, and single cell force spectroscopy, show how the proliferation, adhesion and spreading of cells cultured on coated titanium samples are higher than on uncoated titanium ones. Finally, the chemistry of the layer induces a better formation of blood clots and a higher number of adhered platelets, compared to the uncoated cases, and these are useful features to improve the speed of implant osseointegration. In summary, the nanostructured TiC film, due to its physical and chemical properties, can be used to protect the implants and to improve their acceptance by the bone.

Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy.

PubMed

Rangl, Martina; Leitner, Michael; Riihimäki, Tiina; Lehtonen, Soili; Hytönen, Vesa P; Gruber, Hermann J; Kulomaa, Markku; Hinterdorfer, Peter; Ebner, Andreas

2014-02-01

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (k(off)) and energy landscape distances (x(β)) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the x(β) values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd.

Physical and mechanical characterization of PLLA interference screws produced by two stage injection molding method.

PubMed

Sadeghi-Avalshahr, Ali Reza; Khorsand-Ghayeni, Mohammad; Nokhasteh, Samira; Molavi, Amir Mahdi; Sadeghi-Avalshahr, Mohammad

2016-12-01

The purpose of this study was to produce and evaluate different mechanical, physical and in vitro cell culture characteristics of poly(L-lactic) acid (PLLA) interference screws. This work will focus on evaluating the effect of two important parameters on operation of these screws, first the tunnel diameter which is one of the most important parameters during the operation and second the thermal behavior, the main effective characteristic in production process. In this work, PLLA screws were produced by a two-stage injection molding machine. For mechanical assessment of the produced screws, Polyurethane rigid foam was used as cancellous bone and polypropylene rope as synthetic graft to simulate bone and ligament in real situation. Different tunnel diameters including 7-10 mm were evaluated for fixation strength. When the tunnel diameter was changed from 10 to 9 mm, the pull-out force has increased to about 12 %, which is probably due to the aforementioned frictional forces, however, by reducing the tunnel diameter to 8 and 7 mm, the pull-out force reduced to 16 and 50 % for 8 and 7 mm tunnel diameter, respectively. The minimum and maximum pull-out force was obtained 160.57 and 506.86 N for 7 and 9 mm tunnel diameters, respectively. For physicochemical assay, Fourier transform infrared spectroscopy (FTIR), degradation test and differential scanning calorimetry (DSC) were carried out. The crystallinity (Xc) of samples were decreased considerably from 64.3 % before injection to 32.95 % after injection with two different crystallographic forms α' and α. probably due to the fast cooling rate at room temperature. In addition, MTT and cell attachment assays were utilized by MG63 osteoblast cell line, to evaluate the cytotoxicity of the produced screws. The results revealed no cytotoxicity effect.

Structure and function of Enterotoxigenic Escherichia coli fimbriae from differing assembly pathways

PubMed Central

Mortezaei, Narges; Epler, Chelsea R.; Shao, Paul P.; Shirdel, Mariam; Singh, Bhupender; McVeigh, Annette; Uhlin, Bernt Eric; Savarino, Stephen J.; Andersson, Magnus; Bullitt, Esther

2014-01-01

Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram-negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone-usher pathway and others by the alternate chaperone pathway. Here we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared to CFA/I fimbriae, which are two ETEC fimbriae assembled via different pathways, and to P-fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P-fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling, and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology. PMID:25355550

Towards ultrasound enhanced mid-IR spectroscopy for sensing bacteria in aqueous solutions

NASA Astrophysics Data System (ADS)

Freitag, Stephan; Schwaighofer, Andreas; Radel, Stefan; Lendl, Bernhard

2018-02-01

We employ attenuated total reflection (ATR) mid-IR technology for sensing of bacteria present in aqueous solution. In ATR spectroscopy, the penetration depth of the evanescent field extends to approx. 1-2 micrometers into the aqueous solution depending on the refractive index of the employed materials (Si, ZnS, Ge) used as attenuated total reflection (ATR) element and the geometry of the optical set-up. Due to the flow profile in the microfluidic cell, an additional force is required to bring particles into the evanescent field for measurement. For that purpose, we employ standing ultrasound waves produced between a sound source vibrating at approx. 2 MHz and the ATR crystal acting as a reflector. This ultrasonic trap is integrated into the microfluidic channel. As aqueous solution is passing through that acoustofluidic cell, particles are concentrated in the nodal plane of the standing ultrasound wave, forming particle conglomerates. By selecting appropriate experimental conditions, it is then possible to press bacteria against the crystal surface for interaction with the evanescent wave (as well as to keep them away from the ATR element). Our current work aims at establishing a custommade US-ATR-IR setup for signal enhancement of bacteria (e.g. E. coli, P. aeruginosa as well as Salmonella) in drinking water.

Photoacoustic spectroscopy sample array vessels and photoacoustic spectroscopy methods for using the same

DOEpatents

Amonette, James E.; Autrey, S. Thomas; Foster-Mills, Nancy S.

2006-02-14

Methods and apparatus for simultaneous or sequential, rapid analysis of multiple samples by photoacoustic spectroscopy are disclosed. Particularly, a photoacoustic spectroscopy sample array vessel including a vessel body having multiple sample cells connected thereto is disclosed. At least one acoustic detector is acoustically positioned near the sample cells. Methods for analyzing the multiple samples in the sample array vessels using photoacoustic spectroscopy are provided.

Multistage Force Amplification of Piezoelectric Stacks

NASA Technical Reports Server (NTRS)

Xu, Tian-Bing (Inventor); Siochi, Emilie J. (Inventor); Zuo, Lei (Inventor); Jiang, Xiaoning (Inventor); Kang, Jin Ho (Inventor)

2015-01-01

Embodiments of the disclosure include an apparatus and methods for using a piezoelectric device, that includes an outer flextensional casing, a first cell and a last cell serially coupled to each other and coupled to the outer flextensional casing such that each cell having a flextensional cell structure and each cell receives an input force and provides an output force that is amplified based on the input force. The apparatus further includes a piezoelectric stack coupled to each cell such that the piezoelectric stack of each cell provides piezoelectric energy based on the output force for each cell. Further, the last cell receives an input force that is the output force from the first cell and the last cell provides an output apparatus force In addition, the piezoelectric energy harvested is based on the output apparatus force. Moreover, the apparatus provides displacement based on the output apparatus force.

Contact inhibition of locomotion determines cell-cell and cell-substrate forces in tissues.

PubMed

Zimmermann, Juliane; Camley, Brian A; Rappel, Wouter-Jan; Levine, Herbert

2016-03-08

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell-cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin-Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell-cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge.

Neuron-like PC12 cell patterning on a photoactive self-assembled monolayer.

PubMed

Cheng, Nan; Cao, Xudong

2013-11-01

A new approach to pattern cells using photochemistry and self-assembled monolayer (SAM) was described in this study. Photocleavable 4,5-dimethoxy-2-nitrobenzyl chloroformate (NVOC) protected amine on an alkanethiol-gold SAM was developed for cell patterning. The cleavage of NVOC and the deprotection of amines on the SAM were controlled spatially by two sequential UV exposures with a photomask. Biomolecule patterning was achieved by introducing cell nonadhesive poly(ethylene glycol) after the first exposure and subsequently cell adhesive protein laminin after the second exposure to create surface cell adhesiveness differential for cell patterning. UV-Vis spectrophotometry was used to determine the photolysis of caged self-assembled molecules; in addition, water contact angle, atomic force microscopy, cyclic voltammetry, and X-ray photoelectron spectroscopy were used to characterize properties of different surfaces. To test the efficacy of resulting surfaces in patterning cells, a neuron-like cell line, PC12 cell line, was used. The in vitro cell studies showed successful PC12 cell patterns on the photoactive SAM surfaces. This patterning technique is unique in that it does not rely on cell adhesive or nonadhesive properties of the starting base material as both cell adhesive and cell nonadhesive molecules were individually introduced onto the base material surface through photo-uncaging at preselected regions for the ultimate cell patterning. Copyright © 2013 Wiley Periodicals, Inc.

Measurement of Solution Viscosity via Diffusion-Ordered NMR Spectroscopy (DOSY)

ERIC Educational Resources Information Center

Li, Weibin; Kagan, Gerald; Hopson, Russell; Williard, Paul G.

2011-01-01

Increasingly, the undergraduate chemistry curriculum includes nuclear magnetic resonance (NMR) spectroscopy. Advanced NMR techniques are often taught including two-dimensional gradient-based experiments. An investigation of intermolecular forces including viscosity, by a variety of methods, is often integrated in the undergraduate physical and…

Observation of force-detected nuclear magnetic resonance in a homogeneous field

PubMed Central

Madsen, L. A.; Leskowitz, G. M.; Weitekamp, D. P.

2004-01-01

We report the experimental realization of BOOMERANG (better observation of magnetization, enhanced resolution, and no gradient), a sensitive and general method of magnetic resonance. The prototype millimeter-scale NMR spectrometer shows signal and noise levels in agreement with the design principles. We present 1H and 19F NMR in both solid and liquid samples, including time-domain Fourier transform NMR spectroscopy, multiple-pulse echoes, and heteronuclear J spectroscopy. By measuring a 1H-19F J coupling, this last experiment accomplishes chemically specific spectroscopy with force-detected NMR. In BOOMERANG, an assembly of permanent magnets provides a homogeneous field throughout the sample, while a harmonically suspended part of the assembly, a detector, is mechanically driven by spin-dependent forces. By placing the sample in a homogeneous field, signal dephasing by diffusion in a field gradient is made negligible, enabling application to liquids, in contrast to other force-detection methods. The design appears readily scalable to μm-scale samples where it should have sensitivity advantages over inductive detection with microcoils and where it holds great promise for application of magnetic resonance in biology, chemistry, physics, and surface science. We briefly discuss extensions of the BOOMERANG method to the μm and nm scales. PMID:15326302

Programmable Assembly of Peptide Amphiphile via Noncovalent-to-Covalent Bond Conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sato, Kohei; Ji, Wei; Palmer, Liam C.

Controlling the number of monomers in a supramolecular polymer has been a great challenge in programmable self-assembly of organic molecules. One approach has been to make use of frustrated growth of the supramolecular assembly by tuning the balance of attractive and repulsive intermolecular forces. We report here on the use of covalent bond formation among monomers, compensating for intermolecular electrostatic repulsion, as a mechanism to control the length of a supramolecular nanofiber formed by self-assembly of peptide amphiphiles. Circular dichroism spectroscopy in combination with dynamic light scattering, size-exclusion chromatography, and transmittance electron microscope analyses revealed that hydrogen bonds between peptidesmore » were reinforced by covalent bond formation, enabling the fiber elongation. To examine these materials for their potential biomedical applications, cytotoxicity of nanofibers against C2C12 premyoblast cells was tested. We demonstrated that cell viability increased with an increase in fiber length, presumably because of the suppressed disruption of cell membranes by the fiber end-caps.« less

Programmable Assembly of Peptide Amphiphile via Noncovalent-to-Covalent Bond Conversion

DOE PAGES

Sato, Kohei; Ji, Wei; Palmer, Liam C.; ...

2017-06-22

Controlling the number of monomers in a supramolecular polymer has been a great challenge in programmable self-assembly of organic molecules. One approach has been to make use of frustrated growth of the supramolecular assembly by tuning the balance of attractive and repulsive intermolecular forces. We report here on the use of covalent bond formation among monomers, compensating for intermolecular electrostatic repulsion, as a mechanism to control the length of a supramolecular nanofiber formed by self-assembly of peptide amphiphiles. Circular dichroism spectroscopy in combination with dynamic light scattering, size-exclusion chromatography, and transmittance electron microscope analyses revealed that hydrogen bonds between peptidesmore » were reinforced by covalent bond formation, enabling the fiber elongation. To examine these materials for their potential biomedical applications, cytotoxicity of nanofibers against C2C12 premyoblast cells was tested. We demonstrated that cell viability increased with an increase in fiber length, presumably because of the suppressed disruption of cell membranes by the fiber end-caps.« less

Efficiency Improvement Using Molybdenum Disulphide Interlayers in Single-Wall Carbon Nanotube/Silicon Solar Cells

PubMed Central

Alzahly, Shaykha; Yu, LePing; Gibson, Christopher T.

2018-01-01

Molybdenum disulphide (MoS2) is one of the most studied and widely applied nanomaterials from the layered transition-metal dichalcogenides (TMDs) semiconductor family. MoS2 has a large carrier diffusion length and a high carrier mobility. Combining a layered structure of single-wall carbon nanotube (SWCNT) and MoS2 with n-type silicon (n-Si) provided novel SWCNT/n-Si photovoltaic devices. The solar cell has a layered structure with Si covered first by a thin layer of MoS2 flakes and then a SWCNT film. The films were examined using scanning electron microscopy, atomic force microscopy and Raman spectroscopy. The MoS2 flake thickness ranged from 5 to 90 nm while the nanosheet’s lateral dimensions size ranged up to 1 μm2. This insertion of MoS2 improved the photoconversion efficiency (PCE) of the SWCNT/n-Si solar cells by approximately a factor of 2. PMID:29690503

Identifying the charge generation dynamics in Cs+-based triple cation mixed perovskite solar cells.

PubMed

Salado, Manuel; Kokal, Ramesh K; Calio, Laura; Kazim, Samrana; Deepa, Melepurath; Ahmad, Shahzada

2017-08-30

Triple cation based perovskite solar cells offer enhanced moisture tolerance and stability compared to mixed perovskites. Slight substitution of methyl ammonium or formamidinium cation by cesium (Cs + ), was also reported to eliminate halide segregation due to its smaller size. To elucidate the device kinetics and understand the role of the Cs, we undertook different modes of scanning probe microscopy and electrochemical impedance spectroscopy (EIS) experiments. Kelvin probe force microscopy revealed that the incorporation of the Cs cation increases the contact potential difference (CPD), this CPD further increases when Spiro-OMeTAD is used as a hole transport material. The current at the nanoscale level shows improvement with Cs inclusion and further enhancement by the Spiro-OMeTAD deposition, studied under light illumination, which supports the high photocurrent density obtained from the cells. EIS demonstrates that in a triple cation environment, reduced carrier recombination at the TiO 2 /perovskite interface was also obtained which in turn allow us to achieve a higher V oc value.

Interactions of the anticancer drug tamoxifen with lipid membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khadka, Nawal K.; Cheng, Xiaolin; Ho, Chian Sing

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer areamore » compressibility modulus KA is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Lastly, our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.« less

Optical trapping and Raman spectroscopy of single nanostructures using standing-wave Raman tweezers

NASA Astrophysics Data System (ADS)

Wu, Mu-ying; He, Lin; Chen, Gui-hua; Yang, Guang; Li, Yong-qing

2017-08-01

Optical tweezers integrated with Raman spectroscopy allows analyzing a single trapped micro-particle, but is generally less effective for individual nano-sized objects in the 10-100 nm range. The main challenge is the weak gradient force on nanoparticles that is insufficient to overcome the destabilizing effect of scattering force and Brownian motion. Here, we present standing-wave Raman tweezers for stable trapping and sensitive characterization of single isolated nanostructures with a low laser power by combining a standing-wave optical trap (SWOT) with confocal Raman spectroscopy. This scheme has stronger intensity gradients and balanced scattering forces, and thus is more stable and sensitive in measuring nanoparticles in liquid with 4-8 fold increase in the Raman signals. It can be used to analyze many nanoparticles that cannot be measured with single-beam Raman tweezers, including individual single-walled carbon nanotubes (SWCNT), graphene flakes, biological particles, polystyrene beads (100 nm), SERS-active metal nanoparticles, and high-refractive semiconductor nanoparticles with a low laser power of a few milliwatts. This would enable sorting and characterization of specific SWCNTs and other nanoparticles based on their increased Raman fingerprints.

Interactions of the anticancer drug tamoxifen with lipid membranes

DOE PAGES

Khadka, Nawal K.; Cheng, Xiaolin; Ho, Chian Sing; ...

2015-05-19

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer areamore » compressibility modulus KA is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Lastly, our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.« less

APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

NASA Astrophysics Data System (ADS)

Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

2015-10-01

APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies.

PubMed

Shlyakhtenko, Luda S; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S; Lyubchenko, Yuri L

2015-10-27

APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

Determination of HER2 amplification status in breast cancer cells using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Bi, Xiaohong; Rexer, Brent; Arteaga, Carlos L.; Guo, Mingsheng; Li, Ming; Mahadevan-Jansen, Anita

2010-02-01

The overexpression of HER2 (human epidermal growth factor receptor 2) in breast cancer is associated with increased disease recurrence and worse prognosis. Current diagnosis of HER2 positive breast cancer is time consuming with an estimated 20% inaccuracy. Raman spectroscopy is a proven method for pathological diagnosis based on the molecular composition of tissues. This study aimed to determine the feasibility of Raman spectroscopy to differentially identify the amplification of HER2 in cells. Three cell lines including BT474 (HER2 overexpressing breast cancer cell), MCF-10A (human breast epithelial cell), and MCF-10A with overexpressing HER2, were investigated using a bench top confocal Raman system. A diagnostic algorithm based on generalized linear model (GLM) with elastic-net penalties was established to discriminate 318 spectra collected from the cells, and to identify the spectra regions that differentiate the cell lines. The algorithm was able to differentially identify BT474 breast cancer cells with an overall sensitivity of 100% and specificity of 99%. The results demonstrate the capability of Raman spectroscopy to determine HER2 status in cells. Raman spectroscopy shows promise for application in the diagnosis of HER2 positive breast cancer in clinical practice.

The effects of erythrocyte deformability upon hematocrit assessed by the conductance method.

PubMed

Hayashi, Yoshihito; Katsumoto, Yoichi; Oshige, Ikuya; Omori, Shinji; Yasuda, Akio; Asami, Koji

2009-04-21

A comparative study of centrifugation and conductance methods for the estimation of cell volume fraction (phi) was performed to examine whether the strong forces exerted upon erythrocytes during centrifugation affect their volume, and the results are discussed in terms of erythrocyte deformability. Rabbit erythrocytes of four shapes (spherocytes, echinocytes, stomatocyte-like enlarged erythrocytes and discocytes) were prepared by controlling the pH of the suspending media. The packed cell volumes of the suspensions were measured by standard hematocrit determination methods using centrifugation in capillary tubes. Simultaneously, the same suspensions and their supernatants were used in dielectric spectroscopy measurements, and the low-frequency limits of their conductivities were used for the numerical estimation of phi. The hematocrit values of spherocytes and echinocytes were markedly less than the volume fractions obtained by the conductance method. Namely, the centrifugation reduced the cell volume. For enlarged erythrocytes and discocytes, however, the reduction of cell volume was not observed. These findings showed that phi obtained by the centrifugation method can be greatly affected by the deformability of the cells, but the level of the effect depends on the cell types. Consequently, phi obtained by the centrifugation method should be carefully interpreted.

New advances in probing cell–extracellular matrix interactions

PubMed Central

2017-01-01

The extracellular matrix (ECM) provides structural and biochemical support to cells within tissues. An emerging body of evidence has established that the ECM plays a key role in cell mechanotransduction – the study of coupling between mechanical inputs and cellular phenotype – through either mediating transmission of forces to the cells, or presenting mechanical cues that guide cellular behaviors. Recent progress in cell mechanotransduction research has been facilitated by advances of experimental tools, particularly microtechnologies, engineered biomaterials, and imaging and analytical methods. Microtechnologies have enabled the design and fabrication of controlled physical microenvironments for the study and measurement of cell–ECM interactions. Advances in engineered biomaterials have allowed researchers to develop synthetic ECMs that mimic tissue microenvironments and investigate the impact of altered physicochemical properties on various cellular processes. Finally, advanced imaging and spectroscopy techniques have facilitated the visualization of the complex interaction between cells and ECM in vitro and in living tissues. This review will highlight the application of recent innovations in these areas to probing cell–ECM interactions. We believe cross-disciplinary approaches, combining aspects of the different technologies reviewed here, will inspire innovative ideas to further elucidate the secrets of ECM-mediated cell control. PMID:28352896

Structure of the Global Nanoscience and Nanotechnology Research Literature

DTIC Science & Technology

2006-01-01

Transistors, Nature, 424 (6949): 654-657, 2003. Joannopoulos, JD, Meade, RD, Winn, JN, Photonic Crystals: Molding the Flow of Light, Princeton...1.27 Force Microscopy 40 0.10 0.00 Electron Spectroscopy 40 0.10 0.00 Rutherford backscattering spectrometry 38 0.10 0.00 flow cytometry 36 0.09...Backscattering Spectroscopy/Spectrometry • Flow Cytometry • Spectrophotometry (UV-Visible) • Deep Level Transient Spectroscopy • Inductively

A biotemplated nickel nanostructure: Synthesis, characterization and antibacterial activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashtari, Khadijeh; Fasihi, Javad; Mollania, Nasrin

Highlights: • Nickel nanostructure-encapsulated bacteria were prepared using electroless deposition. • Bacterium surface was activated by red-ox reaction of its surface amino acids. • Interfacial changes at cell surfaces were investigated using fluorescence spectroscopy. • TEM and AFM depicted morphological changes. • Antibacterial activity of nanostructure was examined against different bacteria strains. - Abstract: Nickel nanostructure-encapsulated bacteria were prepared using the electroless deposition procedure and activation of bacterium cell surface by red-ox reaction of surface amino acids. The electroless deposition step occurred in the presence of Ni(II) and dimethyl amine boran (DMAB). Interfacial changes at bacteria cell surfaces during themore » coating process were investigated using fluorescence spectroscopy. Fluorescence of tryptophan residues was completely quenched after the deposition of nickel onto bacteria surfaces. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) depicted morphological changes on the surface of the bacterium. It was found that the Ni coated nanostructure was mechanically stable after ultrasonication for 20 min. Significant increase in surface roughness of bacteria was also observed after deposition of Ni clusters. The amount of coated Ni on the bacteria surface was calculated as 36% w/w. The antibacterial activity of fabricated nanostructure in culture media was examined against three different bacteria strains; Escherichia coli, Bacillus subtilis and Xantomonas campestris. The minimum inhibitory concentrations (MIC) were determined as 500 mg/L, 350 mg/L and 200 mg/L against bacteria, respectively.« less

Evaluation of pulsed laser deposited SrNb0.1Co0.9O3-δ thin films as promising cathodes for intermediate-temperature solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Chen, Dengjie; Chen, Chi; Gao, Yang; Zhang, Zhenbao; Shao, Zongping; Ciucci, Francesco

2015-11-01

SrNb0.1Co0.9O3-δ (SNC) thin films prepared on single-crystal yttria-stabilized zirconia (YSZ) electrolytes are evaluated as promising cathodes for intermediate-temperature solid oxide fuel cells (IT-SOFCs). Geometrically well-defined polycrystalline SNC thin films with low surface roughness and high surface oxygen vacancy concentration are successfully fabricated by pulsed laser deposition. The thin films are characterized by basic techniques, e.g., X-ray diffraction for phase structure identification, scanning electron microscopy and atomic force microscopy for microstructures measurement, and X-ray photoelectron spectroscopy for elements quantification. Electrochemical impedance spectroscopy (EIS) is used to investigate oxygen reduction reaction activities of SNC thin films in symmetric electrochemical cells. Current collectors (Ag paste, Ag strip, and Au strip) are found to have negligible impact on polarization resistances. A slight decrease of the electrode polarization resistances is observed after adding a samarium doped ceria (SDC) buffer layer between SNC and YSZ. SNC thin-film electrodes exhibit low electrode polarization resistances, e.g., 0.237 Ω cm2 (SNC/SDC/YSZ/SDC/SNC) and 0.274 Ω cm2 (SNC/YSZ/SNC) at 700 °C and 0.21 atm, demonstrating the promise of SNC materials for IT-SOFCs. An oxygen reduction reaction mechanism of SNC thin films is also derived by analyzing EIS at temperature of 550-700 °C under oxygen partial pressure range of 0.04-1 atm.

