Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin.

PubMed

Wu, Lei; Yang, Shu-Hou; Xiong, Hao; Yang, Jia-Qian; Guo, Jun; Yang, Wen-Chao; Yang, Guang-Fu

2017-03-21

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by adjusting the recognition group. The refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of chymotrypsin, probe 2 shows about 5-fold higher binding affinity and comparable catalytical efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor characterization. To the best of our knowledge, probe 2 is the first nonpeptide-based small-molecule probe for chymotrypsin, with the advantages of simple structure and high sensitivity compared to the widely used peptide-based substrates. This small-molecule probe is expected to be a useful molecular tool for drug discovery and chymotrypsin-related disease diagnosis.

Polyketide intermediate mimics as probes for revealing cryptic stereochemistry of ketoreductase domains.

PubMed

Li, Yang; Fiers, William D; Bernard, Steffen M; Smith, Janet L; Aldrich, Courtney C; Fecik, Robert A

2014-12-19

Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics 12 and 13 were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide 63 and trans-decalone 64, substrates 12 and 13 showed 2-10 fold lower K(M) values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the k(cat) values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.

Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization.

PubMed

Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio

2011-04-01

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

High temperature integrated ultrasonic shear and longitudinal wave probes

NASA Astrophysics Data System (ADS)

Ono, Y.; Jen, C.-K.; Kobayashi, M.

2007-02-01

Integrated ultrasonic shear wave probes have been designed and developed using a mode conversion theory for nondestructive testing and characterization at elevated temperatures. The probes consisted of metallic substrates and high temperature piezoelectric thick (>40μm) films through a paint-on method. Shear waves are generated due to mode conversion from longitudinal to shear waves because of reflection inside the substrate having a specific shape. A novel design scheme is proposed to reduce the machining time of substrates and thick film fabrication difficulty. A probe simultaneously generating and receiving both longitudinal and shear waves is also developed and demonstrated. In addition, a shear wave probe using a clad buffer rod consisting of an aluminum core and stainless steel cladding has been developed. All the probes were tested and successfully operated at 150°C.

Novel nano-OLED based probes for very high resolution optical microscopy

NASA Astrophysics Data System (ADS)

Zhao, Yiying

Near-field scanning optical microscopy (NSOM) has been applied in the study of nanomaterials, microelectronics, photonics, plasmonics, cells, and molecules. However, conventional NSOM relies on optically pumped probes, suffering low optical transmission, heating of the tip, and poor reproducibility of probe fabrication, increasing the cost, impeding usability, reducing practical imaging resolution, and limiting NSOM's utility. In this thesis, I demonstrate a novel probe based on a nanoscale, electrically pumped organic light-emitting device (OLED) formed on the tip of a low-cost, commercially available atomic force microscopy (AFM) probe. I describe the structure, fabrication, and principles of this novel probe's operation, and discuss its potential to overcome the limitations of conventional NSOM probes. The broader significance of this work in the field of organic optoelectronics is also discussed. Briefly, OLEDs consist of organic thin films sandwiched between two electrodes. Under bias, electrons and holes are injected into the organic layers, leading to radiative recombination. Depositing a small molecular OLED in vacuum onto a pyramid-tipped AFM probe results in a laminar structure that is highly curved at the tip. Simple electrical modeling predicts concentration of electric field and localized electron injection into the organic layers at the tip, improving the local charge balance in an otherwise electron-starved OLED. Utilizing an "inverted" OLED structure (i.e. cathode on the "bottom"), light emission is localized to sub-200 nm sized, green light emitting regions on probe vertices; light output power in the range of 0.1-0.5 nanowatts was observed, comparable to that of typical fiber based NSOM probes but with greater power efficiency. Massive arrays of similar sub-micron OLEDs were also fabricated by depositing onto textured silicon substrates, demonstrating the superior scalability of the probe fabrication process (e.g. relative to pulled glass fibers). The investigation of the effect of non-planar substrate geometry on charge injection, transport and recombination provides broader insights into OLEDs made on rough substrates, general understanding of OLED operation (e.g. filamentary charge conduction) and degradation, and potentially helps to improve technologically important "inverted" OLED structures.

Multispectral Photoacoustic Imaging of Tumor Protease Activity with a Gold Nanocage-Based Activatable Probe.

PubMed

Liu, Cheng; Li, Shiying; Gu, Yanjuan; Xiong, Huahua; Wong, Wing-Tak; Sun, Lei

2018-05-07

Tumor proteases have been recognized as significant regulators in the tumor microenvironment, but the current strategies for in vivo protease imaging have tended to focus on the development of a probe design rather than the investigation of a novel imaging strategy by leveraging the imaging technique and probe. Herein, it is the first report to investigate the ability of multispectral photoacoustic imaging (PAI) to estimate the distribution of protease cleavage sites inside living tumor tissue by using an activatable photoacoustic (PA) probe. The protease MMP-2 is selected as the target. In this probe, gold nanocages (GNCs) with an absorption peak at ~ 800 nm and fluorescent dye molecules with an absorption peak at ~ 680 nm are conjugated via a specific enzymatic peptide substrate. Upon enzymatic activation by MMP-2, the peptide substrate is cleaved and the chromophores are released. Due to the different retention speeds of large GNCs and small dye molecules, the probe alters its intrinsic absorption profile and produces a distinct change in the PA signal. A multispectral PAI technique that can distinguish different chromophores based on intrinsic PA spectral signatures is applied to estimate the signal composition changes and indicate the cleavage interaction sites. Finally, the multispectral PAI technique with the activatable probe is tested in solution, cultured cells, and a subcutaneous tumor model in vivo. Our experiment in solution with enzyme ± inhibitor, cell culture ± inhibitor, and in vivo tumor model with administration of the developed probe ± inhibitor demonstrated the probe was cleaved by the targeted enzyme. Particularly, the in vivo estimation of the cleavage site distribution was validated with the result of ex vivo immunohistochemistry analysis. This novel synergy of the multispectral PAI technique and the activatable probe is a potential strategy for the distribution estimation of tumor protease activity in vivo.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin

2014-01-01

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

Atmospheric pressure plasma jet for biomedical applications characterised by passive thermal probe

NASA Astrophysics Data System (ADS)

Mance, Diana; Wiese, Ruben; Kewitz, Thorben; Kersten, Holger

2018-05-01

Atmospheric pressure plasma jets (APPJs) are a promising tool in medicine with extensive possibilities of utilization. For a safe and therapeutically effective application of APPJs, it is necessary to know in detail the physical processes in plasma as well as possible hazards. In this paper, we focus on plasma thermal energy transferred to the substrate, i.e. to a passive thermal probe acting as substrate dummy. Specifically, we examined the dependence of transferred energy on the distance from the plasma source outlet, on the gas flow rate, and on the length of the visible plasma plume. The plasma plume is the plasma carried by the gas flow from the outlet of the source into the ambient air. The results show the distance between the plasma-generating device and the substrate to be the most important determinant of the transferred thermal energy, among the three examined variables. Most importantly for the end-user, the results also show this relation to be non-linear. To describe this relation, we chose a model based on a Boltzmann type of sigmoid function. Based on the results of our modelling and visual inspection of the plasma, we provide sort of a user guide for the adjustment of a suitable energy flux on the (bio) substrate.

Atomic structure and bonding of the interfacial bilayer between Au nanoparticles and epitaxially regrown MgAl{sub 2}O{sub 4} substrates

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

Zhu, Guo-zhen; Canadian Centre of Electron Microscopy and Department of Materials Science and Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1; Majdi, Tahereh

2014-12-08

A unique metal/oxide interfacial bilayer formed between Au nanoparticles and MgAl{sub 2}O{sub 4} substrates following thermal treatment is reported. Associated with the formation of the bilayer was the onset of an abnormal epitaxial growth of the substrate under the nanoparticle. According to the redistribution of atoms and the changes of their electronic structure probed across the interface by a transmission electron microscopy, we suggest two possible atomic models of the interfacial bilayer.