Organic solar cells with graded absorber layers processed from nanoparticle dispersions.

PubMed

Gärtner, Stefan; Reich, Stefan; Bruns, Michael; Czolk, Jens; Colsmann, Alexander

2016-03-28

The fabrication of organic solar cells with advanced multi-layer architectures from solution is often limited by the choice of solvents since most organic semiconductors dissolve in the same aromatic agents. In this work, we investigate multi-pass deposition of organic semiconductors from eco-friendly ethanol dispersion. Once applied, the nanoparticles are insoluble in the deposition agent, allowing for the application of further nanoparticulate layers and hence for building poly(3-hexylthiophene-2,5-diyl):indene-C60 bisadduct absorber layers with vertically graded polymer and conversely graded fullerene concentration. Upon thermal annealing, we observe some degrees of polymer/fullerene interdiffusion by means of X-ray photoelectron spectroscopy and Kelvin probe force microscopy. Replacing the common bulk-heterojunction by such a graded photo-active layer yields an enhanced fill factor of the solar cell due to an improved charge carrier extraction, and consequently an overall power conversion efficiency beyond 4%. Wet processing of such advanced device architectures paves the way for a versatile, eco-friendly and industrially feasible future fabrication of organic solar cells with advanced multi-layer architectures.

Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

NASA Astrophysics Data System (ADS)

Chen, Huiqing; Li, Xiaojing; Zhao, Yuancong; Li, Jingan; Chen, Jiang; Yang, Ping; Maitz, Manfred F.; Huang, Nan

2015-08-01

A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

Characterization and evaluation of femtosecond laser-induced sub-micron periodic structures generated on titanium to improve osseointegration of implants

NASA Astrophysics Data System (ADS)

Lee, Bryan E. J.; Exir, Hourieh; Weck, Arnaud; Grandfield, Kathryn

2018-05-01

Reproducible and controllable methods of modifying titanium surfaces for dental and orthopaedic applications are of interest to prevent poor implant outcomes by improving osseointegration. This study made use of a femtosecond laser to generate laser-induced periodic surface structures with periodicities of 300, 620 and 760 nm on titanium substrates. The reproducible rippled patterns showed consistent submicron scale roughness and relatively hydrophobic surfaces as measured by atomic force microscopy and contact angle, respectively. Transmission electron microscopy and Auger electron spectroscopy identified a thicker oxide layer on ablated surfaces compared to controls. In vitro testing was conducted using osteosarcoma Saos-2 cells. Cell metabolism on the laser-ablated surfaces was comparable to controls and alkaline phosphatase activity was notably increased at late time points for the 620 and 760 nm surfaces compared to controls. Cells showed a more elongated shape on laser-ablated surfaces compared to controls and showed perpendicular alignment to the periodic structures. This work has demonstrated the feasibility of generating submicron features on an implant material with the ability to influence cell response and improve implant outcomes.

Atomic force measurements of 16-mercaptohexadecanoic acid and its salt with CH 3, OH, and CONHCH 3 functionalized self-assembled monolayers

NASA Astrophysics Data System (ADS)

Morales-Cruz, Angel L.; Tremont, Rolando; Martínez, Ramón; Romañach, Rodolfo; Cabrera, Carlos R.

2005-03-01

Chemical and mechanical properties of different compounds can be elucidated by measuring fundamental forces such as adhesion, attraction and repulsion, between modified surfaces by means of atomic force microscopy (AFM) in force mode calibration. This work presents a combination of AFM, self-assembled monolayers (SAMs), and crystallization techniques to study the forces of interaction between excipients and active ingredients used in pharmaceutical formulations. SAMs of 16-mercaptohexadecanoate, which represent magnesium stereate, were used to modify the probe tip, whereas CH3-, OH- and CONHCH3-functional SAMs were formed on a gold-coated mica substrate, and used as examples of the surfaces of lactose and theophylline. The crystals of lactose and theophylline were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The modification of gold surfaces with 16-mercaptohexadecanoate, 10-mercapto-1-decanol (OH-functional SAM), 1-decanethiol (CH3-functional) and N-methyl-11-mercaptoundecanamide (CONHCH3-functional SAM) was studied by X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES) and Fourier transform-infrared spectroscopy (FT-IR) in specular reflectance mode. XPS and AES results of the modified surfaces showed the presence of sulfur binding, and kinetic energies that correspond to the presence of 10-mercapto-1-decanol, 1-decanethiol, N-methyl-11-mercaptoundecanamide and the salt of 16-mercaptohexadecanoic acid. The absorption bands in the IR spectra further confirm the modification of the gold-coated substrates with these compounds. Force versus distance measurements were performed between the modified tip and the modified gold-coated mica substrates. The mean adhesion forces between the COO-Ca2+ functionalized tip and the CH3-, OH-, and CONHCH3-modified substrates were determined to be 4.5, 8.9 and 6.3 nN, respectively. The magnitude of the adhesion force (ion-dipole) interaction between the modified tip and substrate decreases in the following order: COO-Ca2+/OH > COO-Ca2+/CONHCH3 > COO-Ca2+/CH3.

Probing local bias-induced transitions using photothermal excitation contact resonance atomic force microscopy and voltage spectroscopy

DOE PAGES

Li, Qian; Jesse, Stephen; Tselev, Alexander; ...

2015-01-05

In this paper, nanomechanical properties are closely related to the states of matter, including chemical composition, crystal structure, mesoscopic domain configuration, etc. Investigation of these properties at the nanoscale requires not only static imaging methods, e.g., contact resonance atomic force microscopy (CR-AFM), but also spectroscopic methods capable of revealing their dependence on various external stimuli. Here we demonstrate the voltage spectroscopy of CR-AFM, which was realized by combining photothermal excitation (as opposed to the conventional piezoacoustic excitation method) with the band excitation technique. We applied this spectroscopy to explore local bias-induced phenomena ranging from purely physical to surface electromechanical andmore » electrochemical processes. Our measurements show that the changes in the surface properties associated with these bias-induced transitions can be accurately assessed in a fast and dynamic manner, using resonance frequency as a signature. Finally, with many of the advantages offered by photothermal excitation, contact resonance voltage spectroscopy not only is expected to find applications in a broader field of nanoscience but also will provide a basis for future development of other nanoscale elastic spectroscopies.« less

Green synthesis of silver nanoparticles using Mentha pulegium and investigation of their antibacterial, antifungal and anticancer activity.

PubMed

Kelkawi, Ali Hamad Abd; Abbasi Kajani, Abolghasem; Bordbar, Abdol-Khalegh

2017-06-01

A simple and eco-friendly method for efficient synthesis of stable colloidal silver nanoparticles (AgNPs) using Mentha pulegium extracts is described. A series of reactions was conducted using different types and concentrations of plant extract as well as metal ions to optimize the reaction conditions. AgNPs were characterized by using UV-vis spectroscopy, transmission electron microscopy, atomic force microscopy, dynamic light scattering, zetasizer, energy-dispersive X-ray spectroscopy (EDAX) and Fourier transform infrared spectroscopy (FTIR). At the optimized conditions, plate shaped AgNPs with zeta potential value of -15.7 and plasmon absorption maximum at 450 nm were obtained using high concentration of aqueous extract. Efficient adsorption of organic compounds on the nanoparticles was confirmed by FTIR and EDAX. The biogenic AgNPs displayed promising antibacterial activity on Escherichia coli , Staphylococcus aureus , and Streptococcus pyogenes . The highest antibacterial activity of 25 µg mL-1 was obtained for all the strains using aqueous extract synthesized AgNPs. The aqueous extract synthesised AgNPs also showed considerable antifungal activity against fluconazole resistant Candida albicans . The cytotoxicity assay revealed considerable anticancer activity of AgNPs on HeLa and MCF-7 cancer cells. Overall results indicated high potential of M. pulegium extract to synthesis high quality AgNPs for biomedical applications.

Chemical bath deposited and dip coating deposited CuS thin films - Structure, Raman spectroscopy and surface study

NASA Astrophysics Data System (ADS)

Tailor, Jiten P.; Khimani, Ankurkumar J.; Chaki, Sunil H.

2018-05-01

The crystal structure, Raman spectroscopy and surface microtopography study on as-deposited CuS thin films were carried out. Thin films deposited by two techniques of solution growth were studied. The thin films used in the present study were deposited by chemical bath deposition (CBD) and dip coating deposition techniques. The X-ray diffraction (XRD) analysis of both the as-deposited thin films showed that both the films possess covellite phase of CuS and hexagonal unit cell structure. The determined lattice parameters of both the films are in agreement with the standard JCPDS as well as reported data. The crystallite size determined by Scherrer's equation and Hall-Williamsons relation using XRD data for both the as-deposited thin films showed that the respective values were in agreement with each other. The ambient Raman spectroscopy of both the as-deposited thin films showed major emission peaks at 474 cm-1 and a minor emmision peaks at 265 cm-1. The observed Raman peaks matched with the covellite phase of CuS. The atomic force microscopy of both the as-deposited thin films surfaces showed dip coating thin film to be less rough compared to CBD deposited thin film. All the obtained results are presented and deliberated in details.

Enhancement in photovoltaic properties of silicon solar cells by surface plasmon effect of palladium nanoparticles

NASA Astrophysics Data System (ADS)

Atyaoui, Malek; Atyaoui, Atef; Khalifa, Marwen; Elyagoubi, Jalel; Dimassi, Wissem; Ezzaouia, Hatem

2016-04-01

This work presents the surface Plasmon effect of Palladium nanoparticles (Pd NPs) on the photovoltaic properties of silicon solar cells. Pd NPs were deposited on the p-type silicon base of the n+/p junction using a chemical deposition method in an aqueous solution containing Palladium (II) Nitrate (PdNO3)2 and Ammonium Hydroxide (NH4OH) followed by a thermal treatment at 500 °C under nitrogen atmosphere. Chemical composition and surface morphology of the treated silicon base were examined by energy dispersive X-ray (EDX) spectroscopy, scanning electronic microscopy (SEM) and Atomic Force Microscopy (AFM). The effect of the deposited Pd NPs on the electrical properties was evaluated by the internal quantum efficiency (IQE) and current-voltage (I-V) measurements. The results indicate that the formation of the Pd NPs is accompanied by an enhanced light absorption and improved photovoltaic parameters.

Cytotoxic and antimicrobial effect of biosynthesized silver nanoparticles using the fruit extract of Ribes nigrum

NASA Astrophysics Data System (ADS)

Dobrucka, Renata; Kaczmarek, Mariusz; Dlugaszewska, Jolanta

2018-06-01

The present study reveals the efficiency of the fruit extract of Ribes nigrum in the green synthesis of silver nanoparticles (Ag-NPs). Biosynthesized Ag-NPs were characterized by UV-vis, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The nanoparticles were found to be 5–10 nm. In some places, the particles were agglomerated. The nanoparticles showed strong bactericidal activity and fungicidal activity against dermatophytes Trichophyton rubrum ATCC 28188. Moreover, the A549 and CCD39Lu cells under the influence of the highest concentration of nanoparticles synthesized using the fruit extract of Ribes nigrum showed the maximum mortality. Also, the results indicate that Ag-NPs synthesized using the fruit extract of Ribes nigrum exhibit efficiency in therapy of human non-small cell lung cancer A549.

Effects of Piezoelectric Potential of ZnO on Resistive Switching Characteristics of Flexible ZnO/TiO2 Heterojunction Cells

NASA Astrophysics Data System (ADS)

Li, Hongxia; Zhou, You; Du, Gang; Huang, Yanwei; Ji, Zhenguo

2018-03-01

Flexible resistance random access memory (ReRAM) devices with a heterojunction structure of PET/ITO/ZnO/TiO2/Au were fabricated on polyethylene terephthalate/indium tin oxide (PET/ITO) substrates by different physical and chemical preparation methods. X-ray diffraction, scanning electron microscopy and atomic force microscopy were carried out to investigate the crystal structure, surface topography and cross-sectional structure of the prepared films. X-ray photoelectron spectroscopy was also used to identify the chemical state of Ti, O and Zn elements. Theoretical and experimental analyses were conducted to identify the effect of piezoelectric potential of ZnO on resistive switching characteristics of flexible ZnO/TiO2 heterojunction cells. The results showed a pathway to enhance the performance of ReRAM devices by engineering the interface barrier, which is also feasible for other electronics, optoelectronics and photovoltaic devices.

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

PubMed

Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

2006-07-26

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (Ø <120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

Magnetic core/shell nanoparticle thin films deposited by MAPLE: Investigation by chemical, morphological and in vitro biological assays

NASA Astrophysics Data System (ADS)

Cristescu, R.; Popescu, C.; Socol, G.; Iordache, I.; Mihailescu, I. N.; Mihaiescu, D. E.; Grumezescu, A. M.; Balan, A.; Stamatin, I.; Chifiriuc, C.; Bleotu, C.; Saviuc, C.; Popa, M.; Chrisey, D. B.

2012-09-01

We report on thin film deposition of nanostructured Fe3O4/oleic acid/ceftriaxone and Fe3O4/oleic acid/cefepime nanoparticles (core/shell/adsorption-shell) were fabricated by matrix assisted pulsed laser evaporation (MAPLE) onto inert substrates. The thin films were characterized by profilometry, Fourier transform infrared spectroscopy, atomic force microscopy, and investigated by in vitro biological assays. The biological properties tested included the investigation of the microbial viability and the microbial adherence to the glass coverslip nanoparticle film, using Gram-negative and Gram-positive bacterial strains with known antibiotic susceptibility behavior, the microbial adherence to the HeLa cells monolayer grown on the nanoparticle pellicle, and the cytotoxicity on eukaryotic cells. The proposed system, based on MAPLE, could be used for the development of novel anti-microbial materials or strategies for fighting pathogenic biofilms frequently implicated in the etiology of biofilm associated chronic infections.

Taking Nanomedicine Teaching into Practice with Atomic Force Microscopy and Force Spectroscopy

ERIC Educational Resources Information Center

Carvalho, Filomena A.; Freitas, Teresa; Santos, Nuno C.

2015-01-01

Atomic force microscopy (AFM) is a useful and powerful tool to study molecular interactions applied to nanomedicine. The aim of the present study was to implement a hands-on atomic AFM course for graduated biosciences and medical students. The course comprises two distinct practical sessions, where students get in touch with the use of an atomic…

Optimized Free Energies from Bidirectional Single-Molecule Force Spectroscopy

NASA Astrophysics Data System (ADS)

Minh, David D. L.; Adib, Artur B.

2008-05-01

An optimized method for estimating path-ensemble averages using data from processes driven in opposite directions is presented. Based on this estimator, bidirectional expressions for reconstructing free energies and potentials of mean force from single-molecule force spectroscopy—valid for biasing potentials of arbitrary stiffness—are developed. Numerical simulations on a model potential indicate that these methods perform better than unidirectional strategies.

Spectroellipsometric, AFM and XPS probing of stainless steel surfaces subjected to biological influences

NASA Astrophysics Data System (ADS)

Vinnichenko, M.; Chevolleau, Th; Pham, M. T.; Poperenko, L.; Maitz, M. F.

2002-11-01

Surface modification of austenitic stainless steel (SS) 316L after incubation in growing cell cultures and cell-free media as control has been studied. The following treatments were applied: mouse fibrosarcoma cells L929 for 3 and 7 days, polymorphonuclear neutrophils for 3 and 7 days and human osteosarcoma cells SAOS-2 for 7 and 14 days. Cells were enzymatically removed in all cases. The modified surfaces were probed in comparison with untreated ones by means of spectroscopic ellipsometry (SE), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). XPS shows the appearance of the peak of bonded nitrogen at 400.5 eV characteristic for adsorbed proteins on the surface for each type of cells and for the cell-free medium. Migration of Ni in the adsorbed layer is observed in all cases for samples after the cell cultures. The protein layer thickness is ellipsometrically determined to be within 2.5-6.0 nm for all treated samples with parameterization of its optical constants in Cauchy approach. The study showed that for such biological treatments of the SS the protein layer adsorption is the dominating process in the first 2 weeks, which could play a role in the process of corrosion by complex forming properties with metal ions.

The influence of surface chemistry and topography on the contact guidance of MG63 osteoblast cells.

PubMed

Ismail, F S Magdon; Rohanizadeh, R; Atwa, S; Mason, R S; Ruys, A J; Martin, P J; Bendavid, A

2007-05-01

The purpose of the present study was to determine in vitro the effects of different surface topographies and chemistries of commercially pure titanium (cpTi) and diamond-like carbon (DLC) surfaces on osteoblast growth and attachment. Microgrooves (widths of 2, 4, 8 and 10 microm and a depth of 1.5-2 microm) were patterned onto silicon (Si) substrates using microlithography and reactive ion etching. The Si substrates were subsequently vapor coated with either cpTi or DLC coatings. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Using the MG63 Osteoblast-Like cell line, we determined cell viability, adhesion, and morphology on different substrates over a 3 day culture period. The results showed cpTi surfaces to be significantly more hydrophilic than DLC for groove sizes larger than 2 microm. Cell contact guidance was observed for all grooved samples in comparison to the unpatterned controls. The cell viability tests indicated a significantly greater cell number for 8 and 10 microm grooves on cpTi surfaces compared to other groove sizes. The cell adhesion study showed that the smaller groove sizes, as well as the unpatterned control groups, displayed better cell adhesion to the substrate.

Investigations of the functional states of dendritic cells under different conditioned microenvironments by Fourier transformed infrared spectroscopy.

PubMed

Dong, Rong; Long, Jinhua; Xu, Xiaoli; Zhang, Chunlin; Wen, Zongyao; Li, Long; Yao, Weijuan; Zeng, Zhu

2014-01-10

Dendritic cells are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. The dendritic cell-based vaccination against cancer has been clinically achieved promising successes. But there are still many challenges in its clinical application, especially for how to identify the functional states. The CD14+ monocytes were isolated from human peripheral blood after plastic adherence and purified to approximately 98% with cocktail immunomagnetic beads. The immature dendritic cells and mature dendritic cells were induced by traditional protocols. The resulting dendritic cells were cocultured with normal cells and cancer cells. The functional state of dendritic cells including immature dendritic cells (imDCs) and mature dendritic cells (mDCs) under different conditioned microenvironments were investigated by Fourier transformed infrared spectroscopy (FTIR) and molecular biological methods. The results of Fourier transformed infrared spectroscopy showed that the gene transcription activity and energy states of dendritic cells were specifically suppressed by tumor cells (P < 0.05 or 0.01). The expression levels of NF-kappa B (NF-κB) in dendritic cells were also specifically inhibited by tumor-derived factors (P < 0.05 or 0.01). Moreover, the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were closely correlated with the expression levels of NF-κB (R2:0.69 and R2:0.81, respectively). Our results confirmed that the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were positively correlated with the expression levels of NF-κB, suggesting that Fourier transformed infrared spectroscopy technology could be clinically applied to identify the functional states of dendritic cell when performing dendritic cell-based vaccination. It's significant for the simplification and standardization of dendritic cell-based vaccination clinical preparation protocols.

Antigen-antibody biorecognition events as discriminated by noise analysis of force spectroscopy curves.

PubMed

Bizzarri, Anna Rita; Cannistraro, Salvatore

2014-08-22

Atomic force spectroscopy is able to extract kinetic and thermodynamic parameters of biomolecular complexes provided that the registered unbinding force curves could be reliably attributed to the rupture of the specific complex interactions. To this aim, a commonly used strategy is based on the analysis of the stretching features of polymeric linkers which are suitably introduced in the biomolecule-substrate immobilization procedure. Alternatively, we present a method to select force curves corresponding to specific biorecognition events, which relies on a careful analysis of the force fluctuations of the biomolecule-functionalized cantilever tip during its approach to the partner molecules immobilized on a substrate. In the low frequency region, a characteristic 1/f (α) noise with α equal to one (flickering noise) is found to replace white noise in the cantilever fluctuation power spectrum when, and only when, a specific biorecognition process between the partners occurs. The method, which has been validated on a well-characterized antigen-antibody complex, represents a fast, yet reliable alternative to the use of linkers which may involve additional surface chemistry and reproducibility concerns.

The nature of the force-induced conformation transition of dsDNA studied by using single molecule force spectroscopy.

PubMed

Liu, Ningning; Bu, Tianjia; Song, Yu; Zhang, Wei; Li, Jinjing; Zhang, Wenke; Shen, Jiacong; Li, Hongbin

2010-06-15

Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

Acoustically trapped colloidal crystals that are reconfigurable in real time

PubMed Central

Caleap, Mihai; Drinkwater, Bruce W.

2014-01-01

Photonic and phononic crystals are metamaterials with repeating unit cells that result in internal resonances leading to a range of wave guiding and filtering properties and are opening up new applications such as hyperlenses and superabsorbers. Here we show the first, to our knowledge, 3D colloidal phononic crystal that is reconfigurable in real time and demonstrate its ability to rapidly alter its frequency filtering characteristics. Our reconfigurable material is assembled from microspheres in aqueous solution, trapped with acoustic radiation forces. The acoustic radiation force is governed by an energy landscape, determined by an applied high-amplitude acoustic standing wave field, in which particles move swiftly to energy minima. This creates a colloidal crystal of several milliliters in volume with spheres arranged in an orthorhombic lattice in which the acoustic wavelength is used to control the lattice spacing. Transmission acoustic spectroscopy shows that the new colloidal crystal behaves as a phononic metamaterial and exhibits clear band-pass and band-stop frequencies which are adjusted in real time. PMID:24706925

A Broadband X-Ray Imaging Spectroscopy with High-Angular Resolution: the FORCE Mission

NASA Technical Reports Server (NTRS)

Mori, Koji; Tsuru, Takeshi Go; Nakazawac, Kazuhiro; Ueda, Yoshihiro; Okajima, Takashi; Murakami, Hiroshi; Awaki, Hisamitsu; Matsumoto, Hironori; Fukazawai, Yasushi; Tsunemi, Hiroshi;

A broadband x-ray imaging spectroscopy with high-angular resolution: the FORCE mission

NASA Astrophysics Data System (ADS)

Mori, Koji; Tsuru, Takeshi Go; Nakazawa, Kazuhiro; Ueda, Yoshihiro; Okajima, Takashi; Murakami, Hiroshi; Awaki, Hisamitsu; Matsumoto, Hironori; Fukazawa, Yasushi; Tsunemi, Hiroshi; Takahashi, Tadayuki; Zhang, William W.

2016-07-01

We are proposing FORCE (Focusing On Relativistic universe and Cosmic Evolution) as a future Japan-lead Xray observatory to be launched in the mid 2020s. Hitomi (ASTRO-H) possesses a suite of sensitive instruments enabling the highest energy-resolution spectroscopy in soft X-ray band, a broadband X-ray imaging spectroscopy in soft and hard X-ray bands, and further high energy coverage up to soft gamma-ray band. FORCE is the direct successor to the broadband X-ray imaging spectroscopy aspect of Hitomi (ASTRO-H) with significantly higher angular resolution. The current design of FORCE defines energy band pass of 1-80 keV with angular resolution of < 15 in half-power diameter, achieving a 10 times higher sensitivity above 10 keV compared to any previous missions with simultaneous soft X-ray coverage. Our primary scientific objective is to trace the cosmic formation history by searching for "missing black holes" in various mass-scales: "buried supermassive black holes (SMBHs)" (> 104 M⊙) residing in the center of galaxies in a cosmological distance, "intermediate-mass black holes" (102-104 M⊙) acting as the possible seeds from which SMBHs grow, and "orphan stellar-mass black holes" (< 102 M⊙) without companion in our Galaxy. In addition to these missing BHs, hunting for the nature of relativistic particles at various astrophysical shocks is also in our scope, utilizing the broadband X-ray coverage with high angular-resolution. FORCE are going to open a new era in these fields. The satellite is proposed to be launched with the Epsilon vehicle that is a Japanese current solid-fuel rocket. FORCE carries three identical pairs of Super-mirror and wide-band X-ray detector. The focal length is currently planned to be 10 m. The silicon mirror with multi-layer coating is our primary choice to achieve lightweight, good angular optics. The detector is a descendant of hard X-ray imager onboard Hitomi (ASTRO-H) replacing its silicon strip detector with SOI-CMOS silicon pixel detector, allowing an extension of the low energy threshold down to 1 keV or even less.

Nonparametric density estimation and optimal bandwidth selection for protein unfolding and unbinding data

NASA Astrophysics Data System (ADS)

Bura, E.; Zhmurov, A.; Barsegov, V.

2009-01-01

Dynamic force spectroscopy and steered molecular simulations have become powerful tools for analyzing the mechanical properties of proteins, and the strength of protein-protein complexes and aggregates. Probability density functions of the unfolding forces and unfolding times for proteins, and rupture forces and bond lifetimes for protein-protein complexes allow quantification of the forced unfolding and unbinding transitions, and mapping the biomolecular free energy landscape. The inference of the unknown probability distribution functions from the experimental and simulated forced unfolding and unbinding data, as well as the assessment of analytically tractable models of the protein unfolding and unbinding requires the use of a bandwidth. The choice of this quantity is typically subjective as it draws heavily on the investigator's intuition and past experience. We describe several approaches for selecting the "optimal bandwidth" for nonparametric density estimators, such as the traditionally used histogram and the more advanced kernel density estimators. The performance of these methods is tested on unimodal and multimodal skewed, long-tailed distributed data, as typically observed in force spectroscopy experiments and in molecular pulling simulations. The results of these studies can serve as a guideline for selecting the optimal bandwidth to resolve the underlying distributions from the forced unfolding and unbinding data for proteins.

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.

PubMed

Tang, Jilin; Krajcikova, Daniela; Zhu, Rong; Ebner, Andreas; Cutting, Simon; Gruber, Hermann J; Barak, Imrich; Hinterdorfer, Peter

2007-01-01

Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores. Copyright (c) 2007 John Wiley & Sons, Ltd.

Theoretical Models for Surface Forces and Adhesion and Their Measurement Using Atomic Force Microscopy

PubMed Central

Leite, Fabio L.; Bueno, Carolina C.; Da Róz, Alessandra L.; Ziemath, Ervino C.; Oliveira, Osvaldo N.

2012-01-01

The increasing importance of studies on soft matter and their impact on new technologies, including those associated with nanotechnology, has brought intermolecular and surface forces to the forefront of physics and materials science, for these are the prevailing forces in micro and nanosystems. With experimental methods such as the atomic force spectroscopy (AFS), it is now possible to measure these forces accurately, in addition to providing information on local material properties such as elasticity, hardness and adhesion. This review provides the theoretical and experimental background of AFS, adhesion forces, intermolecular interactions and surface forces in air, vacuum and in solution. PMID:23202925

Multi-Photon Micro-Spectroscopy of Biological Specimens

DTIC Science & Technology

2000-07-01

Micro-spectroscopy, multi-photon fluorescence spectroscopy, second harmonic generation, plant tissues, stem, chloroplast, protoplast, maize, Arabidopsis...harmonic generation (SHG) in the plant cell 5wall. In this case, micro-spectroscopy provides a means of verification that, indeed, SHG occurs in plant ...fluorescence microscopy -the response of plant cells to high intensity illumination," Micron (in press) 2000. 3. H.-C. Huang and C. -C Chen, "Genome

Nanomechanics of Pectin-Linked β-Lactoglobulin Nanofibril Bundles.

PubMed

Loveday, Simon M; Gunning, A Patrick

2018-06-14

Nanofibrils of β-lactoglobulin can be assembled into bundles by site-specific noncovalent cross-linking with high-methoxyl pectin (Hettiarachchi et al. Soft Matter 2016, 12, 756). Here we characterized the nanomechanical properties of bundles using atomic force microscopy and force spectroscopy. Bundles had Gaussian cross sections and a mean height of 17.4 ± 1.4 nm. Persistence lengths were calculated using image analysis with the mean-squared end-to-end model. The relationship between the persistence length and the thickness had exponents of 1.69-2.30, which is consistent with previous reports for other fibril types. In force spectroscopy experiments, the bundles stretched in a qualitatively different manner to fibrils, and some of the force curves were consistent with peeling fibrils away from bundles. The flexibility of pectin-linked nanofibril bundles is likely to be tunable by modulating the stiffness and length of fibrils and the ratio of pectin to fibrils, giving rise to a wide range of structures and functionalities.

Investigating single molecule adhesion by atomic force spectroscopy.

PubMed

Stetter, Frank W S; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

2015-02-27

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

An open source/real-time atomic force microscope architecture to perform customizable force spectroscopy experiments.

PubMed

Materassi, Donatello; Baschieri, Paolo; Tiribilli, Bruno; Zuccheri, Giampaolo; Samorì, Bruno

2009-08-01

We describe the realization of an atomic force microscope architecture designed to perform customizable experiments in a flexible and automatic way. Novel technological contributions are given by the software implementation platform (RTAI-LINUX), which is free and open source, and from a functional point of view, by the implementation of hard real-time control algorithms. Some other technical solutions such as a new way to estimate the optical lever constant are described as well. The adoption of this architecture provides many degrees of freedom in the device behavior and, furthermore, allows one to obtain a flexible experimental instrument at a relatively low cost. In particular, we show how such a system has been employed to obtain measures in sophisticated single-molecule force spectroscopy experiments [Fernandez and Li, Science 303, 1674 (2004)]. Experimental results on proteins already studied using the same methodologies are provided in order to show the reliability of the measure system.

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

PubMed Central

Stetter, Frank W. S.; Kienle, Sandra; Krysiak, Stefanie; Hugel, Thorsten

2015-01-01

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment. PMID:25867282

Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy.

PubMed

Kedrov, Alexej; Janovjak, Harald; Sapra, K Tanuj; Müller, Daniel J

2007-01-01

Molecular interactions are the basic language of biological processes. They establish the forces interacting between the building blocks of proteins and other macromolecules, thus determining their functional roles. Because molecular interactions trigger virtually every biological process, approaches to decipher their language are needed. Single-molecule force spectroscopy (SMFS) has been used to detect and characterize different types of molecular interactions that occur between and within native membrane proteins. The first experiments detected and localized molecular interactions that stabilized membrane proteins, including how these interactions were established during folding of alpha-helical secondary structure elements into the native protein and how they changed with oligomerization, temperature, and mutations. SMFS also enables investigators to detect and locate molecular interactions established during ligand and inhibitor binding. These exciting applications provide opportunities for studying the molecular forces of life. Further developments will elucidate the origins of molecular interactions encoded in their lifetimes, interaction ranges, interplay, and dynamics characteristic of biological systems.

Force Spectroscopy with 9-μs Resolution and Sub-pN Stability by Tailoring AFM Cantilever Geometry.

PubMed

Edwards, Devin T; Faulk, Jaevyn K; LeBlanc, Marc-André; Perkins, Thomas T

2017-12-19

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize the unfolding/refolding dynamics of individual molecules and resolve closely spaced, transiently occupied folding intermediates. On a modern commercial AFM, these applications and others are now limited by the mechanical properties of the cantilever. Specifically, AFM-based SMFS data quality is degraded by a commercial cantilever's limited combination of temporal resolution, force precision, and force stability. Recently, we modified commercial cantilevers with a focused ion beam to optimize their properties for SMFS. Here, we extend this capability by modifying a 40 × 18 μm 2 cantilever into one terminated with a gold-coated, 4 × 4 μm 2 reflective region connected to an uncoated 2-μm-wide central shaft. This "Warhammer" geometry achieved 8.5-μs resolution coupled with improved force precision and sub-pN stability over 100 s when measured on a commercial AFM. We highlighted this cantilever's biological utility by first resolving a calmodulin unfolding intermediate previously undetected by AFM and then measuring the stabilization of calmodulin by myosin light chain kinase at dramatically higher unfolding velocities than in previous AFM studies. More generally, enhancing data quality via an improved combination of time resolution, force precision, and force stability will broadly benefit biological applications of AFM. Published by Elsevier Inc.

Activity in the premotor area related to bite force control--a functional near-infrared spectroscopy study.

PubMed

Takeda, Tomotaka; Shibusawa, Mami; Sudal, Osamu; Nakajima, Kazunori; Ishigami, Keiichi; Sakatani, Kaoru

2010-01-01

The purpose of this study was to elucidate the influence of bite force control on oxygenated hemoglobin (OxyHb) levels in regional cerebral blood flow as an indicator of brain activity in the premotor area. Healthy right-handed volunteers with no subjective or objective symptoms of problems of the stomatognathic system or cervicofacial region were included. Functional near-infrared spectroscopy (fNIRS) was used to determine OxyHb levels in the premotor area during bite force control. A bite block equipped with an occlusal force sensor was prepared to measure clenching at the position where the right upper and lower canine cusps come into contact. Intensity of clenching was shown on a display and feedback was provided to the subjects. Intensity was set at 20, 50 and 80% of maximum voluntary teeth clenching force. To minimize the effect of the temporal muscle on the working side of the jaw, the fNIRS probes were positioned contralaterally, in the left region. The findings of this study are: activation of the premotor area with bite force control was noted in all subjects, and in the group analysis OxyHb in the premotor cortex was significantly increased as the clenching strengthened at 20, 50 and 80% of maximum voluntary clenching force. These results suggest there is a possibility that the premotor area is involved in bite force control.

Contact inhibition of locomotion determines cell–cell and cell–substrate forces in tissues

PubMed Central

Zimmermann, Juliane; Camley, Brian A.; Rappel, Wouter-Jan; Levine, Herbert

2016-01-01

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell–cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin–Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell–cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge. PMID:26903658

αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin

PubMed Central

Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P.; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B.; Fässler, Reinhard; Müller, Daniel J.

2017-01-01

Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix. PMID:28128308

Near infrared light induces post-translational modifications of human red blood cell proteins.

PubMed

Walski, Tomasz; Dyrda, Agnieszka; Dzik, Małgorzata; Chludzińska, Ludmiła; Tomków, Tomasz; Mehl, Joanna; Detyna, Jerzy; Gałecka, Katarzyna; Witkiewicz, Wojciech; Komorowska, Małgorzata

2015-11-01

There is a growing body of evidence that near infrared (NIR) light exerts beneficial effects on cells. Its usefulness in the treatment of cancer, acute brain injuries, strokes and neurodegenerative disorders has been proposed. The mechanism of the NIR action is probably of photochemical nature, however it is not fully understood. Here, using a relatively simple biological model, human red blood cells (RBCs), and a polychromatic non-polarized light source, we investigate the impact of NIR radiation on the oxygen carrier, hemoglobin (Hb), and anion exchanger (AE1, Band 3). The exposure of intact RBCs to NIR light causes quaternary transitions in Hb, dehydration of proteins and decreases the amount of physiologically inactive methemoglobin, as detected by Raman spectroscopy. These effects are accompanied by a lowering of the intracellular pH (pHi) and changes in the cell membrane topography, as documented by atomic force microscopy (AFM). All those changes are in line with our previous studies where alterations of the membrane fluidity and membrane potential were attributed to NIR action on RBCs. The rate of the above listed changes depends strictly on the dose of NIR light that the cells receive, nonetheless it should not be considered as a thermal effect.

Silicon heterojunction solar cells with novel fluorinated n-type nanocrystalline silicon oxide emitters on p-type crystalline silicon

NASA Astrophysics Data System (ADS)

Dhar, Sukanta; Mandal, Sourav; Das, Gourab; Mukhopadhyay, Sumita; Pratim Ray, Partha; Banerjee, Chandan; Barua, Asok Kumar

2015-08-01

A novel fluorinated phosphorus doped silicon oxide based nanocrystalline material have been used to prepare heterojunction solar cells on flat p-type crystalline silicon (c-Si) Czochralski (CZ) wafers. The n-type nc-SiO:F:H material were deposited by radio frequency plasma enhanced chemical vapor deposition. Deposited films were characterized in detail by using atomic force microscopy (AFM), high resolution transmission electron microscopy (HRTEM), Raman, fourier transform infrared spectroscopy (FTIR) and optoelectronics properties have been studied using temperature dependent conductivity measurement, Ellipsometry, UV-vis spectrum analysis etc. It is observed that the cell fabricated with fluorinated silicon oxide emitter showing higher initial efficiency (η = 15.64%, Jsc = 32.10 mA/cm2, Voc = 0.630 V, FF = 0.77) for 1 cm2 cell area compare to conventional n-a-Si:H emitter (14.73%) on flat c-Si wafer. These results indicate that n type nc-SiO:F:H material is a promising candidate for heterojunction solar cell on p-type crystalline wafers. The high Jsc value is associated with excellent quantum efficiencies at short wavelengths (<500 nm).

On the role of extracellular polymeric substances during early stages of Xylella fastidiosa biofilm formation.

PubMed

Lorite, Gabriela S; de Souza, Alessandra A; Neubauer, Daniel; Mizaikoff, Boris; Kranz, Christine; Cotta, Mônica A

2013-02-01

The structural integrity and protection of bacterial biofilms are intrinsically associated with a matrix of extracellular polymeric substances (EPS) produced by the bacteria cells. However, the role of these substances during biofilm adhesion to a surface remains largely unclear. In this study, the influence of EPS on Xylella fastidiosa biofilm formation was investigated. This bacterium is associated with economically important plant diseases; it presents a slow growth rate and thus allows us to pinpoint more precisely the early stages of cell-surface adhesion. Scanning electron microscopy and atomic force microscopy show evidence of EPS production in such early stages and around individual bacteria cells attached to the substrate surface even a few hours after inoculation. In addition, EPS formation was investigated via attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FTIR). To this end, X. fastidiosa cells were inoculated within an ATR liquid cell assembly. IR-ATR spectra clearly reveal EPS formation already during the early stages of X. fastidiosa biofilm formation, thereby providing supporting evidence for the hypothesis of the relevance of the EPS contribution to the adhesion process. Copyright © 2012 Elsevier B.V. All rights reserved.

Raman spectroscopy for cancer detection and characterization in metastasis models

NASA Astrophysics Data System (ADS)

Koga, Shigehiro; Oshima, Yusuke; Sato, Mitsunori; Ishimaru, Kei; Yoshida, Motohira; Yamamoto, Yuji; Matsuno, Yusuke; Watanabe, Yuji

2017-02-01

Raman spectroscopy provides a wealth of diagnostic information to the surgeon with in situ cancer detection and label-free histopathology in clinical practice. Raman spectroscopy is a developing optical technique which can analyze biological tissues with light scattering. The difference in frequencies between the incident light and the scattering light are called Raman shifts, which correspond to the vibrational energy of the molecular bonds. Raman spectrum gives information about the molecular structure and composition in biological specimens. We had been previously reported that Raman spectroscopy could distinguish various histological types of human lung cancer cells from normal cells in vitro. However, to identify and detect cancer diagnostic biomarkers in vivo on Raman spectroscopy is still challenging, because malignancy can be characterized not only by the cancer cells but also by the environmental factors including immune cells, stroma cells, secretion vesicles and extracellular matrix. Here we investigate morphological and molecular dynamics in both cancer cells and their environment in xenograft models and spontaneous metastasis models using Raman spectroscopy combined with fluorescence microscopy and photoluminescence imaging. We are also constructing a custom-designed Raman spectral imaging system for both in vitro and in vivo assay of tumor tissues to reveal the metastasis process and to evaluate therapeutic effects of anti-cancer drugs and their drug delivery toward the clinical application of the technique.

Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

NASA Astrophysics Data System (ADS)

Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

2006-07-01

A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

The interaction of 2-mercaptobenzimidazole with human serum albumin as determined by spectroscopy, atomic force microscopy and molecular modeling.

PubMed

Li, Yuqin; Jia, Baoxiu; Wang, Hao; Li, Nana; Chen, Gaopan; Lin, Yuejuan; Gao, Wenhua

2013-04-01

The interaction of 2-mercaptobenzimidazole (MBI) with human serum albumin (HSA) was studied in vitro by equilibrium dialysis under normal physiological conditions. This study used fluorescence, ultraviolet-visible spectroscopy (UV-vis), Fourier transform infrared (FT-IR), circular dichroism (CD) and Raman spectroscopy, atomic force microscopy (AFM) and molecular modeling techniques. Association constants, the number of binding sites and basic thermodynamic parameters were used to investigate the quenching mechanism. Based on the fluorescence resonance energy transfer, the distance between the HSA and MBI was 2.495 nm. The ΔG(0), ΔH(0), and ΔS(0) values across temperature indicated that the hydrophobic interaction was the predominant binding Force. The UV, FT-IR, CD and Raman spectra confirmed that the HSA secondary structure was altered in the presence of MBI. In addition, the molecular modeling showed that the MBI-HSA complex was stabilized by hydrophobic forces, which resulted from amino acid residues. The AFM results revealed that the individual HSA molecule dimensions were larger after interaction with MBI. Overall, this study suggested a method for characterizing the weak intermolecular interaction. In addition, this method is potentially useful for elucidating the toxigenicity of MBI when it is combined with the biomolecular function effect, transmembrane transport, toxicological testing and other experiments. Copyright © 2012 Elsevier B.V. All rights reserved.

Atomic force microscopy investigation of the interaction of low-level laser irradiation of collagen thin films in correlation with fibroblast response.

PubMed

Stylianou, Andreas; Yova, Dido

2015-12-01

Low-level red laser (LLRL)-tissue interactions have a wide range of medical applications and are garnering increased attention. Although the positive effects of low-level laser therapy (LLLT) have frequently been reported and enhanced collagen accumulation has been identified as one of the most important mechanisms involved, little is known about LLRL-collagen interactions. In this study, we aimed to investigate the influence of LLRL irradiation on collagen, in correlation with fibroblast response. Atomic force microscopy (AFM) and fluorescence spectroscopy were used to characterize surfaces and identify conformational changes in collagen before and after LLRL irradiation. Irradiated and non-irradiated collagen thin films were used as culturing substrates to investigate fibroblast response with fluorescence microscopy. The results demonstrated that LLRL induced small alterations in fluorescence emission and had a negligible effect on the topography of collagen thin films. However, fibroblasts cultured on LLRL-irradiated collagen thin films responded to LRLL. The results of this study show for the first time the effect of LLRL irradiation on pure collagen. Although irradiation did not affect the nanotopography of collagen, it influenced cell behavior. The role of collagen appears to be crucial in the LLLT mechanism, and our results demonstrated that LLRL directly affects collagen and indirectly affects cell behavior.

In vivo and in situ monitoring of the nitric oxide stimulus response of single cancer cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Su, L.; Chen, Y.; Zhang, G. N.; Wang, L. H.; Shen, A. G.; Zhou, X. D.; Wang, X. H.; Hu, J. M.

2013-04-01

Raman spectroscopy is capable of studying time-resolved information of selected biomolecular distributions inside individual cells without labeling. In this study, Raman spectroscopy was for the first time utilized to in vivo and in situ monitor the cellular response to nitric oxide (NO) in single oral squamous cell carcinoma (OSCC) cells over a period of 24 h. Sodium nitroprusside (SNP) was chosen as a NO donor to be incubated with the OSCC cell line (TCA8113) for certain time intervals. In vivo and in situ Raman analysis revealed that the degradation and conformational changes of nucleic acids, lipids and proteins could be directly observed by changes in the characteristic Raman bands. In comparison with conventional flow cytometric analysis, Raman spectroscopy not only detected more subtle NO-induced chemical changes of cells, where the SNP concentration could be even less than 1 mM, but also provided a full view of the whole chemical components of single cells. Raman spectroscopy therefore is an important candidate for label-free, nondestructive and in situ monitoring of cellular changes in response to chemotherapeutic agents, which could potentially be used in rapid screening of novel drugs.

Raman spectral signatures of cervical exfoliated cells from liquid-based cytology samples

NASA Astrophysics Data System (ADS)

Kearney, Padraig; Traynor, Damien; Bonnier, Franck; Lyng, Fiona M.; O'Leary, John J.; Martin, Cara M.