A Crosslinker Based on a Tethered Electrophile for Mapping Kinase-Substrate Networks

PubMed Central

Riel-Mehan, Megan M; Shokat, Kevan M

2014-01-01

SUMMARY Despite the continuing progress made towards mapping kinase signaling networks, there are still many phosphorylation events for which the responsible kinase has not yet been identified. We are interested in addressing this problem through forming covalent crosslinks between a peptide substrate and the corresponding phosphorylating kinase. Previously we reported a dialdehyde-based kinase binding probe capable of such a reaction with a peptide containing a cysteine substituted for the phosphorylatable ser/thr/tyr residue. Here, we examine the yield of a previously reported dialdehyde-based probe, and report that the dialdehyde based probes possesses a significant limitation in terms of crosslinked kinase-substrate product yield. To address this limitation, we develop a crosslinking scheme based on a kinase activity-based probe, and this new cross-linker provides an increase in efficiency and substrate specificity, including in the context of cell lysate. PMID:24746561

Surface enhanced Raman gene probe and methods thereof

DOEpatents

Vo-Dinh, T.

1998-09-29

The subject invention disclosed herein is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

Surface enhanced Raman gene probe and methods thereof

DOEpatents

Vo-Dinh, Tuan

1998-01-01

The subject invention disclosed herein is a new gene probe biosensor and methods thereof based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays.

Surface enhanced Raman gene probe and methods thereof

DOEpatents

Vo-Dinh, T.

1998-07-21

The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

PubMed

Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Monopole antenna in quantitative near-field microwave microscopy of planar structures

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

Reznik, Alexander N.; Korolyov, Sergey A.

We have developed an analytical model of a near-field microwave microscope based on a coaxial resonator with a sharpened tip probe. The probe interacts with a layered sample that features an arbitrary depth distribution of permittivity. The microscopic tip end with the accumulated charge is regarded as a monopole antenna radiating an electric field in near zone. The impedance of such an antenna is determined within a quasi-static approximation. The proposed model is used for calculating the sample-sensitive parameters of the microscope, specifically, resonance frequency f{sub 0} and quality factor Q{sub 0}, as a function of probe-sample distance h. Themore » theory has been verified experimentally in studies of semiconductor structures, both bulk and thin films. For measurements, we built a ∼2.1 GHz microscope with an effective tip radius of about 100 μm. The theoretical and experimental dependences f{sub 0}(h) and Q{sub 0}(h) were found to be in a good agreement. The developed theory underlies the method for determining sheet resistance R{sub sh} of a semiconductor film on a dielectric substrate proposed in this article. Studies were performed on doped n-GaN films on an Al{sub 2}O{sub 3} substrate. The effective radius and height of the probe determined from calibration measurements of etalon samples were used as the model fitting parameters. For etalon samples, we employed homogeneous sapphire and doped silicon plates. We also performed four-probe dc measurements of R{sub sh}. The corresponding values for samples with R{sub sh} > 1 kΩ were found to be 50% to 100% higher than the microwave results, which are attributed to the presence of microdefects in semiconductor films.« less

Inhibition of cytochrome P450 enzymes by saturated and unsaturated fatty acids in human liver microsomes, characterization of enzyme kinetics in the presence of bovine serum albumin (0.1 and 1.0% w/v) and in vitro - in vivo extrapolation of hepatic clearance.

PubMed

Palacharla, Raghava Choudary; Uthukam, Venkatesham; Manoharan, Arunkumar; Ponnamaneni, Ranjith Kumar; Padala, Nagasurya Prakash; Boggavarapu, Rajesh Kumar; Bhyrapuneni, Gopinadh; Ajjala, Devender Reddy; Nirogi, Ramakrishna

2017-04-01

The objective of the study was to determine the effect of fatty acids on CYP enzymes and the effect of BSA on intrinsic clearance of probe substrates. The inhibitory effect of thirteen fatty acids including saturated, mono-unsaturated and polyunsaturated fatty acids on CYP enzymes, kinetic parameters and intrinsic clearance values of nine CYP marker probe substrate reactions in the absence and presence of BSA (0.1 and 1.0% w/v) were characterized in human liver microsomes. The results demonstrate that most of the unsaturated fatty acids showed marked inhibition towards CYP2C8 mediated amodiaquine N-deethylation followed by inhibition of CYP2C9 and CYP2B6 mediated activities. The addition of 0.1% BSA in the incubation markedly improved the unbound intrinsic clearance values of probe substrates by reducing the K m values with little or no effect on maximal velocity. The addition of BSA (0.1 and 1.0% w/v) did not influence the unbound intrinsic clearance of marker reactions for CYP2A6, and CYP3A4 enzymes. The addition of 0.1% w/v BSA is sufficient to determine the intrinsic clearance of marker probe reactions by metabolite formation approach. The predicted hepatic clearance values for the substrates using the well-stirred model, in the presence of BSA (0.1% BSA), are comparable to the in vivo hepatic clearance values. Copyright © 2017 Elsevier B.V. All rights reserved.

Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes

PubMed Central

Poreba, Marcin; Solberg, Rigmor; Rut, Wioletta; Lunde, Ngoc Nguyen; Kasperkiewicz, Paulina; Snipas, Scott J.; Mihelic, Marko; Turk, Dusan; Turk, Boris; Salvesen, Guy S.; Drag, Marcin

2018-01-01

SUMMARY Legumain (AEP) is a lysosomal cysteine protease that is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP up-regulation is linked to a number of diseases including inflammation, arteriosclerosis and tumorigenesis. Thus legumain is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling. PMID:27478158

Carbon nanotube mechanics in scanning probe microscopy

NASA Astrophysics Data System (ADS)

Strus, Mark Christopher

Carbon nanotubes (CNTs) possess unique electrical, thermal, and mechanical properties which have led to the development of novel nanomechanical materials and devices. In this thesis, the mechanical properties of carbon nanotubes are studied with an Atomic Force Microscope (AFM) and, conversely, the use of CNTs to enhance conventional AFM probes is also investigated. First, the performance of AFM probes with multiwalled CNT tips are evaluated during attractive regime AFM imaging of high aspect ratio structures. The presented experimental results show two distinct imaging artifacts, the divot and large ringing artifacts, which are inherent to such CNT AFM probes. Through the adjustment of operating parameters, the connection of these artifacts to CNT bending, adhesion, and stiction is described qualitatively and explained. Next, the adhesion and peeling of CNTs on different substrates is quantitatively investigated with theoretical models and a new AFM mode for nanomechanical peeling. The theoretical model uncovers the rich physics of peeling of CNTs from surfaces, including sudden transitions between different geometric configurations of the nanotube with vastly different interfacial energies. The experimental peeling of CNTs is shown to be capable of resolving differences in CNT peeling energies at attoJoule levels on different materials. AFM peeling force spectroscopy is further studied on a variety of materials, including several polymers, to demonstrate the capability of direct measurement of interfacial energy between an individual nanotube or nanofiber and a given material surface. Theoretical investigations demonstrate that interfacial and flexural energies can be decoupled so that the work of the applied peeling force can be used to estimate the CNT-substrate interfacial fracture energy and nanotube's flexural stiffness. Hundreds of peeling force experiments on graphite, epoxy, and polyimide demonstrate that the peeling force spectroscopy offers a convenient experimental framework to quickly screen different combinations of polymers and functionalized nanotubes for optimal interfacial strength. Finally, multiple CNT AFM probe oscillation states in tapping mode AFM as the cantilever is brought closer to a sample are fully investigated, including two kinds of permanent contact and two types of intermittent contact. Large deformation continuum elastica models of MWCNTs with different end boundary conditions are used to identify whether the CNT remains anchored to the sample in line-contact or in point-contact in the permanent contact regime. Energy dissipation spectroscopy and phase contrast are demonstrated as a way to predict the state of CNT-substrate boundary condition in the intermittent tapping regime on different substrates and to highlight the implications of these different imaging regimes for critical dimension AFM, biological sensing, and nanolithography. Together, this work studies the effect of CNT mechanical interactions in AFM, including artifact-avoidance optimization of and new compositional mapping using CNT AFM probes as well as novel techniques that will potentially enhance the future development of CNT-based nanodevices and materials.

Probing Reversible Chemistry in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase with Kinetic Isotope Effects

PubMed Central

Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

2015-01-01

Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5′-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5′-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5′-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. PMID:25950663

Surface plasmon resonance spectroscopy sensor and methods for using same

DOEpatents

Anderson, Brian Benjamin; Nave, Stanley Eugene

2002-01-01

A surface plasmon resonance ("SPR") probe with a detachable sensor head and system and methods for using the same in various applications is described. The SPR probe couples fiber optic cables directly to an SPR substrate that has a generally planar input surface and a generally curved reflecting surface, such as a substrate formed as a hemisphere. Forming the SPR probe in this manner allows the probe to be miniaturized and operate without the need for high precision, expensive and bulky collimating or focusing optics. Additionally, the curved reflecting surface of the substrate can be coated with one or multiple patches of sensing medium to allow the probe to detect for multiple analytes of interest or to provide multiple readings for comparison and higher precision. Specific applications for the probe are disclosed, including extremely high sensitive relative humidity and dewpoint detection for, e.g., moisture-sensitive environment such as volatile chemical reactions. The SPR probe disclosed operates with a large dynamic range and provides extremely high quality spectra despite being robust enough for field deployment and readily manufacturable.

A novel paramagnetic substrate for detecting myeloperoxidase activity in vivo

PubMed Central

Shazeeb, Mohammed S.; Xie, Yang; Gupta, Suresh; Bogdanov, Alexei A.

2013-01-01

Bis-phenylamides and bis-hydroxyindolamides of DTPA(Gd) are paramagnetic reducing substrates of peroxidases that enable molecular imaging of peroxidase activity in vivo. Specifically, bis-5HT-DTPA(Gd) has been used to image localized inflammation in animal models by detecting neutrophil derived myeloperoxidase (MPO) activity at the inflammation site. However, in other pre-clinical disease models, bis-5HT-DTPA(Gd) presents technical challenges due to its limited solubility in vivo. Here, we report a novel MPO sensing probe obtained by replacing the reducing substrate serotonin (5HT) with 5-hydroxytryptophan (HTrp). Characterization of the resulting probe (bis-HTrp-DTPA(Gd)) in vitro using NMR spectroscopy and enzyme kinetic analysis showed that bis-HTrp-DTPA(Gd): 1) improves solubility in water; 2) acts as a substrate for both HRP and MPO enzymes; 3) induces cross linking of proteins in the presence of MPO; 4) produces oxidation products which bind to plasma proteins and; 5) unlike bis-5HT-DTPA(Gd), does not follow first order reaction kinetics. In vivo MR imaging in mice demonstrated that bis-HTrp-DTPA(Gd) was retained for up to five days in MPO-containing sites and cleared faster than bis-5HT-DTPA(Gd) from MPO-negative sites. In conclusion, bis-HTrp-DTPA(Gd) should offer improvements for MR imaging of MPO-mediated inflammation in vivo especially in high-field MRI, which requires higher dose of contrast agent. PMID:22954188

Local doping of two-dimensional materials

DOEpatents

Wong, Dillon; Velasco, Jr, Jairo; Ju, Long; Kahn, Salman; Lee, Juwon; Germany, Chad E.; Zettl, Alexander K.; Wang, Feng; Crommie, Michael F.

2016-09-20

This disclosure provides systems, methods, and apparatus related to locally doping two-dimensional (2D) materials. In one aspect, an assembly including a substrate, a first insulator disposed on the substrate, a second insulator disposed on the first insulator, and a 2D material disposed on the second insulator is formed. A first voltage is applied between the 2D material and the substrate. With the first voltage applied between the 2D material and the substrate, a second voltage is applied between the 2D material and a probe positioned proximate the 2D material. The second voltage between the 2D material and the probe is removed. The first voltage between the 2D material and the substrate is removed. A portion of the 2D material proximate the probe when the second voltage was applied has a different electron density compared to a remainder of the 2D material.

Deep-Reaching Hydrodynamic Flow Confinement: Micrometer-Scale Liquid Localization for Open Substrates With Topographical Variations.

PubMed

Oskooei, Ali; Kaigala, Govind V

2017-06-01

We present a method for nonintrusive localization and reagent delivery on immersed biological samples with topographical variation on the order of hundreds of micrometers. Our technique, which we refer to as the deep-reaching hydrodynamic flow confinement (DR-HFC), is simple and passive: it relies on a deep-reaching hydrodynamic confinement delivered through a simple microfluidic probe design to perform localized microscale alterations on substrates as deep as 600 μm. Designed to scan centimeter-scale areas of biological substrates, our method passively prevents sample intrusion by maintaining a large gap between the probe and the substrate. The gap prevents collision of the probe and the substrate and reduces the shear stress experienced by the sample. We present two probe designs: linear and annular DR-HFC. Both designs comprise a reagent-injection aperture and aspiration apertures that serve to confine the reagent. We identify the design parameters affecting reagent localization and depth by DR-HFC and study their individual influence on the operation of DR-HFC numerically. Using DR-HFC, we demonstrate localized binding of antihuman immunoglobulin G (IgG) onto an activated substrate at various depths from 50 to 600 μm. DR-HFC provides a readily implementable approach for noninvasive processing of biological samples applicable to the next generation of diagnostic and bioanalytical devices.

Innovative SPM Probes for Energy-Storage Science: MWCNT-Nanopipettes to Nanobattery Probes

NASA Astrophysics Data System (ADS)

Larson, Jonathan; Talin, Alec; Pearse, Alexander; Kozen, Alexander; Reutt-Robey, Janice

As energy-storage materials and designs continue to advance, new tools are needed to direct and explore ion insertion/de-insertion at well-defined battery materials interfaces. Scanned probe tips, assembled from actual energy-storage materials, permit SPM measures of local cathode-anode (tip-sample) interactions, including ion transfer. We present examples of ``cathode'' MWCNT-terminated STM probe tips interacting with Li(s)/Si(111) anode substrates. The MWCNT tip functions as both SPM probe and Li-nanopipette,[1] for controlled transport and manipulation of Li. Local field conditions for lithium ionization and transfer are determined and compared to electrostatic models. Additional lithium metallic and oxide tips have been prepared by thin film deposition on conventional W tips, the latter of which effectively functions as a nanobattery. We demonstrate use of these novel probe materials in the local lithiation of low-index Si anode interfaces, probing local barriers for lithium insertion. Prospects and limitations of these novel SPM probes will be discussed. U.S. Department of Energy Award Number DESC0001160.

Flow-through SIP - A novel stable isotope probing approach limiting cross-feeding

NASA Astrophysics Data System (ADS)

Mooshammer, Maria; Kitzinger, Katharina; Schintlmeister, Arno; Kjedal, Henrik; Nielsen, Jeppe Lund; Nielsen, Per; Wagner, Michael

2017-04-01

Stable isotope probing (SIP) is a widely applied tool to link specific microbial populations to metabolic processes in the environment without the prerequisite of cultivation, which has greatly advanced our understanding of the role of microorganisms in biogeochemical cycling. SIP relies on tracing specific isotopically labeled substrates (e.g., 13C, 15N, 18O) into cellular biomarkers, such as DNA, RNA or phospholipid fatty acids, and is considered to be a robust technique to identify microbial populations that assimilate the labeled substrate. However, cross-feeding can occur when labeled metabolites are released from a primary consumer and then used by other microorganisms. This leads to erroneous identification of organisms that are not directly responsible for the process of interest, but are rather connected to primary consumers via a microbial food web. Here, we introduce a new approach that has the potential to eliminate the effect of cross-feeding in SIP studies and can thus also be used to distinguish primary consumers from other members of microbial food webs. In this approach, a monolayer of microbial cells are placed on a filter membrane, and labeled substrates are supplied by a continuous flow. By means of flow-through, labeled metabolites and degradation products are constantly removed, preventing secondary consumption of the substrate. We present results from a proof-of-concept experiment using nitrifiers from activated sludge as model system, in which we used fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes for identification of nitrifiers in combination with nanoscale secondary ion mass spectrometry (NanoSIMS) for visualization of isotope incorporation at the single-cell level. Our results show that flow-through SIP is a promising approach to significantly reduce cross-feeding and secondary substrate consumption in SIP experiments.

Pharmacophore, QSAR, and binding mode studies of substrates of human cytochrome P450 2D6 (CYP2D6) using molecular docking and virtual mutations and an application to chinese herbal medicine screening.