2017-10-01

It is widely accepted that cervical screening has significantly reduced the incidence of cervical cancer worldwide. The primary screening test for cervical cancer is the Papanicolaou (Pap) test, which has extremely variable specificity and sensitivity. There is an unmet clinical need for methods to aid clinicians in the early detection of cervical precancer. Raman spectroscopy is a label-free objective method that can provide a biochemical fingerprint of a given sample. Compared with studies on infrared spectroscopy, relatively few Raman spectroscopy studies have been carried out to date on cervical cytology. The aim of this study was to define the Raman spectral signatures of cervical exfoliated cells present in liquid-based cytology Pap test specimens and to compare the signature of high-grade dysplastic cells to each of the normal cell types. Raman spectra were recorded from single exfoliated cells and subjected to multivariate statistical analysis. The study demonstrated that Raman spectroscopy can identify biochemical signatures associated with the most common cell types seen in liquid-based cytology samples; superficial, intermediate, and parabasal cells. In addition, biochemical changes associated with high-grade dysplasia could be identified suggesting that Raman spectroscopy could be used to aid current cervical screening tests.

Self-recognition and Ca2+-dependent carbohydrate-carbohydrate cell adhesion provide clues to the cambrian explosion.

PubMed

Fernàndez-Busquets, Xavier; Körnig, André; Bucior, Iwona; Burger, Max M; Anselmetti, Dario

2009-11-01

The Cambrian explosion of life was a relatively short period approximately 540 Ma that marked a generalized acceleration in the evolution of most animal phyla, but the trigger of this key biological event remains elusive. Sponges are the oldest extant Precambrian metazoan phylum and thus a valid model to study factors that could have unleashed the rise of multicellular animals. One such factor is the advent of self-/non-self-recognition systems, which would be evolutionarily beneficial to organisms to prevent germ-cell parasitism or the introduction of deleterious mutations resulting from fusion with genetically different individuals. However, the molecules responsible for allorecognition probably evolved gradually before the Cambrian period, and some other (external) factor remains to be identified as the missing triggering event. Sponge cells associate through calcium-dependent, multivalent carbohydrate-carbohydrate interactions of the g200 glycan found on extracellular proteoglycans. Single molecule force spectroscopy analysis of g200-g200 binding indicates that calcium affects the lifetime (+Ca/-Ca: 680 s/3 s) and bond reaction length (+Ca/-Ca: 3.47 A/2.27 A). Calculation of mean g200 dissociation times in low and high calcium within the theoretical framework of a cooperative binding model indicates the nonlinear and divergent characteristics leading to either disaggregated cells or stable multicellular assemblies, respectively. This fundamental phenomenon can explain a switch from weak to strong adhesion between primitive metazoan cells caused by the well-documented rise in ocean calcium levels at the end of Precambrian time. We propose that stronger cell adhesion allowed the integrity of genetically uniform animals composed only of "self" cells, facilitating genetic constitutions to remain within the metazoan individual and be passed down inheritance lines. The Cambrian explosion might have been triggered by the coincidence in time of primitive animals endowed with self-/non-self-recognition and of a surge in seawater calcium that increased the binding forces between their calcium-dependent cell adhesion molecules.

Watching How Molecules Orient in a Surface Forces Apparatus, Using Confocal Raman Spectroscopy

NASA Astrophysics Data System (ADS)

Jiang, Shan; Kim, Minsu; Bae, Sung Chul; Granick, Steve

2006-03-01

Much is known about surface forces, less about where they come from. This laboratory is engaged in direct vibrational spectroscopic measurements of how molecules orient in confined geometries. Regarding force measurements, PDMS (polydimethylsiloxane) was a model system for many years. In this study, we describe direct experiments using a novel version of confocal Raman spectroscopy. This experiment allows direct measurement of how the PDMS molecules orient under confinment as well as under subsequent shear. When the thickness of the fluid film is less than the unperturbed radius of gyration of the polymer, we obtain two novel findings: (a) linewidth analysis of peaks reveals that vibrational relaxation times are perturbed in this confined geometry; (b) orientation of the chain backbone is not everywhere the same within the molecularly-thin film; domains of various orientation are observed instead.

Single ricin detection by atomic force microscopy chemomechanical mapping

NASA Astrophysics Data System (ADS)

Chen, Guojun; Zhou, Jianfeng; Park, Bosoon; Xu, Bingqian

2009-07-01

The authors report on a study of detecting ricin molecules immobilized on chemically modified Au (111) surface by chemomechanically mapping the molecular interactions with a chemically modified atomic force microscopy (AFM) tip. AFM images resolved the different fold-up conformations of single ricin molecule as well as their intramolecule structure of A- and B-chains. AFM force spectroscopy study of the interaction indicates that the unbinding force has a linear relation with the logarithmic force loading rate, which agrees well with calculations using one-barrier bond dissociation model.

Performance of mid infrared spectroscopy in skin cancer cell type identification

NASA Astrophysics Data System (ADS)

Kastl, Lena; Kemper, Björn; Lloyd, Gavin R.; Nallala, Jayakrupakar; Stone, Nick; Naranjo, Valery; Penaranda, Francisco; Schnekenburger, Jürgen

2017-02-01

Marker free optical spectroscopy is a powerful tool for the rapid inspection of pathologically suspicious skin lesions and the non-invasive detection of early skin tumors. This goal can be reached by the combination of signal localization and the spectroscopical detection of chemical cell signatures. We here present the development and application of mid infrared spectroscopy (midIR) for the analysis of skin tumor cell types and three dimensional tissue phantoms towards the application of midIR spectroscopy for fast and reliable skin diagnostics. We developed standardized in vitro skin systems with increasing complexity, from single skin cell types as fibroblasts, keratinocytes and melanoma cells, to mixtures of these and finally three dimensional skin cancer phantoms. The cell systems were characterized with different systems in the midIR range up to 12 μm. The analysis of the spectra by novel data processing algorithms demonstrated the clear separation of all cell types, especially melanoma cells. Special attention and algorithm training was required for closely related mesenchymal cell types as dedifferentiated melanoma cells and fibroblasts. Proof of concept experiments with mixtures of in vivo fluorescence labelled skin cell types allowed the test of the new algorithms performance for the identification of specific cell types. The intense training of the software systems with various samples resulted in a increased sensitivity and specificity of the combined midIR and software system. These data highlight the potential of midIR spectroscopy as sensitive and specific future optical biopsy technology.

Molecule-Doped Nickel Oxide: Verified Charge Transfer and Planar Inverted Mixed Cation Perovskite Solar Cell.

PubMed

Chen, Wei; Zhou, Yecheng; Wang, Linjing; Wu, Yinghui; Tu, Bao; Yu, Binbin; Liu, Fangzhou; Tam, Ho-Won; Wang, Gan; Djurišić, Aleksandra B; Huang, Li; He, Zhubing

2018-05-01

Both conductivity and mobility are essential to charge transfer by carrier transport layers (CTLs) in perovskite solar cells (PSCs). The defects derived from generally used ionic doping method lead to the degradation of carrier mobility and parasite recombinations. In this work, a novel molecular doping of NiO x hole transport layer (HTL) is realized successfully by 2,2'-(perfluoronaphthalene-2,6-diylidene)dimalononitrile (F6TCNNQ). Determined by X-ray photoelectron spectroscopy and ultraviolet photoelectron spectroscopy, the Fermi level (E F ) of NiO x HTLs is increased from -4.63 to -5.07 eV and valence band maximum (VBM)-E F declines from 0.58 to 0.29 eV after F6TCNNQ doping. The energy level offset between the VBMs of NiO x and perovskites declines from 0.18 to 0.04 eV. Combining with first-principle calculations, electrostatic force microscopy is applied for the first time to verify direct electron transfer from NiO x to F6TCNNQ. The average power conversion efficiency of CsFAMA mixed cation PSCs is boosted by ≈8% depending on F6TCNNQ-doped NiOx HTLs. Strikingly, the champion cell conversion efficiency of CsFAMA mixed cations and MAPbI 3 -based devices gets to 20.86% and 19.75%, respectively. Different from passivation effect, the results offer an extremely promising molecular doping method for inorganic CTLs in PSCs. This methodology definitely paves a novel way to modulate the doping in hybrid electronics more than perovskite and organic solar cells. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

PubMed Central

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology. PMID:29888200

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

PubMed

Ott, Wolfgang; Jobst, Markus A; Bauer, Magnus S; Durner, Ellis; Milles, Lukas F; Nash, Michael A; Gaub, Hermann E

2017-06-27

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

Nanoscale Infrared Spectroscopy of Biopolymeric Materials

Treesearch

Curtis Marcott; Michael Lo; Kevin Kjoller; Craig Prater; Roshan Shetty; Joseph Jakes; Isao Noda

2012-01-01

Atomic Force Microscopy (AFM) and infrared (IR) spectroscopy have been combined in a single instrument capable of producing 100 nm spatial resolution IR spectra and images. This new capability enables the spectroscopic characterization of biomaterial domains at levels not previously possible. A tunable IR laser source generating pulses on the order of 10 ns was used...

Opposite Surface and Bulk Solvatochromic Effects in a Molecular Spin-Crossover Compound Revealed by Ambient Pressure X-ray Absorption Spectroscopy.

PubMed

Borgatti, Francesco; Torelli, Piero; Brucale, Marco; Gentili, Denis; Panaccione, Giancarlo; Castan Guerrero, Celia; Schäfer, Bernhard; Ruben, Mario; Cavallini, Massimiliano

2018-03-27

We investigate the solvatochromic effect of a Fe-based spin-crossover (SCO) compound via ambient pressure soft X-ray absorption spectroscopy (AP-XAS) and atomic force microscopy (AFM). AP-XAS provides the direct evidence of the spin configuration for the Fe(II) 3d states of the SCO material upon in situ exposure to specific gas or vapor mixtures; concurrent changes in nanoscale topography and mechanical characteristics are revealed via AFM imaging and AFM-based force spectroscopy, respectively. We find that exposing the SCO material to gaseous helium promotes an effective decrease of the transition temperature of its surface layers, while the exposure to methanol vapor causes opposite surfacial and bulk solvatochromic effects. Surfacial solvatochromism is accompanied by a dramatic reduction of the surface layers stiffness. We propose a rationalization of the observed effects based on interfacial dehydration and solvation phenomena.

The Evolution of Silica Nanoparticle-polyester Coatings on Surfaces Exposed to Sunlight.

PubMed

Truong, Vi Khanh; Stefanovic, Miljan; Maclaughlin, Shane; Tobin, Mark; Vongsvivut, Jitraporn; Al Kobaisi, Mohammad; Crawford, Russell J; Ivanova, Elena P

2016-10-11

Corrosion of metallic surfaces is prevalent in the environment and is of great concern in many areas, including the military, transport, aviation, building and food industries, amongst others. Polyester and coatings containing both polyester and silica nanoparticles (SiO2NPs) have been widely used to protect steel substrata from corrosion. In this study, we utilized X-ray photoelectron spectroscopy, attenuated total reflection infrared micro-spectroscopy, water contact angle measurements, optical profiling and atomic force microscopy to provide an insight into how exposure to sunlight can cause changes in the micro- and nanoscale integrity of the coatings. No significant change in surface micro-topography was detected using optical profilometry, however, statistically significant nanoscale changes to the surface were detected using atomic force microscopy. Analysis of the X-ray photoelectron spectroscopy and attenuated total reflection infrared micro-spectroscopy data revealed that degradation of the ester groups had occurred through exposure to ultraviolet light to form COO·, -H2C·, -O·, -CO· radicals. During the degradation process, CO and CO2 were also produced.

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation

PubMed Central

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-01-01

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES–TiO2 and PE–TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti4+/Ti3+ redox catalysis occurring on PES–TiO2 and PE–TiO2 during the bacterial inactivation process. On PE–TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C–H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES–TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1–5% wt PES–TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces. PMID:25657831

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation.

PubMed

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-02-06

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES-TiO2 and PE-TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti(4+)/Ti(3+) redox catalysis occurring on PES-TiO2 and PE-TiO2 during the bacterial inactivation process. On PE-TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C-H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES-TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1-5% wt PES-TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces.

Keratocytes Generate Traction Forces in Two PhasesV⃞

PubMed Central

Burton, Kevin; Park, Jung H.; Taylor, D. Lansing

1999-01-01

Forces generated by goldfish keratocytes and Swiss 3T3 fibroblasts have been measured with nanonewton precision and submicrometer spatial resolution. Differential interference contrast microscopy was used to visualize deformations produced by traction forces in elastic substrata, and interference reflection microscopy revealed sites of cell-substratum adhesions. Force ranged from a few nanonewtons at submicrometer spots under the lamellipodium to several hundred nanonewtons under the cell body. As cells moved forward, centripetal forces were applied by lamellipodia at sites that remained stationary on the substratum. Force increased and abruptly became lateral at the boundary of the lamellipodium and the cell body. When the cell retracted at its posterior margin, cell-substratum contact area decreased more rapidly than force, so that stress (force divided by area) increased as the cell pulled away. An increase in lateral force was associated with widening of the cell body. These mechanical data suggest an integrated, two-phase mechanism of cell motility: (1) low forces in the lamellipodium are applied in the direction of cortical flow and cause the cell body to be pulled forward; and (2) a component of force at the flanks pulls the rear margins forward toward the advancing cell body, whereas a large lateral component contributes to detachment of adhesions without greatly perturbing forward movement. PMID:10564269

Keratocytes generate traction forces in two phases.

PubMed

Burton, K; Park, J H; Taylor, D L

1999-11-01

Forces generated by goldfish keratocytes and Swiss 3T3 fibroblasts have been measured with nanonewton precision and submicrometer spatial resolution. Differential interference contrast microscopy was used to visualize deformations produced by traction forces in elastic substrata, and interference reflection microscopy revealed sites of cell-substratum adhesions. Force ranged from a few nanonewtons at submicrometer spots under the lamellipodium to several hundred nanonewtons under the cell body. As cells moved forward, centripetal forces were applied by lamellipodia at sites that remained stationary on the substratum. Force increased and abruptly became lateral at the boundary of the lamellipodium and the cell body. When the cell retracted at its posterior margin, cell-substratum contact area decreased more rapidly than force, so that stress (force divided by area) increased as the cell pulled away. An increase in lateral force was associated with widening of the cell body. These mechanical data suggest an integrated, two-phase mechanism of cell motility: (1) low forces in the lamellipodium are applied in the direction of cortical flow and cause the cell body to be pulled forward; and (2) a component of force at the flanks pulls the rear margins forward toward the advancing cell body, whereas a large lateral component contributes to detachment of adhesions without greatly perturbing forward movement.

Chemically grafted fibronectin for use in QCM-D cell studies

PubMed Central

Sobolewski, Peter; Tomczyk, Nancy; Composto, Russell J.; Eckmann, David M.

2014-01-01

Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using surface ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics. PMID:24657645

MEMS-Based Force-Detected Nuclear Magnetic Resonance (FDNMR) Spectrometer

NASA Technical Reports Server (NTRS)

Lee, Choonsup; Butler, Mark C.; Elgammal, Ramez A.; George, Thomas; Hunt, Brian; Weitekamp, Daniel P.

2006-01-01

Nuclear Magnetic Resonance (NMR) spectroscopy allows assignment of molecular structure by acquiring the energy spectrum of nuclear spins in a molecule, and by interpreting the symmetry and positions of resonance lines in the spectrum. As such, NMR has become one of the most versatile and ubiquitous spectroscopic methods. Despite these tremendous successes, NMR experiments suffer from inherent low sensitivity due to the relatively low energy of photons in the radio frequency (rt) region of the electromagnetic spectrum. Here, we describe a high-resolution spectroscopy in samples with diameters in the micron range and below. We have reported design and fabrication of force-detected nuclear magnetic resonance (FDNMR).

APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

PubMed Central

Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

2015-01-01

APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA. PMID:26503602

Near-infrared Raman spectroscopy of single optically trapped biological cells

NASA Astrophysics Data System (ADS)

Xie, Changan; Dinno, Mumtaz A.; Li, Yong-Qing

2002-02-01

We report on the development and testing of a compact laser tweezers Raman spectroscopy (LTRS) system. The system combines optical trapping and near-infrared Raman spectroscopy for manipulation and identification of single biological cells in solution. A low-power diode laser at 785 nm was used for both trapping and excitation for Raman spectroscopy of the suspended microscopic particles. The design of the LTRS system provides high sensitivity and permits real-time spectroscopic measurements of the biological sample. The system was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different images and Raman spectra were observed for the different cells. The LTRS system may provide a valuable tool for the study of fundamental cellular processes and the diagnosis of cellular disorders.

Monitoring developmental force distributions in reconstituted embryonic epithelia.

PubMed

Przybyla, L; Lakins, J N; Sunyer, R; Trepat, X; Weaver, V M

2016-02-01

The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization. Copyright © 2015 Elsevier Inc. All rights reserved.

A Simple Force-Motion Relation for Migrating Cells Revealed by Multipole Analysis of Traction Stress

PubMed Central

Tanimoto, Hirokazu; Sano, Masaki

2014-01-01

For biophysical understanding of cell motility, the relationship between mechanical force and cell migration must be uncovered, but it remains elusive. Since cells migrate at small scale in dissipative circumstances, the inertia force is negligible and all forces should cancel out. This implies that one must quantify the spatial pattern of the force instead of just the summation to elucidate the force-motion relation. Here, we introduced multipole analysis to quantify the traction stress dynamics of migrating cells. We measured the traction stress of Dictyostelium discoideum cells and investigated the lowest two moments, the force dipole and quadrupole moments, which reflect rotational and front-rear asymmetries of the stress field. We derived a simple force-motion relation in which cells migrate along the force dipole axis with a direction determined by the force quadrupole. Furthermore, as a complementary approach, we also investigated fine structures in the stress field that show front-rear asymmetric kinetics consistent with the multipole analysis. The tight force-motion relation enables us to predict cell migration only from the traction stress patterns. PMID:24411233

Improving Osteoblast Response In Vitro by a Nanostructured Thin Film with Titanium Carbide and Titanium Oxides Clustered around Graphitic Carbon

PubMed Central

Longo, Giovanni; Ioannidu, Caterina Alexandra; Scotto d’Abusco, Anna; Superti, Fabiana; Misiano, Carlo; Zanoni, Robertino; Politi, Laura; Mazzola, Luca; Iosi, Francesca; Mura, Francesco; Scandurra, Roberto

2016-01-01

Introduction Recently, we introduced a new deposition method, based on Ion Plating Plasma Assisted technology, to coat titanium implants with a thin but hard nanostructured layer composed of titanium carbide and titanium oxides, clustered around graphitic carbon. The nanostructured layer has a double effect: protects the bulk titanium against the harsh conditions of biological tissues and in the same time has a stimulating action on osteoblasts. Results The aim of this work is to describe the biological effects of this layer on osteoblasts cultured in vitro. We demonstrate that the nanostructured layer causes an overexpression of many early genes correlated to proteins involved in bone turnover and an increase in the number of surface receptors for α3β1 integrin, talin, paxillin. Analyses at single-cell level, by scanning electron microscopy, atomic force microscopy, and single cell force spectroscopy, show how the proliferation, adhesion and spreading of cells cultured on coated titanium samples are higher than on uncoated titanium ones. Finally, the chemistry of the layer induces a better formation of blood clots and a higher number of adhered platelets, compared to the uncoated cases, and these are useful features to improve the speed of implant osseointegration. Conclusion In summary, the nanostructured TiC film, due to its physical and chemical properties, can be used to protect the implants and to improve their acceptance by the bone. PMID:27031101

Low-energy vibrations of the group 10 metal monocarbonyl MCO (M = Ni, Pd, and Pt): rotational spectroscopy and force field analysis.

PubMed

Okabayashi, Toshiaki; Yamamoto, Takuya; Okabayashi, Emi Y; Tanimoto, Mitsutoshi

2011-03-17

The rotational spectra of NiCO and PdCO in the ground and ν(2) excited vibrational states were observed by employing a source-modulated microwave spectrometer. The NiCO and PdCO molecules were generated in a free space cell by the sputtering reaction of nickel and palladium sheets, respectively, lining the inner surface of a stainless steel cathode with a dc glow plasma of CO and Ar. The molecular constants of NiCO and PdCO were determined by least-squares analysis. By force field analysis for the molecular constants of not only NiCO and PdCO but also of PtCO as previously reported, the harmonic force constants were determined for these three group 10 metal monocarbonyls. The vibrational wavenumbers derived for the lower M-C stretching vibrations were in good agreement with those obtained from the IR spectra in noble gas matrices and those predicted by several quantum chemical calculations published in the past. The bending vibrational wavenumbers derived by the force field analysis were also consistent with most quantum chemical calculations previously reported, but showed systematic discrepancies from the matrix IR values by about 40 cm(-1), even after reassignment (ν(2) band → 2ν(2) band) of the matrix IR spectra of PdCO and PtCO.

Submicrometer fiber-optic chemical sensors: Measuring pH inside single cells

NASA Astrophysics Data System (ADS)

Kopelman, R.

Starting from scratch, we went in two and a half years to 0.04 micron optical microscopy resolution. We have demonstrated the application of near-field scanning optical microscopy to DNA samples and opened the new fields of near-field scanning spectroscopy and submicron opto-chemical sensors. All of these developments have been important steps towards in-situ DNA imaging and characterization on the nanoscale. Our first goal was to make NSOM (near-field scanning optical microscopy) a working enterprise, capable of 'zooming-in' towards a sample and imaging with a resolution exceeding that of traditional microscopy by a factor of ten. This has been achieved. Not only do we have a resolution of about 40 nm but we can image a 1 x 1 micron object in less than 10 seconds. Furthermore, the NSOM is a practical instrument. The tips survive for days or weeks of scanning and new methods of force feedback will soon protect the most fragile samples. Reproducible images of metal gratings, gold particles, dye balls (for calibration) and of several DNA samples have been made, proving the practicality of our approach. We also give highly resolved Force/NSOM images of human blood cells. Our second goal has been to form molecular optics (e.g., exciton donor) tips with a resolution of 2-10 nm for molecular excitation microscopy (MEM). We have produced such tips, and scanned with them, but only with a resolution comparable to that of our standard NSOM tips. However, we have demonstrated their potential for high resolution imaging capabilities: (1) An energy transfer (tip to sample) based feedback capability. (2) A Kasha (external heavy atom) effect based feedback. In addition, a novel and practical opto-chemical sensor that is a billion times smaller than the best ones available has been developed as well. Finally, we have also performed spatially resolved fluorescence spectroscopy.