PubMed

Mo, Sui-Lin; Liu, Wei-Feng; Li, Chun-Guang; Zhou, Zhi-Wei; Luo, Hai-Bin; Chew, Helen; Liang, Jun; Zhou, Shu-Feng

2012-07-01

The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction matched our pharmacophore model for CYP2D6 substrates. Fifty four out of these 60 compounds could be docked into the active site of CYP2D6 and 24 of 54 compounds formed hydrogen bonds with Glu216, Asp301, Ser304, and Ala305 in CYP2D6. These results have provided further insights into the factors that determining the binding modes of substrates to CYP2D6. Screening of high-affinity ligands for CYP2D6 from herbal formula using computational models is a useful approach to identify potential herb-drug interactions.

Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening.

PubMed

Shin, Inchul; Ambler, Brett R; Wherritt, Daniel; Griffith, Wendell P; Maldonado, Amanda C; Altman, Ryan A; Liu, Aimin

2018-03-28

Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe 2 in a significant quantity. Analysis of this new product by HPLC coupled UV-vis spectroscopy, high-resolution mass spectrometry, 1 H NMR, 13 C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases.

Probing the probe: AFM tip-profiling via nanotemplates to determine Hamaker constants from phase-distance curves.

PubMed

Rodriguez, Raul D; Lacaze, Emmanuelle; Jupille, Jacques

2012-10-01

A method to determine the van der Waals forces from phase-distance curves recorded by atomic force microscopy (AFM) in tapping mode is presented. The relationship between the phase shift and the tip-sample distance is expressed as a function of the product of the Hamaker constant by tip radius. Silica-covered silicon tips are used to probe silica-covered silicon substrate in dry conditions to avoid capillary effects. Tips being assumed spherical, radii are determined in situ by averaging profiles recorded in different directions on hematite nanocrystals acting as nanotemplates, thus accounting for tip anisotropy. Through a series of reproducible measurements performed with tips of various radii (including the in-situ characterization of a damaged tip), a value of (6.3±0.4)×10(-20) J is found for the Hamaker constant of interacting silica surfaces in air, in good agreement with tabulated data. The results demonstrate that the onset of the tip-surface interaction is dominated by the van der Waals forces and that the total force can be modeled in the framework of the harmonic approximation. Based on the tip radius and the Hamaker constant associated to the tip-substrate system, the model is quite flexible. Once the Hamaker constant is known, a direct estimate of the tip size can be achieved whereas when the tip size is known, a quantitative evaluation of the van der Waals force becomes possible on different substrates with a spatial resolution at the nanoscale. Copyright © 2012 Elsevier B.V. All rights reserved.

Investigating the interplay between substrate stiffness and ligand chemistry in directing mesenchymal stem cell differentiation within 3D macro-porous substrates.

PubMed

Haugh, Matthew G; Vaughan, Ted J; Madl, Christopher M; Raftery, Rosanne M; McNamara, Laoise M; O'Brien, Fergal J; Heilshorn, Sarah C

2018-07-01

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior. Copyright © 2018 Elsevier Ltd. All rights reserved.

Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis.

PubMed

Lentz, Christian S; Ordonez, Alvaro A; Kasperkiewicz, Paulina; La Greca, Florencia; O'Donoghue, Anthony J; Schulze, Christopher J; Powers, James C; Craik, Charles S; Drag, Marcin; Jain, Sanjay K; Bogyo, Matthew

2016-11-11

Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

PubMed Central

2016-01-01

Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb “Hydrolase important for pathogenesis 1” (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections. PMID:27739665

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

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

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Plasmonic Colloidal Nanoantennas for Tip-Enhanced Raman Spectrocopy

NASA Astrophysics Data System (ADS)

Dill, Tyler J.

Plasmonic nanoantennas that a support localized surface plasmon resonance (LSPR) are capable of confining visible light to subwavelength dimensions due to strong electromagnetic field enhancement at the probe tip. Nanoantenna enable optical methods such as tip-enhanced Raman spectroscopy (TERS), a technique that uses scanning probe microscopy tips to provide chemical information with nanoscale spatial resolution and single-molecule sensitivities. The LSPR supported by the probe tip is extremely sensitive to the nanoscale morphology of the nanoantenna. Control of nanoscale morphology is notoriously difficult to achieve, resulting in TERS probes with poor reproducibility. In my thesis, I demonstrate high-performance, predictable, and broadband nanospectroscopy probes that are fabricated by self-assembly. Shaped metal nanoparticles are organized into dense layers and deposited onto scanning probe tips. When coupled to a metal substrate, these probes support a strong optical resonance in the gap between the substrate and the probe, producing dramatic field enhancements. I show through experiment and electromagnetic modeling that close-packed but electrically isolated nanoparticles are electromagnetically coupled. Hybridized LSPRs supported by self-assembled nanoparticles with a broadband optical response, giving colloidal nanoantenna a high tolerance for geometric variation resulting from fabrication. I find that coupled nanoparticles act as a waveguide, transferring energy from many neighboring nanoparticles towards the active TERS apex. I also use surface-enhanced Raman spectroscopy (SERS) to characterize the effects of nanoparticle polydispersity and gap height on the Raman enhancement. These colloidal probes have consistently achieved dramatic Raman enhancements in the range of 108-109 with sub-50 nm spatial resolution. Furthermore, in contrast to other nanospectroscopy probes, these colloidal probes can be fabricated in a scalable fashion with a batch-to-batch reproducibility of 80%. This body of work serves as an important demonstration that bottom-up engineering can be used for batch fabricatation of high-performance and high-reliability devices using inexpensive equipment and materials.

Controlling nanoscale acoustic strains in silicon using chirped femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Tzianaki, E.; Bakarezos, M.; Tsibidis, G. D.; Petrakis, S.; Loukakos, P. A.; Kosmidis, C.; Tatarakis, M.; Papadogiannis, N. A.

2016-06-01

The influence of femtosecond laser pulse chirp on laser-generated longitudinal acoustic strains in Si (100) monocrystal substrates is studied. Degenerate femtosecond pump-probe transient reflectivity measurements are performed using a layered structure of thin Ti transducer film on an Si substrate. Experimental results show that acoustic strains, manifested as strong Brillouin oscillations, are more effectively induced when negatively chirped femtosecond laser pulses pump the transducer. These results are theoretically supported by a modified thermo-mechanical model based on the combination of a revised two-temperature model and elasticity theory that takes into account the instantaneous frequency of the chirped femtosecond laser pump pulses.

[The evaluation of the error of the thermal pulse technique used to measure moisture content of root substrates in space greenhouse

NASA Technical Reports Server (NTRS)

Podol'skii, I. G.; Norokh, A. A.; Bingham, G. E.; Brigham, G. E. (Principal Investigator); Campbell, W. F. (Principal Investigator)

2002-01-01

Point thermopulse probes were used to monitor moisture level in the root substrates during cultivation of higher plants in a space greenhouse. Investigated were performance data of the thermopulse moisture probe in integration with the space greenhouse. It was shown that within the substrate moisture range from 20 up to 100% of the full saturation the technique error does not exceed 1.5%. The thermopulse technique bears much promise for metrologic monitoring of the root substrate moisture content in space greenhouses no matter water and air supply technology.

A novel paramagnetic substrate for detecting myeloperoxidase activity in vivo.