Nanocomposite of polystyrene foil grafted with metallaboranes for antimicrobial activity

NASA Astrophysics Data System (ADS)

Benkocká, Monika; Kolářová, Kateřina; Matoušek, Jindřich; Semerádtová, Alena; Šícha, Václav; Kolská, Zdeňka

2018-05-01

The surface of polystyrene foil (PS) was chemically modified. Firstly, the surface was pre-treated with Piranha solution. The activated surface was grafted by selected amino-compounds (cysteamine, ethylenediamine or chitosan) and/or subsequently grafted with five members of inorganic metallaboranes. Selected surface properties were studied by using various methods in order to indicate significant changes before and after individual modification steps of polymer foil. Elemental composition of surface was conducted by using X-ray photoelectron spectroscopy, chemistry and polarity by infrared spectroscopy and by electrokinetic analysis, wettability by goniometry, surface morphology by atomic force microscopy. Antimicrobial tests were performed on individual samples in order to confirm antimicrobial impact. Our results show slight antibacterial activity of PS modified with SK5 for Escherichia coli in comparison with the rest of the tested borane. On the other hand molecules of all tested metallaboranes could easier pierce through bacterial cell of Staphylococcus epidermidis due to absence of outer membrane (phospholipid bilayer). Some borane grafted on PS surface embodies the strong activity for Staphylococcus epidermidis and also for Desmodesmus quadricauda growth inhibition.

Photo-thermal and cytotoxic properties of inkjet-printed copper sulfide films on biocompatible latex coated substrates

NASA Astrophysics Data System (ADS)

Sarfraz, Jawad; Borzenkov, Mykola; Niemelä, Erik; Weinberger, Christian; Törngren, Björn; Rosqvist, Emil; Collini, Maddalena; Pallavicini, Piersandro; Eriksson, John; Peltonen, Jouko; Ihalainen, Petri; Chirico, Giuseppe

2018-03-01

Inkjet-printing of metal nanoparticles is a particularly promising technique for the fabrication and modification of surfaces with a multifunctional nature. Recently copper sulfide nanoparticles (CuS NPs) have attracted wide interest due to a range of valuable properties including long term stability, photo-thermal activity, ease of synthesis and low cost. In the present study, printed CuS patterns were successfully fabricated on latex coated paper substrates and characterized by means of atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), UV-Vis-NIR spectroscopy, and grazing incidence X-ray diffraction (GID). The resulted patterns displayed pronounced photo-thermal effect under Near Infrared Irradiation (NIR) even with relatively low laser power. Finally, by utilizing an automated real-time imaging platform it was possible to verify that the CuS printed film was not cytotoxic to human dermal fibroblast cells (HDF). The pronounced photo-thermal properties and nontoxic nature of these printed low-cost flexible CuS films make them promising candidates for fabrication of devices with localized photo-thermal effect suitable for biomedical applications.

Bark extract mediated green synthesis of silver nanoparticles: Evaluation of antimicrobial activity and antiproliferative response against osteosarcoma.

PubMed

Nayak, Debasis; Ashe, Sarbani; Rauta, Pradipta Ranjan; Kumari, Manisha; Nayak, Bismita

2016-01-01

In the current investigation we report the biosynthesis potentials of bark extracts of Ficus benghalensis and Azadirachta indica for production of silver nanoparticle without use of any external reducing or capping agent. The appearance of dark brown color indicated the complete nanoparticle synthesis which was further validated by absorbance peak by UV-vis spectroscopy. The morphology of the synthesized particles was characterized by Field emission- scanning electron microscopy (Fe-SEM) and atomic force microscopy (AFM). The X-ray diffraction (XRD) patterns clearly illustrated the crystalline phase of the synthesized nanoparticles. ATR-Fourier Transform Infrared (ATR-FTIR) spectroscopy was performed to identify the role of various functional groups in the nanoparticle synthesis. The synthesized nanoparticles showed promising antimicrobial activity against Gram negative (Escherichia coli, Pseudomonas aeruginosa and Vibrio cholerae) and Gram positive (Bacillus subtilis) bacteria. The synthesized nano Ag also showed antiproliferative activity against MG-63 osteosarcoma cell line in a dose dependent manner. Thus, these synthesized Ag nanoparticles can be used as a broad spectrum therapeutic agent against osteosarcoma and microorganisms. Copyright © 2015 Elsevier B.V. All rights reserved.

Bulk heterojunction morphology of polymer:fullerene blends revealed by ultrafast spectroscopy

PubMed Central

Serbenta, Almis; Kozlov, Oleg V.; Portale, Giuseppe; van Loosdrecht, Paul H. M.; Pshenichnikov, Maxim S.

2016-01-01

Morphology of organic photovoltaic bulk heterojunctions (BHJs) – a nanoscale texture of the donor and acceptor phases – is one of the key factors influencing efficiency of organic solar cells. Detailed knowledge of the morphology is hampered by the fact that it is notoriously difficult to investigate by microscopic methods. Here we all-optically track the exciton harvesting dynamics in the fullerene acceptor phase from which subdivision of the fullerene domain sizes into the mixed phase (2–15 nm) and large (>50 nm) domains is readily obtained via the Monte-Carlo simulations. These results were independently confirmed by a combination of X-ray scattering, electron and atomic-force microscopies, and time-resolved photoluminescence spectroscopy. In the large domains, the excitons are lost due to the high energy disorder while in the ordered materials the excitons are harvested with high efficiency even from the domains as large as 100 nm due to the absence of low-energy traps. Therefore, optimizing of blend nanomorphology together with increasing the material order are deemed as winning strategies in the exciton harvesting optimization. PMID:27824085

Block copolymer lithography of rhodium nanoparticles for high temperature electrocatalysis.

PubMed

Boyd, David A; Hao, Yong; Li, Changyi; Goodwin, David G; Haile, Sossina M

2013-06-25

We present a method for forming ordered rhodium nanostructures on a solid support. The approach makes use of a block copolymer to create and assemble rhodium chloride nanoparticles from solution onto a surface; subsequent plasma and thermal processing are employed to remove the polymer and fully convert the nanostructures to metallic rhodium. Films cast from a solution of the triblock copolymer poly(styrene-b-2-vinyl pyridine-b-ethylene oxide) dissolved in toluene with rhodium(III) chloride hydrate were capable of producing a monolayer of rhodium nanoparticles of uniform size and interparticle spacing. The nanostructures were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and atomic force microscopy. The electrocatalytic performance of the nanoparticles was investigated with AC impedance spectroscopy. We observed that the addition of the particles to a model solid oxide fuel cell anode provided up to a 14-fold improvement in the anode activity as evidenced by a decrease in the AC impedance resistance. Examination of the anode after electrochemical measurement revealed that the basic morphology and distribution of the particles were preserved.

Modulated Raman Spectroscopy for Enhanced Cancer Diagnosis at the Cellular Level

PubMed Central

De Luca, Anna Chiara; Dholakia, Kishan; Mazilu, Michael

2015-01-01

Raman spectroscopy is emerging as a promising and novel biophotonics tool for non-invasive, real-time diagnosis of tissue and cell abnormalities. However, the presence of a strong fluorescence background is a key issue that can detract from the use of Raman spectroscopy in routine clinical care. The review summarizes the state-of-the-art methods to remove the fluorescence background and explores recent achievements to address this issue obtained with modulated Raman spectroscopy. This innovative approach can be used to extract the Raman spectral component from the fluorescence background and improve the quality of the Raman signal. We describe the potential of modulated Raman spectroscopy as a rapid, inexpensive and accurate clinical tool to detect the presence of bladder cancer cells. Finally, in a broader context, we show how this approach can greatly enhance the sensitivity of integrated Raman spectroscopy and microfluidic systems, opening new prospects for portable higher throughput Raman cell sorting. PMID:26110401

Osteoconductive Potential of Barrier NanoSiO2 PLGA Membranes Functionalized by Plasma Enhanced Chemical Vapour Deposition

PubMed Central

Terriza, Antonia; Vilches-Pérez, Jose I.; de la Orden, Emilio; Yubero, Francisco; Gonzalez-Caballero, Juan L.; González-Elipe, Agustin R.; Vilches, José; Salido, Mercedes

2014-01-01

The possibility of tailoring membrane surfaces with osteoconductive potential, in particular in biodegradable devices, to create modified biomaterials that stimulate osteoblast response should make them more suitable for clinical use, hopefully enhancing bone regeneration. Bioactive inorganic materials, such as silica, have been suggested to improve the bioactivity of synthetic biopolymers. An in vitro study on HOB human osteoblasts was performed to assess biocompatibility and bioactivity of SiO2 functionalized poly(lactide-co-glycolide) (PLGA) membranes, prior to clinical use. A 15 nm SiO2 layer was deposited by plasma enhanced chemical vapour deposition (PECVD), onto a resorbable PLGA membrane. Samples were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, scanning electron microscopy, and infrared spectroscopy (FT-IR). HOB cells were seeded on sterilized test surfaces where cell morphology, spreading, actin cytoskeletal organization, and focal adhesion expression were assessed. As proved by the FT-IR analysis of samples, the deposition by PECVD of the SiO2 onto the PLGA membrane did not alter the composition and other characteristics of the organic membrane. A temporal and spatial reorganization of cytoskeleton and focal adhesions and morphological changes in response to SiO2 nanolayer were identified in our model. The novedous SiO2 deposition method is compatible with the standard sterilization protocols and reveals as a valuable tool to increase bioactivity of resorbable PLGA membranes. PMID:24883304

Inhibition of Alzheimer’s Amyloid Toxicity with a Tricyclic Pyrone Molecule In Vitro and In Vivo

PubMed Central

Hong, Hyun-Seok; Rana, Sandeep; Barrigan, Lydia; Shi, Aibin; Zhang, Yi; Zhou, Feimeng; Jin, Lee-Way; Hua, Duy H.

2009-01-01

Small amyloid β 1–42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer’s disease (AD). Methods to reduce the level of Aβ, prevent Aβ aggregation, and eliminate existing Aβ aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Aβ oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Aβ42 oligomer. Circular dichroism spectroscopy reveals monomeric Aβ42 peptide remains as a random coil/α-helix structure in the presence of CP2 over 48 h. Atomic force microscopy (AFM) studies show CP2 exhibits similar ability to inhibit Aβ42 aggregation as that of Congo Red and curcumin. AFM closed-fluid cell study demonstrates that CP2 disaggregates Aβ42 oligomers and protofibrils. CP2 also blocks Aβ fibrillations using a protein quantification method. Treatment of 5x FAD mice, a robust Aβ42-producing animal model of AD, with a two-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Aβ species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Aβ aggregation and disaggregating existing Aβ oligomers and protofibrils. PMID:19141069

Atomic force microscope with combined FTIR-Raman spectroscopy having a micro thermal analyzer

DOEpatents

Fink, Samuel D [Aiken, SC; Fondeur, Fernando F [North Augusta, SC

2011-10-18

An atomic force microscope is provided that includes a micro thermal analyzer with a tip. The micro thermal analyzer is configured for obtaining topographical data from a sample. A raman spectrometer is included and is configured for use in obtaining chemical data from the sample.

Single cell manipulation utilizing femtosecond laser-induced shock and stress waves

NASA Astrophysics Data System (ADS)

Hosokawa, Yoichiroh

2017-02-01

When an intense femtosecond laser pulse is focused into a culture medium through an objective lens, an impulsive force is loaded on the cells with generations of the shock and stress waves at the laser focal point. The shock and stress waves were acted to single cells in the vicinity of the laser focal point as an impulsive force. We have applied the impulsive force to manipulate single cells. As the transient intensity of the impulsive force is over 1000 times stronger than the force due to optical tweezers, drastic single manipulation which is difficult by the optical tweezers can be realized. The generation process of the impulsive force and behavior of animal cell after loading the impulsive force were reviewed, and then our original quantification method of the impulsive force utilizing atomic force microscope (AFM) was introduced with its applications for evaluating adhesions between animal cells and between sub-organelles in plant cell.

Differences in Physical and Biochemical Properties of Thermus scotoductus SA-01 Cultured with Dielectric or Convection Heating.

PubMed

Cockrell, Allison L; Fitzgerald, Lisa A; Cusick, Kathleen D; Barlow, Daniel E; Tsoi, Stanislav D; Soto, Carissa M; Baldwin, Jeffrey W; Dale, Jason R; Morris, Robert E; Little, Brenda J; Biffinger, Justin C

2015-09-01

A thermophile, Thermus scotoductus SA-01, was cultured within a constant-temperature (65°C) microwave (MW) digester to determine if MW-specific effects influenced the growth and physiology of the organism. As a control, T. scotoductus cells were also cultured using convection heating at the same temperature as the MW studies. Cell growth was analyzed by optical density (OD) measurements, and cell morphologies were characterized using electron microscopy imaging (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]), dynamic light scattering (DLS), and atomic force microscopy (AFM). Biophysical properties (i.e., turgor pressure) were also calculated with AFM, and biochemical compositions (i.e., proteins, nucleic acids, fatty acids) were analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the fatty acid methyl esters extracted from cell membranes. Here we report successful cultivation of a thermophile with only dielectric heating. Under the MW conditions for growth, cell walls remained intact and there were no indications of membrane damage or cell leakage. Results from these studies also demonstrated that T. scotoductus cells grown with MW heating exhibited accelerated growth rates in addition to altered cell morphologies and biochemical compositions compared with oven-grown cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Tension Monitoring during Epithelial-to-Mesenchymal Transition Links the Switch of Phenotype to Expression of Moesin and Cadherins in NMuMG Cells

PubMed Central

Schneider, David; Baronsky, Thilo; Pietuch, Anna; Rother, Jan; Oelkers, Marieelen; Fichtner, Dagmar; Wedlich, Doris; Janshoff, Andreas

2013-01-01

Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT. PMID:24339870

The substrate matters in the Raman spectroscopy analysis of cells

PubMed Central

Mikoliunaite, Lina; Rodriguez, Raul D.; Sheremet, Evgeniya; Kolchuzhin, Vladimir; Mehner, Jan; Ramanavicius, Arunas; Zahn, Dietrich R.T.

2015-01-01

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research. PMID:26310910

The substrate matters in the Raman spectroscopy analysis of cells

NASA Astrophysics Data System (ADS)

Mikoliunaite, Lina; Rodriguez, Raul D.; Sheremet, Evgeniya; Kolchuzhin, Vladimir; Mehner, Jan; Ramanavicius, Arunas; Zahn, Dietrich R. T.

2015-08-01

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.

Development of silver nanoparticles loaded chitosan-alginate constructs with biomedical potentialities.

PubMed

Bilal, Muhammad; Rasheed, Tahir; Iqbal, Hafiz M N; Li, Chuanlong; Hu, Hongbo; Zhang, Xuehong

2017-12-01

Herein, a facile biosynthesis of silver nanoparticles (AgNPs) and AgNPs-loaded chitosan-alginate constructs with biomedical potentialities is reported. The UV-vis spectroscopic profile confirmed the synthesis of AgNPs using methanolic leaves extract of Euphorbia helioscopia. The newly developed AgNPs were characterized using various analytical and imaging techniques including UV-vis and FT-IR spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). The optimally yielded AgNPs at 24h reaction period were loaded onto various chitosan-alginate constructs. A maximum of 95% loading efficiency (LE) was recorded with a chitosan: alginate ratio at 2:1, followed by 81% at 2:2 ratios. The anti-bacterial activities of AgNPs and AgNPs loaded chitosan-alginate constructs were tested against six bacterial strains i.e. Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Morganella morganii and Haemophilus influenza. A significant reduction in the log values was recorded for all test constructs, in comparison to the initial bacterial count (control value, i.e., 1.5×10 8 CFU/mL). The cytotoxicity profile revealed complete biocompatibility against normal cell line i.e. L929. Almost all constructs showed considerable cytotoxicity up to certain extant against human epithelial cells (HeLa) cancer cells. In summary, the highest antibacterial activities along with anti-cancer behavior both suggest the biomedical potentialities of newly engineered AgNPs and AgNPs-loaded chitosan-alginate constructs. Copyright © 2017 Elsevier B.V. All rights reserved.

Titanium dioxide nanotubes functionalized with Cratylia mollis seed lectin, Cramoll, enhanced osteoblast-like cells adhesion and proliferation.

PubMed

Oliveira, Weslley F; Silva, Germana M M; Cabral Filho, Paulo E; Fontes, Adriana; Oliveira, Maria D L; Andrade, César A S; Silva, Márcia V; Coelho, Luana C B B; Machado, Giovanna; Correia, Maria T S

2018-09-01

An alternative to accelerate the osseointegration on titanium dioxide nanotubes (TNTs) used in osseointegrated implants is through the functionalization of these nanostructured surfaces with biomolecules. In this work, we immobilized a lectin with recognized mitogenic activity, the Cramoll lectin, extracted from Cratylia mollis seeds, on surfaces modified by TNTs. For the immobilization of Cramoll on TNTs surfaces, we used the Layer-by-Layer technique (LbL) by growing five alternate layers of poly(allylamine) hydrochloride (PAH) and poly(acrylic) acid (PAA); lastly we incubated the lectin, at different concentrations, with the TNTs-LbL. Before and after the immobilization procedures, the substrate surfaces were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and, electrochemical impedance spectroscopy (EIS). We also evaluated the Cramoll activity after immobilization on TNTs by using the lectin interaction with ovalbumin. The lectin did not lose its biological activity, even after immobilization onto nanotubular arrays. In addition, we observed an increase osteoblast-like cell adhesion on the TNTs-LbL-Cramoll system when compared to the bare TNTs surfaces. Moreover, a significative cell proliferation was identified on the substrates when Cramoll was immobilized at concentrations of 80, 160 and 320 μg/mL after 48 h of incubation by using the resazurin assay. Our results suggest that Cramoll was efficiently immobilized on a nanotubular array and this new platform presents a great potential to be tested in implantology. Copyright © 2018 Elsevier B.V. All rights reserved.

Force Dynamics During T Cell Activation

NASA Astrophysics Data System (ADS)

Garcia, David A.; Upadhyaya, Arpita

T cell activation is an essential step in the adaptive immune response. The binding of the T cell receptor (TCR) with antigen triggers signaling cascades and cell spreading. Physical forces exerted on the TCR by the cytoskeleton have been shown to induce signaling events. While cellular forces are known to depend on the mechanical properties of the cytoskeleton, the biophysical mechanisms underlying force induced activation of TCR-antigen interactions unknown. Here, we use traction force microscopy to measure the force dynamics of activated Jurkat T cells. The movements of beads embedded in an elastic gel serve as a non-invasive reporter of cytoskeletal and molecular motor dynamics. We examined the statistical structure of the force profiles throughout the cell during signaling activation. We found two spatially distinct active regimes of force generation characterized by different time scales. Typically, the interior of the cells was found to be more active than the periphery. Inhibition of myosin motor activity altered the correlation time of the bead displacements indicating additional sources of stochastic force generation. Our results indicate a complex interaction between myosin activity and actin polymerization dynamics in producing cellular forces in immune cells.

Mechanical control of mitotic progression in single animal cells

PubMed Central

Cattin, Cedric J.; Düggelin, Marcel; Martinez-Martin, David; Gerber, Christoph; Müller, Daniel J.; Stewart, Martin P.

2015-01-01

Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback–controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces where cells resist confinement (50–100 nN), and yield forces (>100 nN) where a significant decline in cell height impinges on microtubule spindle function, thereby inhibiting mitotic progression. Yield forces are coincident with a nonlinear drop in cell height potentiated by persistent blebbing and loss of cortical F-actin homogeneity. Our results suggest that a buildup of actomyosin-dependent cortical tension and intracellular pressure precedes mechanical failure, or herniation, of the cell cortex at the yield force. Thus, we reveal how the mechanical properties of mitotic cells and their response to external forces are linked to mitotic progression under conditions of mechanical confinement. PMID:26305930

Probing static disorder in Arrhenius kinetics by single-molecule force spectroscopy.

PubMed

Kuo, Tzu-Ling; Garcia-Manyes, Sergi; Li, Jingyuan; Barel, Itay; Lu, Hui; Berne, Bruce J; Urbakh, Michael; Klafter, Joseph; Fernández, Julio M

2010-06-22

The widely used Arrhenius equation describes the kinetics of simple two-state reactions, with the implicit assumption of a single transition state with a well-defined activation energy barrier DeltaE, as the rate-limiting step. However, it has become increasingly clear that the saddle point of the free-energy surface in most reactions is populated by ensembles of conformations, leading to nonexponential kinetics. Here we present a theory that generalizes the Arrhenius equation to include static disorder of conformational degrees of freedom as a function of an external perturbation to fully account for a diverse set of transition states. The effect of a perturbation on static disorder is best examined at the single-molecule level. Here we use force-clamp spectroscopy to study the nonexponential kinetics of single ubiquitin proteins unfolding under force. We find that the measured variance in DeltaE shows both force-dependent and independent components, where the force-dependent component scales with F(2), in excellent agreement with our theory. Our study illustrates a novel adaptation of the classical Arrhenius equation that accounts for the microscopic origins of nonexponential kinetics, which are essential in understanding the rapidly growing body of single-molecule data.

High-throughput single-molecule force spectroscopy for membrane proteins

NASA Astrophysics Data System (ADS)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

Molecular energy dissipation in nanoscale networks of dentin matrix protein 1 is strongly dependent on ion valence

NASA Astrophysics Data System (ADS)

Adams, J.; Fantner, G. E.; Fisher, L. W.; Hansma, P. K.

2008-09-01

The fracture resistance of biomineralized tissues such as bone, dentin, and abalone is greatly enhanced through the nanoscale interactions of stiff inorganic mineral components with soft organic adhesive components. A proper understanding of the interactions that occur within the organic component, and between the organic and inorganic components, is therefore critical for a complete understanding of the mechanics of these tissues. In this paper, we use atomic force microscope (AFM) force spectroscopy and dynamic force spectroscopy to explore the effect of ionic interactions within a nanoscale system consisting of networks of dentin matrix protein 1 (DMP1) (a component of both bone and dentin organic matrix), a mica surface and an AFM tip. We find that DMP1 is capable of dissipating large amounts of energy through an ion-mediated mechanism, and that the effectiveness increases with increasing ion valence.

Vibration-rotation-tunneling spectroscopy of the van der Waals Bond: A new look at intermolecular forces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cohen, R.C.; Saykally, R.J.

Measurements of the low-frequency van der Waals vibrations in weakly bound complexes by high-resolution laser spectroscopy provide a means to probe intermolecular forces at unprecedented levels of detail and precision. Several new methods are presently being used to record vibration/rotation-tunneling (VRT) transitions associated with the motions of the weak bonds in van der Waals clusters. The most direct measurements are those probing only the van der Waals modes themselves, which occur at far-infrared wavelengths. This article presents a review of the information on both intramolecular forces and intramolecular dynamics that has been obtained from far-infrared VRT spectra of 18 complexesmore » during the past several years. Some rotationally resolved measurements of van der Waals modes observed in combination with electronic or vibrational excitation are also discussed. 185 refs., 15 figs., 1 tab.« less

Solvation of fluoro-acetonitrile in water by 2D-IR spectroscopy: A combined experimental-computational study

NASA Astrophysics Data System (ADS)

Cazade, Pierre-André; Tran, Halina; Bereau, Tristan; Das, Akshaya K.; Kläsi, Felix; Hamm, Peter; Meuwly, Markus

2015-06-01

The solvent dynamics around fluorinated acetonitrile is characterized by 2-dimensional infrared spectroscopy and atomistic simulations. The lineshape of the linear infrared spectrum is better captured by semiempirical (density functional tight binding) mixed quantum mechanical/molecular mechanics simulations, whereas force field simulations with multipolar interactions yield lineshapes that are significantly too narrow. For the solvent dynamics, a relatively slow time scale of 2 ps is found from the experiments and supported by the mixed quantum mechanical/molecular mechanics simulations. With multipolar force fields fitted to the available thermodynamical data, the time scale is considerably faster—on the 0.5 ps time scale. The simulations provide evidence for a well established CF-HOH hydrogen bond (population of 25%) which is found from the radial distribution function g(r) from both, force field and quantum mechanics/molecular mechanics simulations.

Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

2015-05-01

Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

Matrix Effects in Quantitative Assessment of Pharmaceutical Tablets Using Transmission Raman and Near-Infrared (NIR) Spectroscopy.

PubMed

Sparén, Anders; Hartman, Madeleine; Fransson, Magnus; Johansson, Jonas; Svensson, Olof

2015-05-01

Raman spectroscopy can be an alternative to near-infrared spectroscopy (NIR) for nondestructive quantitative analysis of solid pharmaceutical formulations. Compared with NIR spectra, Raman spectra have much better selectivity, but subsampling was always an issue for quantitative assessment. Raman spectroscopy in transmission mode has reduced this issue, since a large volume of the sample is measured in transmission mode. The sample matrix, such as particle size of the drug substance in a tablet, may affect the Raman signal. In this work, matrix effects in transmission NIR and Raman spectroscopy were systematically investigated for a solid pharmaceutical formulation. Tablets were manufactured according to an experimental design, varying the factors particle size of the drug substance (DS), particle size of the filler, compression force, and content of drug substance. All factors were varied at two levels plus a center point, except the drug substance content, which was varied at five levels. Six tablets from each experimental point were measured with transmission NIR and Raman spectroscopy, and their concentration of DS was determined for a third of those tablets. Principal component analysis of NIR and Raman spectra showed that the drug substance content and particle size, the particle size of the filler, and the compression force affected both NIR and Raman spectra. For quantitative assessment, orthogonal partial least squares regression was applied. All factors varied in the experimental design influenced the prediction of the DS content to some extent, both for NIR and Raman spectroscopy, the particle size of the filler having the largest effect. When all matrix variations were included in the multivariate calibrations, however, good predictions of all types of tablets were obtained, both for NIR and Raman spectroscopy. The prediction error using transmission Raman spectroscopy was about 30% lower than that obtained with transmission NIR spectroscopy.

A simple force-motion relation for migrating cells revealed by multipole analysis of traction stress.

PubMed

Tanimoto, Hirokazu; Sano, Masaki

2014-01-07

For biophysical understanding of cell motility, the relationship between mechanical force and cell migration must be uncovered, but it remains elusive. Since cells migrate at small scale in dissipative circumstances, the inertia force is negligible and all forces should cancel out. This implies that one must quantify the spatial pattern of the force instead of just the summation to elucidate the force-motion relation. Here, we introduced multipole analysis to quantify the traction stress dynamics of migrating cells. We measured the traction stress of Dictyostelium discoideum cells and investigated the lowest two moments, the force dipole and quadrupole moments, which reflect rotational and front-rear asymmetries of the stress field. We derived a simple force-motion relation in which cells migrate along the force dipole axis with a direction determined by the force quadrupole. Furthermore, as a complementary approach, we also investigated fine structures in the stress field that show front-rear asymmetric kinetics consistent with the multipole analysis. The tight force-motion relation enables us to predict cell migration only from the traction stress patterns. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single cell active force generation under dynamic loading - Part I: AFM experiments.

PubMed

Weafer, P P; Reynolds, N H; Jarvis, S P; McGarry, J P

2015-11-01

A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Measured forces for the untreated cells are dramatically different to cytochalasin-D (cyto-D) treated cells, indicating that the contractile actin cytoskeleton plays a critical role in the response of cells to dynamic loading. Following a change in applied strain magnitude, while maintaining a constant applied strain rate, the compression force for contractile cells recovers to 88.9±7.8% of the steady state force. In contrast, cyto-D cell compression forces recover to only 38.0±6.7% of the steady state force. Additionally, untreated cells exhibit strongly negative (pulling) forces during unloading half-cycles when the probe is retracted. In comparison, negligible pulling forces are measured for cyto-D cells during probe retraction. The current study demonstrates that active contractile forces, generated by actin-myosin cross-bridge cycling, dominate the response of single cells to dynamic loading. Such active force generation is shown to be independent of applied strain magnitude. Passive forces generated by the applied deformation are shown to be of secondary importance, exhibiting a high dependence on applied strain magnitude, in contrast to the active forces in untreated cells. A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Contractile cells, which contain the active force generation machinery of the actin cytoskeleton, are shown to be insensitive to applied strain magnitude, exhibiting high resistance to dynamic compression and stretching. Such trends are not observed for cells in which the actin cytoskeleton has been chemically disrupted. These biomechanical insights have not been previously reported. This detailed characterisation of single cell active and passive stress during dynamic loading has important implications for tissue engineering strategies, where applied deformation has been reported to significantly affect cell mechanotransduction and matrix synthesis. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Raman spectroscopy for grading of live osteosarcoma cells.

PubMed

Chiang, Yi-Hung; Wu, Stewart H; Kuo, Yi-Chun; Chen, How-Foo; Chiou, Arthur; Lee, Oscar K

2015-04-18

Osteosarcoma is the most common primary malignant bone tumor, and the grading of osteosarcoma cells relies on traditional histopathology and molecular biology methods, which require RNA extraction, protein isolation and immunohistological staining. All these methods require cell isolation, lysis or fixation, which is time-consuming and requires certain amount of tumor specimen. In this study, we report the use of Raman spectroscopy for grading of malignant osteosarcoma cells. We demonstrate that, based on the detection of differential production of mineral species, Raman spectroscopy can be used as a live cell analyzer to accurately assess the grades of osteosarcoma cells by evaluating their mineralization levels. Mineralization level was assessed by measuring amount of hydroxyapatite (HA), which is highly expressed in mature osteoblasts, but not in poorly differentiated osteosarcoma cell or mesenchymal stem cells, the putative cell-of-origin of osteosarcoma. We found that under Raman spectroscopy, the level of HA production was high in MG-63 cells, which are low-grade. Moreover, hydroxyapatite production was low in high-grade osteosarcoma cells such as 143B and SaOS2 cells (p < 0.05). Matrix metalloproteinase MMP2, MMP9 were highly expressed in SaOS2, 143B and MSCs and decreased in human fetal osteoblast (FOB) and MG-63 cells as expected (p < 0.05). These results may highlight the inverse correlation between HA level and prognosis of osteosarcoma. The use of Raman spectroscopy for the measurement of HA production by the protocol reported in this study may serve as a useful tool to rapidly and accurately assess the degree of malignancy in osteosarcoma cells in a label-free manner. Such application may shorten the period of pathological diagnosis and may benefit patients who are inflicted with osteosarcoma.

Maximum likelihood estimation of protein kinetic parameters under weak assumptions from unfolding force spectroscopy experiments

NASA Astrophysics Data System (ADS)

Aioanei, Daniel; Samorì, Bruno; Brucale, Marco

2009-12-01

Single molecule force spectroscopy (SMFS) is extensively used to characterize the mechanical unfolding behavior of individual protein domains under applied force by pulling chimeric polyproteins consisting of identical tandem repeats. Constant velocity unfolding SMFS data can be employed to reconstruct the protein unfolding energy landscape and kinetics. The methods applied so far require the specification of a single stretching force increase function, either theoretically derived or experimentally inferred, which must then be assumed to accurately describe the entirety of the experimental data. The very existence of a suitable optimal force model, even in the context of a single experimental data set, is still questioned. Herein, we propose a maximum likelihood (ML) framework for the estimation of protein kinetic parameters which can accommodate all the established theoretical force increase models. Our framework does not presuppose the existence of a single force characteristic function. Rather, it can be used with a heterogeneous set of functions, each describing the protein behavior in the stretching time range leading to one rupture event. We propose a simple way of constructing such a set of functions via piecewise linear approximation of the SMFS force vs time data and we prove the suitability of the approach both with synthetic data and experimentally. Additionally, when the spontaneous unfolding rate is the only unknown parameter, we find a correction factor that eliminates the bias of the ML estimator while also reducing its variance. Finally, we investigate which of several time-constrained experiment designs leads to better estimators.

Emerging technology: applications of Raman spectroscopy for prostate cancer.

PubMed

Kast, Rachel E; Tucker, Stephanie C; Killian, Kevin; Trexler, Micaela; Honn, Kenneth V; Auner, Gregory W

2014-09-01

There is a need in prostate cancer diagnostics and research for a label-free imaging methodology that is nondestructive, rapid, objective, and uninfluenced by water. Raman spectroscopy provides a molecular signature, which can be scaled from micron-level regions of interest in cells to macroscopic areas of tissue. It can be used for applications ranging from in vivo or in vitro diagnostics to basic science laboratory testing. This work describes the fundamentals of Raman spectroscopy and complementary techniques including surface enhanced Raman scattering, resonance Raman spectroscopy, coherent anti-Stokes Raman spectroscopy, confocal Raman spectroscopy, stimulated Raman scattering, and spatially offset Raman spectroscopy. Clinical applications of Raman spectroscopy to prostate cancer will be discussed, including screening, biopsy, margin assessment, and monitoring of treatment efficacy. Laboratory applications including cell identification, culture monitoring, therapeutics development, and live imaging of cellular processes are discussed. Potential future avenues of research are described, with emphasis on multiplexing Raman spectroscopy with other modalities.

Force transmission in epithelial tissues.

PubMed

Vasquez, Claudia G; Martin, Adam C

2016-03-01

In epithelial tissues, cells constantly generate and transmit forces between each other. Forces generated by the actomyosin cytoskeleton regulate tissue shape and structure and also provide signals that influence cells' decisions to divide, die, or differentiate. Forces are transmitted across epithelia because cells are mechanically linked through junctional complexes, and forces can propagate through the cell cytoplasm. Here, we review some of the molecular mechanisms responsible for force generation, with a specific focus on the actomyosin cortex and adherens junctions. We then discuss evidence for how these mechanisms promote cell shape changes and force transmission in tissues. © 2016 Wiley Periodicals, Inc.

Characterization of mechanical unfolding intermediates of membrane proteins by coarse grained molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Yamada, Tatsuya; Mitaku, Shigeki; Yamato, Takahisa

2018-01-01

Single-molecule force spectroscopy by atomic force microscopy allows us to get insight into the mechanical unfolding of membrane proteins, and a typical experiment exhibits characteristic patterns on the force distance curves. The origin of these patterns, however, has not been fully understood yet. We performed coarse-grained simulation of the forced unfolding of halorodopsin, reproduced the characteristic features of the experimental force distance curves. A further examination near the membrane-water interface indicated the existence of a motif for the force peak formation, i.e., the occurrence of hydrophobic residues in the upper interface region and hydrophilic residues below the lower interface region.

Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters

PubMed Central

Ng, Mei Rosa; Besser, Achim; Brugge, Joan S; Danuser, Gaudenz

2014-01-01

Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions. DOI: http://dx.doi.org/10.7554/eLife.03282.001 PMID:25479385

Dielectric spectroscopy platform to measure MCF10A epithelial cell aggregation as a model for spheroidal cell cluster analysis.

PubMed

Heileman, K L; Tabrizian, M

2017-05-02

3-Dimensional cell cultures are more representative of the native environment than traditional cell cultures on flat substrates. As a result, 3-dimensional cell cultures have emerged as a very valuable model environment to study tumorigenesis, organogenesis and tissue regeneration. Many of these models encompass the formation of cell aggregates, which mimic the architecture of tumor and organ tissue. Dielectric impedance spectroscopy is a non-invasive, label free and real time technique, overcoming the drawbacks of established techniques to monitor cell aggregates. Here we introduce a platform to monitor cell aggregation in a 3-dimensional extracellular matrix using dielectric spectroscopy. The MCF10A breast epithelial cell line serves as a model for cell aggregation. The platform maintains sterile conditions during the multi-day assay while allowing continuous dielectric spectroscopy measurements. The platform geometry optimizes dielectric measurements by concentrating cells within the electrode sensing region. The cells show a characteristic dielectric response to aggregation which corroborates with finite element analysis computer simulations. By fitting the experimental dielectric spectra to the Cole-Cole equation, we demonstrated that the dispersion intensity Δε and the characteristic frequency f c are related to cell aggregate growth. In addition, microscopy can be performed directly on the platform providing information about cell position, density and morphology. This platform could yield many applications for studying the electrophysiological activity of cell aggregates.

Raman spectroscopy in biomedicine – non-invasive in vitro analysis of cells and extracellular matrix components in tissues

PubMed Central

Brauchle, Eva; Schenke-Layland, Katja

2013-01-01

Raman spectroscopy is an established laser-based technology for the quality assurance of pharmaceutical products. Over the past few years, Raman spectroscopy has become a powerful diagnostic tool in the life sciences. Raman spectra allow assessment of the overall molecular constitution of biological samples, based on specific signals from proteins, nucleic acids, lipids, carbohydrates, and inorganic crystals. Measurements are non-invasive and do not require sample processing, making Raman spectroscopy a reliable and robust method with numerous applications in biomedicine. Moreover, Raman spectroscopy allows the highly sensitive discrimination of bacteria. Rama spectra retain information on continuous metabolic processes and kinetics such as lipid storage and recombinant protein production. Raman spectra are specific for each cell type and provide additional information on cell viability, differentiation status, and tumorigenicity. In tissues, Raman spectroscopy can detect major extracellular matrix components and their secondary structures. Furthermore, the non-invasive characterization of healthy and pathological tissues as well as quality control and process monitoring of in vitro-engineered matrix is possible. This review provides comprehensive insight to the current progress in expanding the applicability of Raman spectroscopy for the characterization of living cells and tissues, and serves as a good reference point for those starting in the field. PMID:23161832

Synthesis and characterization of designed BMHP1-derived self-assembling peptides for tissue engineering applications

NASA Astrophysics Data System (ADS)

Silva, Diego; Natalello, Antonino; Sanii, Babak; Vasita, Rajesh; Saracino, Gloria; Zuckermann, Ronald N.; Doglia, Silvia Maria; Gelain, Fabrizio

2012-12-01

The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π-π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces. BMHP1-derived SAPs is a class of biotinylated oligopeptides, which self-assemble in β-structured fibers to form a self-healing hydrogel. In the current study, selected modifications in previously described BMHP1-derived SAPs were designed in order to investigate the influence of modified residues on self-assembly kinetics and scaffold formation properties. The Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis demonstrated the secondary structure (β-sheet) formation in all modified SAP sequences, whereas atomic force microscopy (AFM) analysis further confirmed the presence of nanofibers. Furthermore, the fiber shape and dimension analysis by AFM showed flattened and twisted fiber morphology ranging from ~8 nm to ~70 nm. The mechanical properties of the pre-assembled and post assembled solution were investigated by rheometry. The shear-thinning behavior and rapid re-healing properties of the pre-assembled solutions make them a preferable choice for injectable scaffolds. The wide range of stiffnesses (G') -from ~1000 to ~27 000 Pa - exhibited by the post-assembled scaffolds demonstrated their potential for a variety of tissue engineering applications. The extra cellular matrix (ECM) mimicking (physically and chemically) properties of SAP scaffolds enhanced cell adhesion and proliferation. The capability of the scaffold to facilitate murine neural stem cell (mNSC) proliferation was evaluated in vitro: the increased mNSCs adhesion and proliferation demonstrated the potential of newly synthesized SAPs for regenerative medicine approaches.The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π-π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces. BMHP1-derived SAPs is a class of biotinylated oligopeptides, which self-assemble in β-structured fibers to form a self-healing hydrogel. In the current study, selected modifications in previously described BMHP1-derived SAPs were designed in order to investigate the influence of modified residues on self-assembly kinetics and scaffold formation properties. The Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis demonstrated the secondary structure (β-sheet) formation in all modified SAP sequences, whereas atomic force microscopy (AFM) analysis further confirmed the presence of nanofibers. Furthermore, the fiber shape and dimension analysis by AFM showed flattened and twisted fiber morphology ranging from ~8 nm to ~70 nm. The mechanical properties of the pre-assembled and post assembled solution were investigated by rheometry. The shear-thinning behavior and rapid re-healing properties of the pre-assembled solutions make them a preferable choice for injectable scaffolds. The wide range of stiffnesses (G') -from ~1000 to ~27 000 Pa - exhibited by the post-assembled scaffolds demonstrated their potential for a variety of tissue engineering applications. The extra cellular matrix (ECM) mimicking (physically and chemically) properties of SAP scaffolds enhanced cell adhesion and proliferation. The capability of the scaffold to facilitate murine neural stem cell (mNSC) proliferation was evaluated in vitro: the increased mNSCs adhesion and proliferation demonstrated the potential of newly synthesized SAPs for regenerative medicine approaches. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32656f

Green biosynthesis of silver nanoparticles using leaves extract of Artemisia vulgaris and their potential biomedical applications.

PubMed

Rasheed, Tahir; Bilal, Muhammad; Iqbal, Hafiz M N; Li, Chuanlong

2017-10-01

Biosynthesis of nanoparticles from plant extracts is receiving enormous interest due to their abundant availability and a broad spectrum of bioactive reducing metabolites. In this study, the reducing potential of Artemisia vulgaris leaves extract (AVLE) was investigated for synthesizing silver nanoparticles without the addition of any external reducing or capping agent. The appearance of blackish brown color evidenced the complete synthesis of nanoparticles. The synthesized silver nanoparticles were characterized by UV-vis spectroscopy, scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscope (TEM), atomic force microscopy (AFM) and Fourier transforms infrared spectroscopy (FT-IR) analysis. UV-vis absorption profile of the bio-reduced sample elucidated the main peak around 420nm, which correspond to the surface plasmon resonance of silver nanoparticles. SEM and AFM analyses confirmed the morphology of the synthesized nanoparticles. Similarly, particles with a distinctive peak of silver were examined with EDX. The average diameter of silver nanoparticles was about 25nm from Transmission Electron Microscopy (TEM). FTIR spectroscopy scrutinized the involvement of various functional groups during nanoparticle synthesis. The green synthesized nanoparticles presented effective antibacterial activity against pathogenic bacteria than AVLE alone. In-vitro antioxidant assays revealed that silver nanoparticles (AV-AgNPs) exhibited promising antioxidant properties. The nanoparticles also displayed a potent cytotoxic effect against HeLa and MCF-7 cell lines. In conclusion, the results supported the advantages of employing a bio-green approach for developing silver nanoparticles with antimicrobial, antioxidant, and antiproliferative activities in a simple and cost- competitive manner. Copyright © 2017 Elsevier B.V. All rights reserved.

Raman spectroscopy differentiates between sensitive and resistant multiple myeloma cell lines

NASA Astrophysics Data System (ADS)

Franco, Domenico; Trusso, Sebastiano; Fazio, Enza; Allegra, Alessandro; Musolino, Caterina; Speciale, Antonio; Cimino, Francesco; Saija, Antonella; Neri, Fortunato; Nicolò, Marco S.; Guglielmino, Salvatore P. P.

2017-12-01

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific and biologically perturbing. Here, we show that single-cell micro-Raman spectroscopy can be used to discriminate between resistant and sensitive multiple myeloma cell lines based on their highly reproducible biomolecular spectral signatures. In order to demonstrate robustness of the proposed approach, we used two different cell lines of multiple myeloma, namely MM.1S and U266B1, and their counterparts MM.1R and U266/BTZ-R subtypes, resistant to dexamethasone and bortezomib, respectively. Then, micro-Raman spectroscopy provides an easily accurate and noninvasive method for cancer detection for both research and clinical environments. Characteristic peaks, mostly due to different DNA/RNA ratio, nucleic acids, lipids and protein concentrations, allow for discerning the sensitive and resistant subtypes. We also explored principal component analysis (PCA) for resistant cell identification and classification. Sensitive and resistant cells form distinct clusters that can be defined using just two principal components. The identification of drug-resistant cells by confocal micro-Raman spectroscopy is thus proposed as a clinical tool to assess the development of resistance to glucocorticoids and proteasome inhibitors in myeloma cells.

Biofunctionalization of a “Clickable” Organic Layer Photochemically Grafted on Titanium Substrates

PubMed Central

Li, Yan; Zhao, Meirong; Wang, Jun; Liu, Kai; Cai, Chengzhi

2011-01-01

We have developed a general method combining photochemical grafting and copper-catalyzed click chemistry for biofunctionalization of titanium substrates. The UV-activated grafting of an α,ω-alkenyne onto TiO2/Ti substrates provided a “clickable” thin film platform. The selective attachment of the vinyl end of the molecule to the surface was achieved by masking the alkynyl end with a trimethylgermanyl (TMG) protecting group. Subsequently, various oligo(ethylene glycol) (OEG) derivatives terminated with an azido group were attached to the TMG-alkynyl modified titanium surface via a one-pot deprotection/click reaction. The films were characterized by X-ray photoelectron spectroscopy (XPS), contact angle goniometry, ellipsometry, and atomic force microscopy (AFM). We showed that the titanium surface presenting click-immobilized OEG substantially suppressed the nonspecific attachment of protein and cells as compared to the unmodified titanium substrate. Furthermore, glycine-arginine-glycine-aspartate (GRGD), a cell adhesion peptide, was coimmobilized with OEG on the platform. We demonstrated that the resultant GRGD-presenting thin film on Ti substrates can promote the specific adhesion and spreading of AsPC-1 cells. PMID:21417429

In Situ Generation of Two-Dimensional Au–Pt Core–Shell Nanoparticle Assemblies

PubMed Central

2010-01-01

Two-dimensional assemblies of Au–Pt bimetallic nanoparticles are generated in situ on polyethyleneimmine (PEI) silane functionalized silicon and indium tin oxide (ITO) coated glass surfaces. Atomic force microscopy (AFM), UV–Visible spectroscopy, and electrochemical measurements reveal the formation of core–shell structure with Au as core and Pt as shell. The core–shell structure is further supported by comparing with the corresponding data of Au nanoparticle assemblies. Static contact angle measurements with water show an increase in hydrophilic character due to bimetallic nanoparticle generation on different surfaces. It is further observed that these Au–Pt core–shell bimetallic nanoparticle assemblies are catalytically active towards methanol electro-oxidation, which is the key reaction for direct methanol fuel cells (DMFCs). PMID:20651923

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function

PubMed Central

Gahl, Trevor J.; Kunze, Anja

2018-01-01

Cellular processes like membrane deformation, cell migration, and transport of organelles are sensitive to mechanical forces. Technically, these cellular processes can be manipulated through operating forces at a spatial precision in the range of nanometers up to a few micrometers through chaperoning force-mediating nanoparticles in electrical, magnetic, or optical field gradients. But which force-mediating tool is more suitable to manipulate cell migration, and which, to manipulate cell signaling? We review here the differences in forces sensation to control and engineer cellular processes inside and outside the cell, with a special focus on neuronal cells. In addition, we discuss technical details and limitations of different force-mediating approaches and highlight recent advancements of nanomagnetics in cell organization, communication, signaling, and intracellular trafficking. Finally, we give suggestions about how force-mediating nanoparticles can be used to our advantage in next-generation neurotherapeutic devices. PMID:29867315

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function.

PubMed

Gahl, Trevor J; Kunze, Anja

2018-01-01

Cellular processes like membrane deformation, cell migration, and transport of organelles are sensitive to mechanical forces. Technically, these cellular processes can be manipulated through operating forces at a spatial precision in the range of nanometers up to a few micrometers through chaperoning force-mediating nanoparticles in electrical, magnetic, or optical field gradients. But which force-mediating tool is more suitable to manipulate cell migration, and which, to manipulate cell signaling? We review here the differences in forces sensation to control and engineer cellular processes inside and outside the cell, with a special focus on neuronal cells. In addition, we discuss technical details and limitations of different force-mediating approaches and highlight recent advancements of nanomagnetics in cell organization, communication, signaling, and intracellular trafficking. Finally, we give suggestions about how force-mediating nanoparticles can be used to our advantage in next-generation neurotherapeutic devices.

Metallodrug induced apoptotic cell death and survival attempts are characterizable by Raman spectroscopy

NASA Astrophysics Data System (ADS)

le Roux, K.; Prinsloo, L. C.; Meyer, D.

2014-09-01

Chrysotherapeutics are under investigation as new or additional treatments for different types of cancers. In this study, gold complexes were investigated for their anticancer potential using Raman spectroscopy. The aim of the study was to determine whether Raman spectroscopy could be used for the characterization of metallodrug-induced cell death. Symptoms of cell death such as decreased peak intensities of proteins bonds and phosphodiester bonds found in deoxyribose nucleic acids were evident in the principal component analysis of the spectra. Vibrational bands around 761 cm-1 and 1300 cm-1 (tryptophan, ethanolamine group, and phosphatidylethanolamine) and 1720 cm-1 (ester bonds associated with phospholipids) appeared in the Raman spectra of cervical adenocarcinoma (HeLa) cells after metallodrug treatment. The significantly (p < 0.05, one way analysis of variance) increased intensity of phosphatidylethanolamine after metallodrug treatment could be a molecular signature of induced apoptosis since both the co-regulated phosphatidylserine and phosphatidylethanolamine are externalized during cell death. Treated cells had significantly higher levels of glucose and glycogen vibrational peaks, indicative of a survival mechanism of cancer cells under chemical stress. Cancer cells excrete chemotherapeutics to improve their chances of survival and utilize glucose to achieve this. Raman spectroscopy was able to monitor a survival strategy of cancer cells in the form of glucose uptake to alleviate chemical stress. Raman spectroscopy was invaluable in obtaining molecular information generated by biomolecules affected by anticancer metallodrug treatments and presents an alternative to less reproducible, conventional biochemical assays for cytotoxicity analyses.

Milk caseins as useful vehicle for delivery of dipyridamole drug.

PubMed

Dezhampanah, Hamid; Esmaili, Masoomeh; Hasani, Leila

2018-05-01

The interaction of bovine milk α- and β-caseins as an efficient drug carrier system with Dipyridamole (DIP) was investigated using spectroscopy and molecular docking studies at different temperatures (20-37 °C). FTIR, CD, and fluorescence spectroscopy methods demonstrated that α- and β-caseins interact with DIP molecule mainly via hydrophobic and hydrophilic interactions and change in secondary structure of α- and β-caseins. DIP showed a higher quenching efficiency and binding constant of α-casein than β-casein. There was only one binding site for DIP and it was located on the surface of the protein molecule. The thermodynamic parameters of calculation showed that the binding process occurs spontaneously and demonstrated that α- and β-caseins provide very good binding and entrapment to DIP via hydrogen bonds, Van der Waals forces, and hydrophobic interactions. Fluorescence resonance energy transfer, synchronous fluorescence spectroscopy, and docking study showed that DIP binds to the Trp residues of α- and β-casein molecules with short distances. Docking study showed that DIP molecule made several hydrogen bonds and van der Waals interactions with α- and β-caseins. The study of cell culture and micellar solubility of DIP demonstrated α- and β-caseins relatively the same helping in delivery of DIP. Milk α- and β-caseins are considered as a useful vehicle for the solublization and stabilization of DIP in aqueous solution at natural pH.

Monitoring of live cell cultures during apoptosis by phase imaging and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Sharikova, Anna; Saide, George; Sfakis, Lauren; Park, Jun Yong; Desta, Habben; Maloney, Maxwell C.; Castracane, James; Mahajan, Supriya D.; Khmaladze, Alexander

2017-02-01

Non-invasive live cell measurements are an important tool in biomedical research. We present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy records an interference pattern between object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information about the sample. When the phase is mapped across the sample and converted into height information for each pixel, a three dimensional image is obtained. The measurement of live cell cultures by digital holographic microscopy yields information about cell shape and volume, changes to which are reflective of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy, on the other hand, is sensitive to rotational and vibrational molecular transitions, as well as intermolecular vibrations. Therefore, Raman spectroscopy provides complementary information about cells, such as protein, lipid and nucleic acid content, and, particularly, the spectral signatures associated with structural changes in molecules. The cell cultures are kept in the temperature-controlled environmental chamber during the experiment, which allows monitoring over multiple cell cycles. The DHM system combines a visible (red) laser source with conventional microscope base, and LabVIEW-run data processing. We analyzed and compared cell culture information obtained by these two methods.

In vitro testing of curcumin based composites coatings as antitumoral systems against osteosarcoma cells

NASA Astrophysics Data System (ADS)

Tirca, I.; Mitran, V.; Marascu, V.; Brajnicov, S.; Ion, V.; Stokker-Cheregi, F.; Popovici, I. A.; Cimpean, A.; Dinca, V.; Dinescu, M.

2017-12-01

In this work, we propose a new design for biodegradable composite coatings obtained by laser methods, which are aimed at evaluating the effects of active antitumoral elements on osteosarcoma cells. Our approach relies on embedding curcumin, which is a natural polyphenol having antitumoral properties, within biodegradable copolymer coatings (i.e. polyvinyl alcohol-polyethylene glycol - PVA-PEG) by using matrix assisted pulsed laser evaporation (MAPLE). The structural and morphological characteristics of the coatings were tailored by using different solvents (water, ethanol, benzene, dimethylsufoxide) as deposition matrix. The morphological characteristics of the resulting films were investigated by atomic force microscopy (AFM), whereas their chemical composition was characterized by Fourier transform infrared spectroscopy (FTIR). These characteristics were correlated with the degradation behavior by using ellipsometry (SE) and AFM measurements data. The in vitro study of the MG-63 osteosarcoma cell behavior indicates that the developed hybrid coatings significantly decreased osteosarcoma cell viability and proliferation potential. The physico-chemical characteristics of the thin films, along with the preliminary in vitro analyses, suggest that our developed polymeric hybrid coatings represent an efficient way to tackle the design of antitumoral surfaces, with applications in biomedicine.

Characterization of novel sufraces by FTIR spectroscopy and atomic force microscopy for food pathogen detection

USDA-ARS?s Scientific Manuscript database

Single molecular detection of pathogens and toxins of interest to food safety is within grasp using technology such as Atomic Force Microscopy. Using antibodies or specific aptamers connected to the AFM tip make it possible to detect a pathogen molecule on a surface. However, it also becomes necess...

Reconsideration of dynamic force spectroscopy analysis of streptavidin-biotin interactions.

PubMed

Taninaka, Atsushi; Takeuchi, Osamu; Shigekawa, Hidemi

2010-05-13

To understand and design molecular functions on the basis of molecular recognition processes, the microscopic probing of the energy landscapes of individual interactions in a molecular complex and their dependence on the surrounding conditions is of great importance. Dynamic force spectroscopy (DFS) is a technique that enables us to study the interaction between molecules at the single-molecule level. However, the obtained results differ among previous studies, which is considered to be caused by the differences in the measurement conditions. We have developed an atomic force microscopy technique that enables the precise analysis of molecular interactions on the basis of DFS. After verifying the performance of this technique, we carried out measurements to determine the landscapes of streptavidin-biotin interactions. The obtained results showed good agreement with theoretical predictions. Lifetimes were also well analyzed. Using a combination of cross-linkers and the atomic force microscope that we developed, site-selective measurement was carried out, and the steps involved in bonding due to microscopic interactions are discussed using the results obtained by site-selective analysis.

Spatiotemporally and Mechanically Controlled Triggering of Mast Cells using Atomic Force Microscopy

PubMed Central

Hu, Kenneth K.; Bruce, Marc A.; Butte, Manish J.

2014-01-01

Mast cells are thought to be sensitive to mechanical forces, for example, coughing in asthma or pressure in “physical urticarias”. Conversion of mechanical forces to biochemical signals could potentially augment antigenic signaling. Studying the combined effects of mechanical and antigenic cues on mast cells and other hematopoietic cells has been elusive. Here, we present an approach using a modified atomic force microscope cantilever to deliver antigenic signals to mast cells while simultaneously applying mechanical forces. We developed a strategy to concurrently record degranulation events by fluorescence microscopy during antigenic triggering. Finally, we also measured the mechanical forces generated by mast cells while antigen receptors are ligated. We showed that mast cells respond to antigen delivered by the AFM cantilever with prompt degranulation and the generation of strong pushing and pulling forces. We did not discern any relationship between applied mechanical forces and the kinetics of degranulation. These experiments present a new method for dissecting the interactions of mechanical and biochemical cues in signaling responses of immune cells. PMID:24777418

Traction force dynamics predict gap formation in activated endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valent, Erik T.; Nieuw Amerongen, Geerten P. van; Hinsbergh, Victor W.M. van

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneousmore » distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. - Highlights: • Endothelial monolayers exert dynamic- and heterogeneous traction forces. • High traction forces correlate with junctional areas and the F-actin cytoskeleton. • Newly formed inter-endothelial gaps are characterized by opposing traction forces. • Force stability is a key feature controlling endothelial permeability.« less

Biocompatibility of Hydrogen-Diluted Amorphous Silicon Carbide Thin Films for Artificial Heart Valve Coating

NASA Astrophysics Data System (ADS)

Rizal, Umesh; Swain, Bhabani S.; Rameshbabu, N.; Swain, Bibhu P.

2018-01-01

Amorphous silicon carbide (a-SiC:H) thin films were synthesized using trichloromethylsilane by a hot wire chemical vapor deposition process. The deposited films were characterized by Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, x-ray diffraction and x-ray photoelectron spectroscopy to confirm its chemical bonding, structural network and composition of the a-SiC:H films. The optical microscopy images reveal that hydrogen dilution increased the surface roughness and pore density of a-SiC:H thin film. The Raman spectroscopy and FTIR spectra reveal chemical network consisting of Si-Si, C-C and Si-C bonds, respectively. The XRD spectroscopy and Raman spectroscopy indicate a-SiC:H still has short-range order. In addition, in vitro cytotoxicity test ensures the behavior of cell-semiconductor hybrid to monitor the proper coordination. The live-dead assays and MTT assay reveal an increase in green nucleus cell, and cell viability is greater than 88%, respectively, showing non-toxic nature of prepared a-SiC:H film. Moreover, the result indicated by direct contact assay, and cell prefers to adhere and proliferate on a-SiC:H thin films having a positive effect as artificial heart valve coating material.

Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

PubMed Central

Bizarro, C. V.; Alemany, A.; Ritort, F.

2012-01-01

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na+]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg2+ salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs. PMID:22492710

Optimal Control-Enabled Imaging and Spectroscopy using a Nanowire Magnetic Resonance Force Microscope

NASA Astrophysics Data System (ADS)

Rose, William; Haas, Holger; Chen, Angela; Cory, David; Budakian, Raffi

Magnetic resonance imaging (MRI) is a powerful non-invasive technique that has transformed our ability to study the structure and function of biological systems. Key to its success has been the unique ability to combine imaging with magnetic resonance spectroscopy. Although it remains a significant challenge, there is considerable interest in extending MRI spectroscopy to the nanometer scale because it would provide a fundamentally new route for determining the structure and function of complex biomolecules. We present data taken with a nanowire magnetic resonance force microscopy (MRFM) setup. We show how the capabilities of this very sensitive spin-detection system can be extended to include spectroscopy and nanometer-scale imaging by combining optimal control theory (OCT) techniques with magic echo sequences. We apply OCT-based dynamical-decoupling pulses to nanoscale ensembles of proton spins in polystyrene, and demonstrate a 500-fold line-narrowing of the proton spin resonance, from 30 kHz to 60 Hz. We further demonstrate 1-D imaging over a 35-nm region with an average voxel size of 2.2 nm. Funding provided by the U.S. Army Research Office, Grant No. W911NF-12-1-0341.

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

PubMed Central

Arnaut, Vera; Langecker, Martin; Simmel, Friedrich C.

2013-01-01

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified. PMID:24010663

Micropipette force probe to quantify single-cell force generation: application to T-cell activation

PubMed Central

Sawicka, Anna; Babataheri, Avin; Dogniaux, Stéphanie; Barakat, Abdul I.; Gonzalez-Rodriguez, David; Hivroz, Claire; Husson, Julien

2017-01-01

In response to engagement of surface molecules, cells generate active forces that regulate many cellular processes. Developing tools that permit gathering mechanical and morphological information on these forces is of the utmost importance. Here we describe a new technique, the micropipette force probe, that uses a micropipette as a flexible cantilever that can aspirate at its tip a bead that is coated with molecules of interest and is brought in contact with the cell. This technique simultaneously allows tracking the resulting changes in cell morphology and mechanics as well as measuring the forces generated by the cell. To illustrate the power of this technique, we applied it to the study of human primary T lymphocytes (T-cells). It allowed the fine monitoring of pushing and pulling forces generated by T-cells in response to various activating antibodies and bending stiffness of the micropipette. We further dissected the sequence of mechanical and morphological events occurring during T-cell activation to model force generation and to reveal heterogeneity in the cell population studied. We also report the first measurement of the changes in Young’s modulus of T-cells during their activation, showing that T-cells stiffen within the first minutes of the activation process. PMID:28931600

In Vitro Evaluation of PCL and P(3HB) as Coating Materials for Selective Laser Melted Porous Titanium Implants.

PubMed

Grau, Michael; Matena, Julia; Teske, Michael; Petersen, Svea; Aliuos, Pooyan; Roland, Laura; Grabow, Niels; Murua Escobar, Hugo; Gellrich, Nils-Claudius; Haferkamp, Heinz; Nolte, Ingo

2017-11-23

Titanium is widely used as a bone implant material due to its biocompatibility and high resilience. Since its Young's modulus differs from bone tissue, the resulting "stress shielding" could lead to scaffold loosening. However, by using a scaffold-shaped geometry, the Young's modulus can be adjusted. Also, a porous geometry enables vascularisation and bone ingrowth inside the implant itself. Additionally, growth factors can improve these effects. In order to create a deposit and release system for these factors, the titanium scaffolds could be coated with degradable polymers. Therefore, in the present study, synthetic poly-ε-caprolactone (PCL) and the biopolymer poly(3-hydroxybutyrate) (P(3HB)) were tested for coating efficiency, cell adhesion, and biocompatibility to find a suitable coating material. The underlying scaffold was created from titanium by Selective Laser Melting (SLM) and coated with PCL or P(3HB) via dip coating. To test the biocompatibility, Live Cell Imaging (LCI) as well as vitality and proliferation assays were performed. In addition, cell adhesion forces were detected via Single Cell Force Spectroscopy, while the coating efficiency was observed using environmental scanning electron microscopy (ESEM) and energy-dispersive X-ray (EDX) analyses. Regarding the coating efficiency, PCL showed higher values in comparison to P(3HB). Vitality assays revealed decent vitality values for both polymers, while values for PCL were significantly lower than those for blank titanium. No significant differences could be observed between PCL and P(3HB) in proliferation and cell adhesion studies. Although LCI observations revealed decreasing values in cell number and populated area over time on both polymer-coated scaffolds, these outcomes could be explained by the possibility of coating diluent residues accumulating in the culture medium. Overall, both polymers fulfill the requirements regarding biocompatibility. Nonetheless, since only PCL coating ensured the maintenance of the porous implant structure, it is preferable to be used as a coating material for creating a deposit and release system for growth factors.

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

PubMed

Sarkar, Anwesha; Zhao, Yuanchang; Wang, Yongliang; Wang, Xuefeng

2018-06-25

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging. The FAC molecule consists of an adhesive domain for surface coating and a force-reporting domain which can be activated to fluoresce by integrin molecular tension. The tension threshold required for FAC activation is tunable in 10-60 piconewton (pN), allowing the selective imaging of cellular force contributed by integrin tension at different force levels. We tested the performance of two FACs with tension thresholds of 12 and 54 pN (nominal values), respectively, on both glass and polystyrene surfaces. Cellular forces were successfully mapped by fluorescence imaging on all the surfaces. FAC-coated surfaces also enable co-imaging of cellular forces and cell structures in both live cells and immunostained cells, therefore opening a new avenue for the study of the interplay of force and structure. We demonstrated the co-imaging of integrin tension and talin clustering in live cells, and concluded that talin clustering always occurs before the generation of integrin tension above 54 pN, reinforcing the notion that talin is an important adaptor protein for integrin tension transmission. Overall, FAC provides a highly convenient approach that is accessible to general biological laboratories for the study of cellular forces with high sensitivity and resolution, thus holding the potential to greatly boost the research of cell mechanobiology.

Actin stress in cell reprogramming

PubMed Central

Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

2014-01-01

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

Biophysical force regulation in 3D tumor cell invasion

NASA Astrophysics Data System (ADS)

Wu, Mingming

When embedded within 3D extracellular matrices (ECM), animal cells constantly probe and adapt to the ECM locally (at cell length scale) and exert forces and communicate with other cells globally (up to 10 times of cell length). It is now well accepted that mechanical crosstalk between animal cells and their microenvironment critically regulate cell function such as migration, proliferation and differentiation. Disruption of the cell-ECM crosstalk is implicated in a number of pathologic processes including tumor progression and fibrosis. Central to the problem of cell-ECM crosstalk is the physical force that cells generate. By measuring single cell generated force within 3D collagen matrices, we revealed a mechanical crosstalk mechanism between the tumor cells and the ECM. Cells generate sufficient force to stiffen collagen fiber network, and stiffer matrix, in return promotes larger cell force generation. Our work highlights the importance of fibrous nonlinear elasticity in regulating tumor cell-ECM interaction, and results may have implications in the rapid tissue stiffening commonly found in tumor progression and fibrosis. This work is partially supported by NIH Grants R21RR025801 and R21GM103388.

Interfacial interaction track of amorphous solid dispersions established by water-soluble polymer and indometacin.

PubMed

Li, Jing; Fan, Na; Wang, Xin; Li, Chang; Sun, Mengchi; Wang, Jian; Fu, Qiang; He, Zhonggui

2017-08-30

The present work studied interfacial interactions of amorphous solid dispersions matrix of indometacin (IMC) that established using PVP K30 (PVP) and PEG 6000 (PEG) by focusing on their interaction forces and wetting process. Infrared spectroscopy (IR), raman spectroscopy, X-ray photoelectron spectra and contact angle instrument were used throughout the study. Hydrogen bond energy formed between PEG and IMC were stronger than that of PVP and IMC evidenced by molecular modeling measurement. The blue shift of raman spectroscopy confirmed that hydrogen bonding forces were formed between IMC and two polymers. The contact angle study can be used as an easy method to determine the dissolution mechanism of amorphous solid dispersions through fitting the profile of contact angle of water on a series of tablets. It is believed that the track of interfacial interactions will certainly become powerful tools to for designing and evaluating amorphous solid dispersions. Copyright © 2017 Elsevier B.V. All rights reserved.

Thermal infrared near-field spectroscopy.

PubMed

Jones, Andrew C; Raschke, Markus B

2012-03-14

Despite the seminal contributions of Kirchhoff and Planck describing far-field thermal emission, fundamentally distinct spectral characteristics of the electromagnetic thermal near-field have been predicted. However, due to their evanescent nature their direct experimental characterization has remained elusive. Combining scattering scanning near-field optical microscopy with Fourier-transform spectroscopy using a heated atomic force microscope tip as both a local thermal source and scattering probe, we spectroscopically characterize the thermal near-field in the mid-infrared. We observe the spectrally distinct and orders of magnitude enhanced resonant spectral near-field energy density associated with vibrational, phonon, and phonon-polariton modes. We describe this behavior and the associated distinct on- and off-resonance nanoscale field localization with model calculations of the near-field electromagnetic local density of states. Our results provide a basis for intrinsic and extrinsic resonant manipulation of optical forces, control of nanoscale radiative heat transfer with optical antennas, and use of this new technique of thermal infrared near-field spectroscopy for broadband chemical nanospectroscopy. © 2012 American Chemical Society

Influence of atomic tip structure on the intensity of inelastic tunneling spectroscopy data analyzed by combined scanning tunneling spectroscopy, force microscopy, and density functional theory

NASA Astrophysics Data System (ADS)

Okabayashi, Norio; Gustafsson, Alexander; Peronio, Angelo; Paulsson, Magnus; Arai, Toyoko; Giessibl, Franz J.

2016-04-01

Achieving a high intensity in inelastic scanning tunneling spectroscopy (IETS) is important for precise measurements. The intensity of the IETS signal can vary by up to a factor of 3 for various tips without an apparent reason accessible by scanning tunneling microscopy (STM) alone. Here, we show that combining STM and IETS with atomic force microscopy enables carbon monoxide front-atom identification, revealing that high IETS intensities for CO/Cu(111) are obtained for single-atom tips, while the intensity drops sharply for multiatom tips. Adsorption of the CO molecule on a Cu adatom [CO/Cu/Cu(111)] such that the molecule is elevated over the substrate strongly diminishes the tip dependence of IETS intensity, showing that an elevated position channels most of the tunneling current through the CO molecule even for multiatom tips, while a large fraction of the tunneling current bypasses the CO molecule in the case of CO/Cu(111).

Investigating tautomeric polymorphism in crystalline anthranilic acid using terahertz spectroscopy and solid-state density functional theory.