PubMed

Shazeeb, Mohammed S; Xie, Yang; Gupta, Suresh; Bogdanov, Alexei A

2012-01-01

Bis-phenylamides and bis-hydroxyindolamides of diethylenetriaminepentaacetic acid-gadolinium (DTPA(Gd)) are paramagnetic reducing substrates of peroxidases that enable molecular imaging of peroxidase activity in vivo. Specifically, gadolinium chelates of bis-5-hydroxytryptamide-DTPA (bis-5HT-DTPA(Gd)) have been used to image localized inflammation in animal models by detecting neutrophil-derived myeloperoxidase (MPO) activity at the inflammation site. However, in other preclinical disease models, bis-5HT-DTPA(Gd) presents technical challenges due to its limited solubility in vivo. Here we report a novel MPO-sensing probe obtained by replacing the reducing substrate serotonin (5-HT) with 5-hydroxytryptophan (HTrp). Characterization of the resulting probe (bis-HTrp-DTPA(Gd)) in vitro using nuclear magnetic resonance spectroscopy and enzyme kinetic analysis showed that bis-HTrp-DTPA(Gd) (1) improves solubility in water; (2) acts as a substrate for both horseradish peroxidase and MPO enzymes; (3) induces cross-linking of proteins in the presence of MPO; (4) produces oxidation products, which bind to plasma proteins; and (5) unlike bis-5HT-DTPA(Gd), does not follow first-order reaction kinetics. In vivo magnetic resonance imaging (MRI) in mice demonstrated that bis-HTrp-DTPA(Gd) was retained for up to 5 days in MPO-containing sites and cleared faster than bis-5HT-DTPA(Gd) from MPO-negative sites. Bis-HTrp-DTPA(Gd) should offer improvements for MRI of MPO-mediated inflammation in vivo, especially in high-field MRI, which requires a higher dose of contrast agent.

Radical probing of spliceosome assembly.

PubMed

Grewal, Charnpal S; Kent, Oliver A; MacMillan, Andrew M

2017-08-01

Here we describe the synthesis and use of a directed hydroxyl radical probe, tethered to a pre-mRNA substrate, to map the structure of this substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly. This methodology may be adapted to the synthesis of a wide variety of modified RNAs for use as probes of RNA structure and RNA-protein interaction. Copyright © 2017 Elsevier Inc. All rights reserved.

Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site.

PubMed

Roston, Daniel; Demapan, Darren; Cui, Qiang

2016-06-15

A reaction's transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here, we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS, while substrates with poor LGs have substantial cleavage of that bond. The results predict nonlinear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorus-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, and thus classical experimental approaches such as free energy relations should be interpreted with care.

Development of Substrate-Selective Probes for Affinity Pulldown of Histone Demethylases

PubMed Central

2015-01-01

JmjC-domain containing histone demethylases (JHDMs) play critical roles in many key cellular processes and have been implicated in multiple disease conditions. Each enzyme within this family is known to have a strict substrate scope, specifically the position of the lysine within the histone and its degree of methylation. While much progress has been made in determining the substrates of each enzyme, new methods with which to systematically profile each histone mark are greatly needed. Novel chemical tools have the potential to fill this role and, furthermore, can be used as probes to answer fundamental questions about these enzymes and serve as potential therapeutic leads. In this work, we first investigated three small-molecule probes differing in the degree of “methylation state” and their differential bindings to JHDM1A (an H3K36me1/2 demethylase) using a fluorescence polarization-based competition assay. We then applied this specificity toward the “methylation state” and combined it with specificity toward lysine position in the design and synthesis of a peptidic probe targeting H3K36me2 JHDMs. The probe is further functionalized with a benzophenone cross-linking moiety and a biotin for affinity purification. Results showed binding of the peptidic probe to JHDM1A and specific enrichment of this protein in the presence of its native histone substrates. Affinity purification pulldown experiments from nuclear lysate coupled with mass spectrometry revealed the capability of the probe to pull out and enrich JHDMs along with other epigenetic proteins and transcriptional regulators. PMID:25335116

Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

PubMed

Zanetti-Domingues, Laura C; Tynan, Christopher J; Rolfe, Daniel J; Clarke, David T; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

PubMed Central

Rolfe, Daniel J.; Clarke, David T.; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion. PMID:24066121

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor.

PubMed

Perrone, Maria Grazia; Inglese, Carmela; Berardi, Francesco; Leopoldo, Marcello; Perrone, Roberto; Colabufo, Nicola Antonio

2013-01-01

P-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood-brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1-40) HiLyte Fluor™ TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine. Male Wistar rats' everted intestines were divided into sacs, each sac was filled with 10μM Ab-HiLyte with or without 50μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594nm at each appropriate time. The Ab-HiLyte P-gp mediated efflux had a K=1.00×10(-2)min(-1) and t(1/2)=68.74min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K=1.65×10(-3)min(-1)) and the half life is extremely increased (t(1/2)=419min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K=1.81×10(-2)min(-1) and t(1/2)=38.28min). The results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound. Copyright © 2013 Elsevier Inc. All rights reserved.

A fluorogenic substrate of beta-lactamases and its potential as a probe to detect the bacteria resistant to the third-generation oxyimino-cephalosporins.

PubMed

Thai, Hien Bao Dieu; Yu, Jin Kyung; Park, Byung Sun; Park, Yeon-Joon; Min, Sun-Joon; Ahn, Dae-Ro

2016-03-15

We devised and synthesized a fluorogenic substrate of β-lactamases as a probe to detect the activity of the enzymes. Fluorescence of the probe emitted upon treatment of a β-lactamase and increased proportionally to the concentration of the enzyme, demonstrating its sensing property for the activity of the enzyme. We also showed that the probe could be utilized to assay the enzyme and to determine kinetic parameters of the enzyme. Moreover, the probe was able to detect resistance to the third-generation oxyimino-cephalosporin-derived antibiotics such as cefotaxime and ceftazidime. In particular, the probe could identify the ceftazidime-resistance in bacteria that was not detectable using conventional pH-sensing materials, indicating the practical utility of the probe. Copyright © 2015 Elsevier B.V. All rights reserved.

Vacuum scanning capillary photoemission microscopy.

PubMed

Aseyev, S A; Cherkun, A P; Mironov, B N; Petrunin, V V; Chekalin, S V

2017-08-01

We demonstrate the use of a conical capillary in a scanning probe microscopy for surface analysis. The probe can measure photoemission from a substrate by transmitting photoelectrons along the capillary as a function of probe position. The technique is demonstrated on a model substrate consisting of a gold reflecting layer on a compact disc which has been illuminated by an unfocused laser beam with a wavelength 400nm, from a femtosecond laser with a beam size of 4mm. A quartz capillary with a 2-µm aperture has been used in the experiments. The period of gold microstructure, shown to be 1.6µ, was measured by the conical probe operating in shear force mode. In shear force regime, the dielectric capillary has been used as a "classical" SPM tip, which provided images reflecting the surface topology. In a photoelectron regime photoelectrons passed through hollow tip and entered a detector. The spatial distribution of the recorded photoelectrons consisted of periodic mountain-valley strips, resembling the surface profile of the sample. Submicron spatial resolution has been achieved. This approach paves the way to study pulsed photodesorption of large organic molecular ions with high spatial and element resolution using the combination of a hollow-tip scanner with time-of-flight technique. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of a biomimetic 3D substrate based on the Human Elastin-like Polypeptides (HELPs) model system for elastolytic activity detection.

PubMed

Corich, Lucia; Busetti, Marina; Petix, Vincenzo; Passamonti, Sabina; Bandiera, Antonella

2017-08-10

Elastin is a fibrous protein that confers elasticity to tissues such as skin, arteries and lung. It is extensively cross-linked, highly hydrophobic and insoluble. Nevertheless, elastin can be hydrolysed by bacterial proteases in infectious diseases, resulting in more or less severe tissue damage. Thus, development of substrates able to reliably and specifically detect pathogen-secreted elastolytic activity is needed to improve the in vitro evaluation of the injury that bacterial proteases may provoke. In this work, two human biomimetic elastin polypeptides, HELP and HELP1, as well as the matrices derived from HELP, have been probed as substrates for elastolytic activity detection. Thirty strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were analyzed in parallel with standard substrates, to detect proteolytic and elastolytic activity. Results point to the HELP-based 3D matrix as an interesting biomimetic model of elastin to assess bacterial elastolytic activity in vitro. Moreover, this model substrate enables to further elucidate the mechanism underlying elastin degradation at molecular level, as well as to develop biomimetic material-based devices responsive to external stimuli. Copyright © 2017 Elsevier B.V. All rights reserved.

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

PubMed

Lopes, Jose L S; Beltramini, Leila M; Wallace, Bonnie A; Araujo, Ana P U

2015-01-01

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Brominated Luciferins Are Versatile Bioluminescent Probes

DOE PAGES

Steinhardt, Rachel C.; Rathbun, Colin M.; Krull, Brandon T.; ...

2016-12-08

Here, we report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.

Substrate-dependent denitrification of abundant probe-defined denitrifying bacteria in activated sludge.

PubMed

Morgan-Sagastume, Fernando; Nielsen, Jeppe Lund; Nielsen, Per Halkjaer

2008-11-01

The denitrification capacity of different phylogenetic bacterial groups was investigated on addition of different substrates in activated sludge from two nutrient-removal plants. Nitrate/nitrite consumption rates (CRs) were calculated from nitrate and nitrite biosensor, in situ measurements. The nitrate/nitrite CRs depended on the substrate added, and acetate alone or combined with other substrates yielded the highest rates (3-6 mg N gVSS(-1) h(-1)). The nitrate CRs were similar to the nitrite CRs for most substrates tested. The structure of the active denitrifying population was investigated using heterotrophic CO2 microautoradiography (HetCO2-MAR) and FISH. Probe-defined denitrifiers appeared as specialized substrate utilizers despite acetate being preferentially used by most of them. Azoarcus and Accumulibacter abundance in the two different sludges was related to differences in their substrate-specific nitrate/nitrite CRs. Aquaspirillum-related bacteria were the most abundant potential denitrifiers (c. 20% of biovolume); however, Accumulibacter (3-7%) and Azoarcus (2-13%) may have primarily driven denitrification by utilizing pyruvate, ethanol, and acetate. Activated sludge denitrification was potentially conducted by a diverse, versatile population including not only Betaproteobacteria (Aquaspirillum, Thauera, Accumulibacter, and Azoarcus) but also some Alphaproteobacteria and Gammaproteobacteria, as indicated by the assimilation of 14CO2 by these probe-defined groups with a complex substrate mixture as an electron donor and nitrite as an electron acceptor in HetCO2-MAR-FISH tests.

Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria.

PubMed

Alcaine, S D; Pacitto, D; Sela, D A; Nugen, S R

2015-11-21

Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes.

PubMed

Yeung, John H K; Or, Penelope M Y

2012-03-15

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms. Copyright © 2011 Elsevier GmbH. All rights reserved.

TMG-chitotriomycin as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases.

PubMed

Shiota, Hiroto; Kanzaki, Hiroshi; Hatanaka, Tadashi; Nitoda, Teruhiko

2013-06-28

TMG-chitotriomycin (1) produced by the actinomycete Streptomyces annulatus NBRC13369 was examined as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases (HexNAcases). According to the results of inhibition assays, 14 GH20 HexNAcases from various organisms were divided into 1-sensitive and 1-insensitive enzymes. Three representatives of each group were investigated for their substrate specificity. The 1-sensitive HexNAcases hydrolyzed N-acetylchitooligosaccharides but not N-glycan-type oligosaccharides, whereas the 1-insensitive enzymes hydrolyzed N-glycan-type oligosaccharides but not N-acetylchitooligosaccharides, indicating that TMG-chitotriomycin can be used as a molecular probe to distinguish between chitin-degrading HexNAcases and glycoconjugate-processing HexNAcases. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Phagostimulant Blend for the Asian Citrus Psyllid.

PubMed

Lapointe, Stephen L; Hall, David G; George, Justin

2016-09-01

Chemical cues that elicit orientation by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), are of interest because it is the primary vector of the causal pathogen of citrus greening disease. Non-pesticidal control methods for D. citri remain a high priority for the citrus industry. While searching for semiochemicals that may be involved in orientation to host plants, we previously identified a blend of formic and acetic acids that stimulated substrate probing by D. citri. Here, we applied geometric mixture designs and response surface modeling to identify and optimize a 3-component blend that further increased the number of salivary sheaths produced by D. citri on a wax substrate containing a 3.5:1.6:1 blend of formic acid, acetic acid, and p-cymene, respectively. No evidence was found for remote orientation by D. citri adults through olfaction to the phagostimulant blends. Increased probing in response to the presence of phagostimulants in the wax matrix occurred after contact with the substrate. Yellow wax beads always attracted more D. citri adults and received more probes compared with white wax beads. Yellow beads containing the 3-component blend of phagostimulants were probed by D. citri 2 to 3 times more often compared with yellow beads alone. The phagostimulant effect also was tested by covering wax beads containing the 3-component blend with a plastic film to minimize olfaction or contact chemoreception by antennation. The plastic film did not affect the probing response, thus suggesting that chemosensation was associated with mouthparts and not olfactory receptors. Salivary sheaths produced in wax beads containing the phagostimulant blend were 4.5 times longer than sheaths produced in beads without tastants. This phenomenon might be used to improve a trap, design an attract-and-kill product, or enhance other means of managing D. citri and citrus greening disease.

Determining confounding sensitivities in eddy current thin film measurements

NASA Astrophysics Data System (ADS)

Gros, Ethan; Udpa, Lalita; Smith, James A.; Wachs, Katelyn

2017-02-01

Eddy current (EC) techniques are widely used in industry to measure the thickness of non-conductive films on a metal substrate. This is done by using a system whereby a coil carrying a high-frequency alternating current is used to create an alternating magnetic field at the surface of the instrument's probe. When the probe is brought near a conductive surface, the alternating magnetic field will induce ECs in the conductor. The substrate characteristics and the distance of the probe from the substrate (the coating thickness) affect the magnitude of the ECs. The induced currents load the probe coil affecting the terminal impedance of the coil. The measured probe impedance is related to the lift off between coil and conductor as well as conductivity of the test sample. For a known conductivity sample, the probe impedance can be converted into an equivalent film thickness value. The EC measurement can be confounded by a number of measurement parameters. It was the goal of this research to determine which physical properties of the measurement set-up and sample can adversely affect the thickness measurement. The eddy-current testing was performed using a commercially available, hand-held eddy-current probe (ETA3.3H spring-loaded eddy probe running at 8 MHz) that comes with a stand to hold the probe. The stand holds the probe and adjusts the probe on the z-axis to help position the probe in the correct area as well as make precise measurements. The signal from the probe was sent to a hand-held readout, where the results are recorded directly in terms of liftoff or film thickness. Understanding the effect of certain factors on the measurements of film thickness, will help to evaluate how accurate the ETA3.3H spring-loaded eddy probe was at measuring film thickness under varying experimental conditions. This research studied the effects of a number of factors such as i) conductivity, ii) edge effect, iii) surface finish of base material and iv) cable condition.

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells.

PubMed

Zhang, Jingtuo; Li, Cong; Dutta, Colina; Fang, Mingxi; Zhang, Shuwei; Tiwari, Ashutosh; Werner, Thomas; Luo, Fen-Tair; Liu, Haiying

2017-05-22

A novel near-infrared fluorescent probe for β-galactosidase has been developed based on a hemicyanine skeleton, which is conjugated with a d-galactose residue via a glycosidic bond. The probe serves as a substrate of β-galactosidase and displays rapid and sensitive turn-on fluorescent responses to β-galactosidase in aqueous solution. A 12.8-fold enhancement of fluorescence intensity at 703 nm was observed after incubation of 10 nM of β-galactosidase with 5 μM probe for 10 min. The probe can sensitively detect as little as 0.1 nM of β-galactosidase and shows linear responses to the enzyme concentration below 1.4 nM. The kinetic study showed that the probe has high binding affinity to β-galactosidase with K m  = 3.6 μM. The probe was used to detect β-galactosidase in living cells by employing the premature cell senescence model. The probe exhibited strong fluorescent signals in senescent cells but not in normal cells, which demonstrates that the probe is able to detect the endogenous senescence-associated β-galactosidase in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

A micro-mechanical model to determine changes of collagen fibrils under cyclic loading

NASA Astrophysics Data System (ADS)

Chen, Michelle L.; Susilo, Monica E.; Ruberti, Jeffrey A.; Nguyen, Thao D.