PubMed

Delaney, Sean P; Witko, Ewelina M; Smith, Tiffany M; Korter, Timothy M

2012-08-02

Terahertz spectroscopy is sensitive to the interactions between molecules in the solid-state and recently has emerged as a new analytical tool for investigating polymorphism. Here, this technique is applied for the first time to the phenomenon of tautomeric polymorphism where the crystal structures of anthranilic acid (2-aminobenzoic acid) have been investigated. Three polymorphs of anthranilic acid (denoted Forms I, II and III) were studied using terahertz spectroscopy and the vibrational modes and relative polymorph stabilities analyzed using solid-state density functional theory calculations augmented with London dispersion force corrections. Form I consists of both neutral and zwitterionic molecules and was found to be the most stable polymorph as compared to Forms II and III (both containing only neutral molecules). The simulations suggest that a balance between steric interactions and electrostatic forces is responsible for the favoring of the mixed neutral/zwitterion solid over the all neutral or all zwitterion crystalline arrangements.

Development of functional biointerfaces by surface modification of polydimethylsiloxane with bioactive chlorogenic acid.

PubMed

Wu, Ming; He, Jia; Ren, Xiao; Cai, Wen-Sheng; Fang, Yong-Chun; Feng, Xi-Zeng

2014-04-01

The effect of physicochemical surface properties and chemical structure on the attachment and viability of bacteria and mammalian cells has been extensively studied for the development of biologically relevant applications. In this study, we report a new approach that uses chlorogenic acid (CA) to modify the surface wettability, anti-bacterial activity and cell adhesion properties of polydimethylsiloxane (PDMS). The chemical structure of the surface was obtained by X-ray photoelectron spectroscopy (XPS), the roughness was measured by atomic force microscopy (AFM), and the water contact angle was evaluated for PDMS substrates both before and after CA modification. Molecular modelling showed that the modification was predominately driven by van der Waals and electrostatic interactions. The exposed quinic-acid moiety improved the hydrophilicity of CA-modified PDMS substrates. The adhesion and viability of E. coli and HeLa cells were investigated using fluorescence and phase contrast microscopy. Few viable bacterial cells were found on CA-coated PDMS surfaces compared with unmodified PDMS surfaces. Moreover, HeLa cells exhibited enhanced adhesion and increased spreading on the modified PDMS surface. Thus, CA-coated PDMS surfaces reduced the ratio of viable bacterial cells and increased the adhesion of HeLa cells. These results contribute to the purposeful design of anti-bacterial surfaces for medical device use. Copyright © 2013 Elsevier B.V. All rights reserved.

Tuning Surface Chemistry of Polyetheretherketone by Gold Coating and Plasma Treatment

NASA Astrophysics Data System (ADS)

Novotná, Zdeňka; Rimpelová, Silvie; Juřík, Petr; Veselý, Martin; Kolská, Zdeňka; Hubáček, Tomáš; Borovec, Jakub; Švorčík, Václav

2017-06-01

Polyetheretherketone (PEEK) has good chemical and biomechanical properties that are excellent for biomedical applications. However, PEEK exhibits hydrophobic and other surface characteristics which cause limited cell adhesion. We have investigated the potential of Ar plasma treatment for the formation of a nanostructured PEEK surface in order to enhance cell adhesion. The specific aim of this study was to reveal the effect of the interface of plasma-treated and gold-coated PEEK matrices on adhesion and spreading of mouse embryonic fibroblasts. The surface characteristics (polarity, surface chemistry, and structure) before and after treatment were evaluated by various experimental techniques (gravimetry, goniometry, X-ray photoelectron spectroscopy (XPS), and electrokinetic analysis). Further, atomic force microscopy (AFM) was employed to examine PEEK surface morphology and roughness. The biological response of cells towards nanostructured PEEK was evaluated in terms of cell adhesion, spreading, and proliferation. Detailed cell morphology was evaluated by scanning electron microscopy (SEM). Compared to plasma treatment, gold coating improved PEEK wettability. The XPS method showed a decrease in the carbon concentration with increasing time of plasma treatment. Cell adhesion determined on the interface between plasma-treated and gold-coated PEEK matrices was directly proportional to the thickness of a gold layer on a sample. Our results suggest that plasma treatment in a combination with gold coating could be used in biomedical applications requiring enhanced cell adhesion.

Elucidation of intermolecular interaction of bovine serum albumin with Fenhexamid: A biophysical prospect.

PubMed

Shi, Jie-Hua; Lou, Yan-Yue; Zhou, Kai-Li; Pan, Dong-Qi

2018-03-01

Fenhexamid, as a hydroxyanilide, is widely applied to control Botrytis cinerea for protecting crops and fruits. But it could adversely affect human and animals health due to accumulation of residues in food production. Here, the affinity characteristics of fenhexamid on bovine serum albumin (BSA) was studied via a series of spectroscopic methods such as steady-state fluorescence spectroscopy, ultraviolet spectroscopy (UV), synchronous fluorescence spectroscopy (SFS), 3D fluorescence spectroscopy, and fourier transform infrared spectroscopy (FT-IR). The experimental results illustrated that the fluorescence quenching mechanism of BSA induced by fenhexamid was a static quenching. The binding constant (K b ) of fenhexamid with BSA was 2.399 × 10 4  M -1 at 298 K and the combination ratio was about 1:1. The competitive experiment demonstrated that fenhexamid was binding on the BSA at site II (subdomain IIIA), which was confirmed by the molecular docking studies. The negative values of thermodynamic parameter (ΔH 0 , ΔS 0 and ΔG 0 ) revealed that the reaction of fenhexamid with BSA could proceed spontaneously, the van der Waals force and hydrogen bonding interaction conducted the main effect, and the binding process was enthalpy-driven. What's more, the 8-Anilino-1-naphthalenesulfonate (ANS) and sucrose binding studies were also performed and further verified the binding force between BSA and fenhexamid. Copyright © 2018 Elsevier B.V. All rights reserved.

Accelerated cell sheet detachment by copolymerizing hydrophilic PEG side chains into PNIPAm nanocomposite hydrogels.

PubMed

Liu, Dan; Wang, Tao; Liu, Xinxing; Tong, Zhen

2012-10-01

One-end-connected short poly(ethylene glycol) (PEG) side chains were facilely introduced into the poly(N-isopropylacrylamide) (PNIPAm) nanocomposite hydrogel (NC gel) via in situ copolymerization of NIPAm monomer and PEG macromonomer in the aqueous suspension of hectorite clay Laponite XLS. The NC gels were characterized with Fourier transform infrared and x-ray photoelectron spectroscopy for the composition, DSC and transmittance for the phase separation temperature, dynamic mechanical spectra and swelling ratio for the interaction. Increasing the PEG content led to a small increase in the storage modulus and the lower critical solution temperature (LCST) of the copolymerized NC gels, and the LCST of the copolymerized NC gels was still below 37 °C. The L929 cell adhesion and proliferation on the surface of these NC gels were not suppressed by the incorporation of hydrophilic PEG side chains. By lowering temperature below the LCST, the cell sheet spontaneously detached from the copolymerized NC gels. The surface morphology and surface wettability of the NC gels were detected by atom force microscope and contact angle measurement. A rough and hydrophilic surface induced by a small amount of PEG side chains was found to be favorable to accelerate the cell sheet detachment, probably due to the enhanced water permeation into the gel-cell sheet interface.

A durable and biocompatible ascorbic acid-based covalent coating method of polydimethylsiloxane for dynamic cell culture.

PubMed

Leivo, Joni; Virjula, Sanni; Vanhatupa, Sari; Kartasalo, Kimmo; Kreutzer, Joose; Miettinen, Susanna; Kallio, Pasi

2017-07-01

Polydimethylsiloxane (PDMS) is widely used in dynamic biological microfluidic applications. As a highly hydrophobic material, native PDMS does not support cell attachment and culture, especially in dynamic conditions. Previous covalent coating methods use glutaraldehyde (GA) which, however, is cytotoxic. This paper introduces a novel and simple method for binding collagen type I covalently on PDMS using ascorbic acid (AA) as a cross-linker instead of GA. We compare the novel method against physisorption and GA cross-linker-based methods. The coatings are characterized by immunostaining, contact angle measurement, atomic force microscopy and infrared spectroscopy, and evaluated in static and stretched human adipose stem cell (hASC) cultures up to 13 days. We found that AA can replace GA as a cross-linker in the covalent coating method and that the coating is durable after sonication and after 6 days of stretching. Furthermore, we show that hASCs attach and proliferate better on AA cross-linked samples compared with physisorbed or GA-based methods. Thus, in this paper, we provide a new PDMS coating method for studying cells, such as hASCs, in static and dynamic conditions. The proposed method is an important step in the development of PDMS-based devices in cell and tissue engineering applications. © 2017 The Author(s).

Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe

PubMed Central

Seddon, James R. T.; Lai, Stanley C. S.; Lemay, Serge G.; Bridle, Helen L.

2016-01-01

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions. PMID:26938220

Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness

PubMed Central

Zeng, Guanghong; Vad, Brian S.; Dueholm, Morten S.; Christiansen, Gunna; Nilsson, Martin; Tolker-Nielsen, Tim; Nielsen, Per H.; Meyer, Rikke L.; Otzen, Daniel E.

2015-01-01

The success of Pseudomonas species as opportunistic pathogens derives in great part from their ability to form stable biofilms that offer protection against chemical and mechanical attack. The extracellular matrix of biofilms contains numerous biomolecules, and it has recently been discovered that in Pseudomonas one of the components includes β-sheet rich amyloid fibrils (functional amyloid) produced by the fap operon. However, the role of the functional amyloid within the biofilm has not yet been investigated in detail. Here we investigate how the fap-based amyloid produced by Pseudomonas affects biofilm hydrophobicity and mechanical properties. Using atomic force microscopy imaging and force spectroscopy, we show that the amyloid renders individual cells more resistant to drying and alters their interactions with hydrophobic probes. Importantly, amyloid makes Pseudomonas more hydrophobic and increases biofilm stiffness 20-fold. Deletion of any one of the individual members of in the fap operon (except the putative chaperone FapA) abolishes this ability to increase biofilm stiffness and correlates with the loss of amyloid. We conclude that amyloid makes major contributions to biofilm mechanical robustness. PMID:26500638

Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness.

PubMed

Zeng, Guanghong; Vad, Brian S; Dueholm, Morten S; Christiansen, Gunna; Nilsson, Martin; Tolker-Nielsen, Tim; Nielsen, Per H; Meyer, Rikke L; Otzen, Daniel E

2015-01-01

The success of Pseudomonas species as opportunistic pathogens derives in great part from their ability to form stable biofilms that offer protection against chemical and mechanical attack. The extracellular matrix of biofilms contains numerous biomolecules, and it has recently been discovered that in Pseudomonas one of the components includes β-sheet rich amyloid fibrils (functional amyloid) produced by the fap operon. However, the role of the functional amyloid within the biofilm has not yet been investigated in detail. Here we investigate how the fap-based amyloid produced by Pseudomonas affects biofilm hydrophobicity and mechanical properties. Using atomic force microscopy imaging and force spectroscopy, we show that the amyloid renders individual cells more resistant to drying and alters their interactions with hydrophobic probes. Importantly, amyloid makes Pseudomonas more hydrophobic and increases biofilm stiffness 20-fold. Deletion of any one of the individual members of in the fap operon (except the putative chaperone FapA) abolishes this ability to increase biofilm stiffness and correlates with the loss of amyloid. We conclude that amyloid makes major contributions to biofilm mechanical robustness.

Dye-sensitized solar cells based on purple corn sensitizers

NASA Astrophysics Data System (ADS)

Phinjaturus, Kawin; Maiaugree, Wasan; Suriharn, Bhalang; Pimanpaeng, Samuk; Amornkitbamrung, Vittaya; Swatsitang, Ekaphan

2016-09-01

Natural dye extracted from husk, cob and silk of purple corn, were used for the first time as photosensitizers in dye sensitized solar cells (DSSCs). The dye sensitized solar cells fabrication process has been optimized in terms of solvent extraction. The resulting maximal efficiency of 1.06% was obtained from purple corn husk extracted by acetone. The ultraviolet-visible (UV-vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), electrochemical impedance spectroscopy (EIS) and incident photon-to-current efficiency (IPCE) were employed to characterize the natural dye and the DSSCs.

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

PubMed

Dufrêne, Y F

2001-02-01

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.

Cytotoxic T cells use mechanical force to potentiate target cell killing

PubMed Central

Basu, Roshni; Whitlock, Benjamin M.; Husson, Julien; Le Floc’h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C.; Huse, Morgan

2016-01-01

SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. PMID:26924577

Electrochemical behavior and biological response of Mesenchymal Stem Cells on cp-Ti after N-ions implantation

NASA Astrophysics Data System (ADS)

Rizwan, M.; Ahmad, A.; Deen, K. M.; Haider, W.

2014-11-01

Titanium and its alloys are most widely used as implant materials due to their excellent biocompatibility, mechanical properties and chemical stability. In this study Nitrogen ions of known dosage were implanted over cp-Ti by Pelletron accelerator with beam energy of 0.25 MeV.The atomic force microscopy of bare and nitrogen implanted specimens confirmed increase in surface roughness with increase in nitrogen ions concentration. X-ray diffraction patterns of ions implanted surfaces validated the formation of TiN0.3 and Ti3N2-xnitride phases. The tendency to form passive film and electrochemical behavior of these surfaces in ringer lactate (RL) solution was evaluated by Potentiodynamic polarization and electrochemical impedance spectroscopy respectively. It is proved that nitrogen ions implantation was beneficial to reduce corrosion rate and stabilizing passive film by increasing charge transfer resistance in RL. It was concluded that morphology and proliferation of Mesenchymal Stem Cells on nitrogen ions implanted surfaces strongly depends on surface roughness and nitride phases.

Deposition and characterization of spray pyrolysed p-type Cu2SnS3 thin film for potential absorber layer of solar cell

NASA Astrophysics Data System (ADS)

Thiruvenkadam, S.; Sakthi, P.; Prabhakaran, S.; Chakravarty, Sujay; Ganesan, V.; Rajesh, A. Leo

2018-06-01

Thin film of ternary Cu2SnS3 (CTS), a potential absorber layer for solar cells was successfully deposited by chemical spray pyrolysis technique. The GIXRD pattern revealed that the film having tetragonal Cu2SnS3 phase with the preferential orientation along (112), (200), (220) and (312) plane and it is further confirmed using Raman spectroscopy by the existence of Raman peak at 320 cm-1. Atomic Force Microscopy (AFM) was used to estimate the surface roughness of 28.8 nm. The absorption coefficient was found to be greater than the order of 105 cm-1 and bandgap of 1.70 eV. Hall effect measurement indicates the p type nature of the film with a hole concentration of 1.03 × 1016cm-3 and a hall mobility of 404 cm2/V. The properties of CTS thin film confirmed suitable to be a potential absorber layer material for photovoltaic applications.

Influence of Crystallinity and Energetics on Charge Separation in Polymer–Inorganic Nanocomposite Films for Solar Cells

PubMed Central

Bansal, Neha; Reynolds, Luke X.; MacLachlan, Andrew; Lutz, Thierry; Ashraf, Raja Shahid; Zhang, Weimin; Nielsen, Christian B.; McCulloch, Iain; Rebois, Dylan G.; Kirchartz, Thomas; Hill, Michael S.; Molloy, Kieran C.; Nelson, Jenny; Haque, Saif A.

2013-01-01

The dissociation of photogenerated excitons and the subsequent spatial separation of the charges are of crucial importance to the design of efficient donor-acceptor heterojunction solar cells. While huge progress has been made in understanding charge generation at all-organic junctions, the process in hybrid organic:inorganic systems has barely been addressed. Here, we explore the influence of energetic driving force and local crystallinity on the efficiency of charge pair generation at hybrid organic:inorganic semiconductor heterojunctions. We use x-ray diffraction, photoluminescence quenching, transient absorption spectroscopy, photovoltaic device and electroluminescence measurements to demonstrate that the dissociation of photogenerated polaron pairs at hybrid heterojunctions is assisted by the presence of crystalline electron acceptor domains. We propose that such domains encourage delocalization of the geminate pair state. The present findings suggest that the requirement for a large driving energy for charge separation is relaxed when a more crystalline electron acceptor is used. PMID:23524906

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork

PubMed Central

Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

2006-01-01

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane. PMID:16858413

Adhesion of Photoactive Microalgae to Surfaces is Switchable by Light

NASA Astrophysics Data System (ADS)

Bäumchen, Oliver; Kreis, Christian; Le Blay, Marine; Linne, Christine; Makowski, Marcin

The natural habitats of many microorganisms are confined geometries, such as the interstitial space of rocks and soil, where interactions with interfaces and surfaces are of paramount importance. We performed in vivo force spectroscopy experiments on the unicellular biflagellated microalga Chlamydomonas, a prime model organism in cell- and microbiology, and discovered that the flagella-mediated adhesion to surfaces can be switched on and off by light. Time-resolved micropipette experiments show that the light-switchable adhesiveness of the flagella is a completely reversible process that is based on a redistribution of adhesion-promoting flagella-membrane proteins within seconds. Light-switchable adhesion enables the cell to regulate the transition between planktonic and surface-associated state, which possibly represents a significant biological advantage for photoactive microorganisms. In terms of the colonization of surfaces and the formation of biofilms, the findings might have immediate economic and environmental relevance in biotechnological settings, such as photo-bioreactors for the sustainable production of biofuels.

Cell force mapping using a double-sided micropillar array based on the moiré fringe method

NASA Astrophysics Data System (ADS)

Zhang, F.; Anderson, S.; Zheng, X.; Roberts, E.; Qiu, Y.; Liao, R.; Zhang, X.

2014-07-01

The mapping of traction forces is crucial to understanding the means by which cells regulate their behavior and physiological function to adapt to and communicate with their local microenvironment. To this end, polymeric micropillar arrays have been used for measuring cell traction force. However, the small scale of the micropillar deflections induced by cell traction forces results in highly inefficient force analyses using conventional optical approaches; in many cases, cell forces may be below the limits of detection achieved using conventional microscopy. To address these limitations, the moiré phenomenon has been leveraged as a visualization tool for cell force mapping due to its inherent magnification effect and capacity for whole-field force measurements. This Letter reports an optomechanical cell force sensor, namely, a double-sided micropillar array (DMPA) made of poly(dimethylsiloxane), on which one side is employed to support cultured living cells while the opposing side serves as a reference pattern for generating moiré patterns. The distance between the two sides, which is a crucial parameter influencing moiré pattern contrast, is predetermined during fabrication using theoretical calculations based on the Talbot effect that aim to optimize contrast. Herein, double-sided micropillar arrays were validated by mapping mouse embryo fibroblast contraction forces and the resulting force maps compared to conventional microscopy image analyses as the reference standard. The DMPA-based approach precludes the requirement for aligning two independent periodic substrates, improves moiré contrast, and enables efficient moiré pattern generation. Furthermore, the double-sided structure readily allows for the integration of moiré-based cell force mapping into microfabricated cell culture environments or lab-on-a-chip devices.

Sub-Thz Vibrational Spectroscopy for Analysis of Ovarian Cancer Cells

NASA Astrophysics Data System (ADS)

Ferrance, Jerome P.; Sizov, Igor; Jazaeri, Amir; Moyer, Aaron; Gelmont, Boris; Globus, Tatiana

2016-06-01

Sub-THz vibrational spectroscopy utilizes wavelengths in the submillimeter-wave range ( 1.5-30 wn), beyond those traditionally used for chemical and biomolecular analysis. This low energy radiation excites low-frequency internal molecular motions (vibrations) involving hydrogen bonds and other weak connections within these molecules. The ability of sub-THz spectroscopy to identify and quantify biological molecules is based on detection of signature resonance absorbance at specific frequencies between 0.05 and 1 THz, for each molecule. The long wavelengths of this radiation, mean that it can even pass through entire cells, detecting the combinations of proteins and nucleic acids that exist within the cell. This research introduces a novel sub-THz resonance spectroscopy instrument with spectral resolution sufficient to identify individual resonance absorption peaks, for the analysis of ovarian cancer cells. In vitro cell cultures of SK-OV-3 and ES-2 cells, two human ovarian cancer subtypes, were characterized and compared with a normal non-transformed human fallopian tube epithelial cell line (FT131). A dramatic difference was observed between the THz absorption spectra of the cancer and normal cell sample materials with much higher absorption intensity and a very strong absorption peak at a frequency of 13 wn dominating the cancer sample spectra. Comparison of experimental spectra with molecular dynamic simulated spectroscopic signatures suggests that the high intensity spectral peak could originate from overexpressed mi-RNA molecules specific for ovarian cancer. Ovarian cancer cells are utilized as a proof of concept, but the sub-THz spectroscopy method is very general and could also be applied to other types of cancer.

Forces in yeast flocculation

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

2015-01-01

In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

Force spectroscopy of quadruple H-bonded dimers by AFM: dynamic bond rupture and molecular time-temperature superposition.

PubMed

Zou, Shan; Schönherr, Holger; Vancso, G Julius

2005-08-17

We report on the application of the time-temperature superposition principle to supramolecular bond-rupture forces on the single-molecule level. The construction of force-loading rate master curves using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) experiments carried out in situ at different temperatures allows one to extend the limited range of the experimentally accessible loading rates and hence to cross from thermodynamic nonequilibrium to quasi-equilibrium states. The approach is demonstrated for quadruple H-bonded ureido-4[1H]-pyrimidinone (UPy) moieties studied by variable-temperature SMFS in organic media. The unbinding forces of single quadruple H-bonding (UPy)2 complexes, which were identified based on a polymeric spacer strategy, were found to depend on the loading rate in the range of 5 nN/s to 500 nN/s at 301 K in hexadecane. By contrast, these rupture forces were independent of the loading rate from 5 to 200 nN/s at 330 K. These results indicate that the unbinding behavior of individual supramolecular complexes can be directly probed under both thermodynamic nonequilibrium and quasi-equilibrium conditions. On the basis of the time-temperature superposition principle, a master curve was constructed for a reference temperature of 301 K, and the crossover force (from loading-rate independent to -dependent regimes) was determined as approximately 145 pN (at a loading rate of approximately 5.6 nN/s). This approach significantly broadens the accessible loading-rate range and hence provides access to fine details of potential energy landscape of supramolecular complexes based on SMFS experiments.

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Seunghwan; Hashizume, Yui; Mishima, Mari

Graphical abstract: - Highlights: • We developed a method to measure cell adhesion force by detaching cell using an arrowhead nanoneedle and AFM. • A nanofilm consisting of fibronectin and gelatin was formed on cell surface to reinforce the cell cortex. • By the nanofilm lamination, detachment efficiencies of strongly adherent cell lines were improved markedly. - Abstract: The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered–integrin bindings link cells mechanically to the ECM. In order tomore » quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin–substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.« less