Dynamic mechanical loading induces growth and remodeling in biological tissues. It can alter the degradation rate and intrinsic mechanical properties of collagen through cellular activity. Experiments showed that repeated cyclic loading of a dense collagen fibril substrate increased collagen stiffness and strength, lengthened the substrate, but did not significantly change the fibril areal fraction or fibril anisotropy (Susilo, et al. ``Collagen Network Hardening Following Cyclic Tensile Loading'', Interface Focus, submitted). We developed a model for the collagen fibril substrate (Tonge, et al. ``A micromechanical modeling study of the mechanical stabilization of enzymatic degradation of collagen tissues'', Biophys J, in press.) to probe whether changes in the fibril morphology and mechanical properties can explain the tissue-level properties observed during cyclic loading. The fibrils were modeled as a continuous distribution of wavy elastica, based on experimental measurements of fibril density and collagen anisotropy, and can experience damage after a critical stress threshold. Other mechanical properties in the model were fit to the stress response measured before and after the extended cyclic loading to determine changes in the strength and stiffness of collagen fibrils.

Multi-Wall Carbon Nanotubes as Lithium Nanopipettes and SPM Probes

NASA Astrophysics Data System (ADS)

Larson, Jonathan; Bharath, Satyaveda; Cullen, William; Reutt-Robey, Janice

2014-03-01

A multi-walled carbon nanotube (MWCNT) - terminated SPM cantilever, was utilized to perform nanolithography and surface diffusion measurements on a thin film of vapor-deposited lithium atop a silicon (111) substrate under ultra-high vacuum conditions. In these investigations the MWCNT tip was shown to act as both a lithium nanopipette and a probe for non-contact atomic force microscopy (NC-AFM) measurements. With the application of appropriate bias conditions, the MWCNT could site-selectively extract (expel) nano-scale amounts of lithium from (to) the sample surface. Depressions, mounds, and spikes were generated on the surface in this way and were azimuthally symmetric about the selected point of pipetting. Following lithium transfer to/from the substrate, the MWCNT pipette-induced features were sequentially imaged with NC-AFM using the MWCNT as the probe. Vacancy pits of ca. 300 nm diameter and 1.5 nm depth were observed to decay on a timescale of hours at room temperature, through diffusion-limited decay processes. A continuum model was utilized to simulate the island decay rates, and the lithium surface diffusion coefficient of D =7.5 (+/-1.3)*10-15 cm2/s was extracted. U.S. Department of Energy Award Number DESC0001160.

Application of spatial time domain reflectometry measurements in heterogeneous, rocky substrates

NASA Astrophysics Data System (ADS)

Gonzales, C.; Scheuermann, A.; Arnold, S.; Baumgartl, T.

2016-10-01

Measurement of soil moisture across depths using sensors is currently limited to point measurements or remote sensing technologies. Point measurements have limitations on spatial resolution, while the latter, although covering large areas may not represent real-time hydrologic processes, especially near the surface. The objective of the study was to determine the efficacy of elongated soil moisture probes—spatial time domain reflectometry (STDR)—and to describe transient soil moisture dynamics of unconsolidated mine waste rock materials. The probes were calibrated under controlled conditions in the glasshouse. Transient soil moisture content was measured using the gravimetric method and STDR. Volumetric soil moisture content derived from weighing was compared with values generated from a numerical model simulating the drying process. A calibration function was generated and applied to STDR field data sets. The use of elongated probes effectively assists in the real-time determination of the spatial distribution of soil moisture. It also allows hydrologic processes to be uncovered in the unsaturated zone, especially for water balance calculations that are commonly based on point measurements. The elongated soil moisture probes can potentially describe transient substrate processes and delineate heterogeneity in terms of the pore size distribution in a seasonally wet but otherwise arid environment.

Correlation between He-Ne scatter and 2.7-microm pulsed laser damage at coating defects.

PubMed

Porteus, J O; Spiker, C J; Franck, J B

1986-11-01

A reported correlation between defect-initiated pulsed laser damage and local predamage scatter in multilayer infrared mirror coatings has been analyzed in detail. Examination of a much larger data base confirms the previous result on dielectric-enhanced reflectors with polished substrates over a wide range of energy densities above the damage onset. Scatter signals from individual undamaged defects were detected using a He-Ne scatter probe with a focal spot that nearly coincides with the 150-microm-diam (D1/e(2)) focal spot of the damage-probe beam. Subsequent damage frequency measurements (1-on-1) were made near normal or at 45 degrees incidence with 100-ns pulses at 2.7-microm wavelength. The correlation is characterized by an increase in damage frequency with increasing predamage scatter signal and by equivalence of the defect densities indicated by the two probes. Characteristics of the correlation are compared with a simple model based on focal spot intensity profiles. Conditions that limit correlation are discussed, including variable scatter from defects and background scatter from diamond-turned substrates. Results have implication for nondestructive defect detection and coating quality control.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe.

PubMed

Lee, Hyeran; Akers, Walter J; Cheney, Philip P; Edwards, W Barry; Liang, Kexian; Culver, Joseph P; Achilefu, Samuel

2009-01-01

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with (64)Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters k(cat) and K(M) of 0.55+/-0.01 s(-1) and 1.12+/-0.06 microM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled (64)Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

NASA Astrophysics Data System (ADS)

Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

2009-07-01

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55+/-0.01 s-1 and 1.12+/-0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Study the friction behaviour of poly[2-(dimethylamino)ethyl methacrylate] brush with AFM probes in contact mechanics

NASA Astrophysics Data System (ADS)

Raftari, Maryam; Zhang, Zhenyu; Leggett, Graham J.; Geoghegan, Mark

2011-10-01

We have studied the frictional behaviour of grafted poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) films using friction force microscopy (FFM). The films were prepared on native oxide-terminated silicon substrates using the technique of atom transfer radical polymerization (ATRP). We show that single asperity contact mechanics (Johnson-Kendall-Roberts(JKR) and Derjaguin-Muller-Toporov(DMT)) as well as a linear (Amontons) relation between applied load and frictional load depending on the pH of the FFM probe. Measurements were made using functionalized and unfunctionalized silicon nitride triangular probes. Functionalized probes included gold-coated probes, and ones coated with a self-assembled monolayer of dodecanethiol (DDT). The frictional behaviour between PDMAEMA and all tips immersed in pH from 3 to 11 are corresponded to the DMT or JKR model and are linear in pH=1, 2, and 12. These results show that contact mechanics of polyelectrolytes in water is complex and strongly dependent on the environmental pH.

Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis

PubMed Central

Abe, Kouki; Katsuno, Hiroko; Toriyama, Michinori; Baba, Kentarou; Mori, Tomoyuki; Hakoshima, Toshio; Kanemura, Yonehiro; Watanabe, Rikiya; Inagaki, Naoyuki

2018-01-01

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia. PMID:29483251

Novel SiO2-deposited CaF2 substrate for vibrational sum-frequency generation (SFG) measurements of chemisorbed monolayers in an aqueous environment.

PubMed

Padermshoke, Adchara; Konishi, Shouta; Ara, Masato; Tada, Hirokazu; Ishibashi, Taka-Aki

2012-06-01

A novel SiO(2)-deposited CaF(2) (SiO(2)/CaF(2)) substrate for measuring vibrational sum-frequency generation (SFG) spectra of silane-based chemisorbed monolayers in aqueous media has been developed. The substrate is suitable for silanization and transparent over a broad range of the infrared (IR) probe. The present work demonstrates the practical application of the SiO(2)/CaF(2) substrate and, to our knowledge, the first SFG spectrum at the solid/water interface of a silanized monolayer observed over the IR fingerprint region (1780-1400 cm(-1)) using a back-side probing geometry. This new substrate can be very useful for SFG studies of various chemisorbed organic molecules, particularly biological compounds, in aqueous environments.

Detection of protease activity in cells and animals.

PubMed

Verdoes, Martijn; Verhelst, Steven H L

2016-01-01

Proteases are involved in a wide variety of biologically and medically important events. They are entangled in a complex network of processes that regulate their activity, which makes their study intriguing, but challenging. For comprehensive understanding of protease biology and effective drug discovery, it is therefore essential to study proteases in models that are close to their complex native environments such as live cells or whole organisms. Protease activity can be detected by reporter substrates and activity-based probes, but not all of these reagents are suitable for intracellular or in vivo use. This review focuses on the detection of proteases in cells and in vivo. We summarize the use of probes and substrates as molecular tools, discuss strategies to deliver these tools inside cells, and describe sophisticated read-out techniques such as mass spectrometry and various imaging applications. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Local electrical properties of n-AlInAs/i-GaInAs electron channel structures characterized by the probe-electron-beam-induced current technique.

PubMed

Watanabe, Kentaro; Nokuo, Takeshi; Chen, Jun; Sekiguchi, Takashi

2014-04-01

We developed a probe-electron-beam-induced current (probe-EBIC) technique to investigate the electrical properties of n-Al(0.48)In(0.52)As/i-Ga(0.30)In(0.70)As electron channel structures for a high-electron-mobility transistor, grown on a lattice-matched InP substrate and lattice-mismatched GaAs (001) and Si (001) substrates. EBIC imaging of planar surfaces at low magnifications revealed misfit dislocations originating from the AlInAs-graded buffer layer. The cross-sections of GaInAs channel structures on an InP substrate were studied by high-magnification EBIC imaging as well as cathodoluminescence (CL) spectroscopy. EBIC imaging showed that the structure is nearly defect-free and the carrier depletion zone extends from the channel toward the i-AlInAs buffer layer.

Electrochemical biosensor based on enzyme substrate as a linker: Application for aldolase activity with pectin-thionine complex as recognization element and signal amplification probe.

PubMed

Wang, Xiaonan; Wang, Meiwen; Zhang, Yuanyuan; Miao, Xiaocao; Huang, Yuanyuan; Zhang, Juan; Sun, Lizhou

2016-09-15

A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate, thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe. The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p-phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability. Copyright © 2016 Elsevier B.V. All rights reserved.

Surface potential measurement of n-type organic semiconductor thin films by mist deposition via Kelvin probe microscopy

NASA Astrophysics Data System (ADS)

Odaka, Akihiro; Satoh, Nobuo; Katori, Shigetaka

2017-08-01

We partially deposited fullerene (C60) and phenyl-C61-butyric acid methyl ester thin films that are typical n-type semiconductor materials on indium-tin oxide by mist deposition at various substrate temperatures. The topographic and surface potential images were observed via dynamic force microscopy/Kelvin probe force microscopy with the frequency modulation detection method. We proved that the area where a thin film is deposited depends on the substrate temperature during deposition from the topographic images. It was also found that the surface potential depends on the substrate temperature from the surface potential images.

Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells.

PubMed

Kim, Sung Bae; Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

2016-02-17

Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.

Pump-probe surface photovoltage spectroscopy measurements on semiconductor epitaxial layers.

PubMed

Jana, Dipankar; Porwal, S; Sharma, T K; Kumar, Shailendra; Oak, S M

2014-04-01

Pump-probe Surface Photovoltage Spectroscopy (SPS) measurements are performed on semiconductor epitaxial layers. Here, an additional sub-bandgap cw pump laser beam is used in a conventional chopped light geometry SPS setup under the pump-probe configuration. The main role of pump laser beam is to saturate the sub-bandgap localized states whose contribution otherwise swamp the information related to the bandgap of material. It also affects the magnitude of Dember voltage in case of semi-insulating (SI) semiconductor substrates. Pump-probe SPS technique enables an accurate determination of the bandgap of semiconductor epitaxial layers even under the strong influence of localized sub-bandgap states. The pump beam is found to be very effective in suppressing the effect of surface/interface and bulk trap states. The overall magnitude of SPV signal is decided by the dependence of charge separation mechanisms on the intensity of the pump beam. On the contrary, an above bandgap cw pump laser can be used to distinguish the signatures of sub-bandgap states by suppressing the band edge related feature. Usefulness of the pump-probe SPS technique is established by unambiguously determining the bandgap of p-GaAs epitaxial layers grown on SI-GaAs substrates, SI-InP wafers, and p-GaN epilayers grown on Sapphire substrates.

Discovery of Selective, Substrate-Competitive, and Passive Membrane Permeable Glycogen Synthase Kinase-3β Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling of New C-Glycosylflavones.

PubMed

Liang, Zhibin; Li, Qing X

2018-05-16

Glycogen synthase kinase-3β (GSK-3β) is a key enzyme responsible for tau hyperphosphorylation and is a viable therapeutic target of Alzheimer's disease (AD). We developed a new class of GSK-3β inhibitors based on the 6- C-glycosylflavone isoorientin (1). The new inhibitors are passive membrane permeable and constitutively attenuate GSK-3β mediated tau hyperphosphorylation and amyloid neurotoxicity in an AD cellular model. Enzymatic assays and kinetic studies demonstrated that compound 30 is a GSK-3β substrate-competitive inhibitor with distinct kinase selectivity, isoform-selectivity and over 310-fold increased potency as compared to 1. Structure-activity relationship analyses and in silico modeling suggest the mechanism of actions by which the hydrophobic, π-cation, and orthogonal multipolar interactions of 30 with the substrate site are critical for the GSK-3β inhibition and selectivity. The results provide new insights into GSK-3β drug discovery. The new inhibitors are valuable chemical probes and drug leads with therapeutic potential to tackle AD and other GSK-3β relevant diseases.

Comparison of silicon, nickel, and nickel silicide (Ni 3Si) as substrates for epitaxial diamond growth

NASA Astrophysics Data System (ADS)

Tucker, D. A.; Seo, D.-K.; Whangbo, M.-H.; Sivazlian, F. R.; Stoner, B. R.; Bozeman, S. P.; Sowers, A. T.; Nemanich, R. J.; Glass, J. T.

1995-07-01

We carried out experimental and theoretical studies aimed at probing interface interactions of diamond with Si, Ni, and Ni 3Si substrates. Oriented diamond films deposited on (100) silicon were characterized by polar Raman, polar XRD, and cross-sectional HRTEM. These studies show that the diamond-(100)/Si(100) interface does not adopt the 45°-rotation but the 3 : 2-match arrangement. Our extended Hückel tight-binding (EHTB) electronic structure calculations for a model system show that the interface interaction favors the 3 : 2-match arrangement. Growth on polycrystalline Ni 3Si resulted in oriented diamond particles while, under the same growth conditions, largely graphite was formed on the nickel substrate. Our EHTB electronic structure calculations for model systems show that the (111) and (100) surfaces of Ni 3Si have a strong preference for diamond-nucleation over graphite-nucleation, but this is not the case for the (111) and (100) surfaces of Ni.

Heme Structural Perturbation of PEG-Modified Horseradish Peroxidase C in Aromatic Organic Solvents Probed by Optical Absorption and Resonance Raman Dispersion Spectroscopy

PubMed Central

Huang, Qing; Al-Azzam, Wasfi; Griebenow, Kai; Schweitzer-Stenner, Reinhard

2003-01-01

The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B1g distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme's pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B2g-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP. PMID:12719258

Probing the bulk ionic conductivity by thin film hetero-epitaxial engineering

NASA Astrophysics Data System (ADS)

Pergolesi, Daniele; Roddatis, Vladimir; Fabbri, Emiliana; Schneider, Christof W.; Lippert, Thomas; Traversa, Enrico; Kilner, John A.

2015-02-01

Highly textured thin films with small grain boundary regions can be used as model systems to directly measure the bulk conductivity of oxygen ion conducting oxides. Ionic conducting thin films and epitaxial heterostructures are also widely used to probe the effect of strain on the oxygen ion migration in oxide materials. For the purpose of these investigations a good lattice matching between the film and the substrate is required to promote the ordered film growth. Moreover, the substrate should be a good electrical insulator at high temperature to allow a reliable electrical characterization of the deposited film. Here we report the fabrication of an epitaxial heterostructure made with a double buffer layer of BaZrO3 and SrTiO3 grown on MgO substrates that fulfills both requirements. Based on such template platform, highly ordered (001) epitaxially oriented thin films of 15% Sm-doped CeO2 and 8 mol% Y2O3 stabilized ZrO2 are grown. Bulk conductivities as well as activation energies are measured for both materials, confirming the success of the approach. The reported insulating template platform promises potential application also for the electrical characterization of other novel electrolyte materials that still need a thorough understanding of their ionic conductivity.

Monitoring transients in low inductance circuits

DOEpatents

Guilford, Richard P.; Rosborough, John R.

1987-01-01

A pair of flat cable transmission lines are monitored for transient current spikes by using a probe connected to a current transformer by a pickup loop and monitoring the output of the current transformer. The approach utilizes a U-shaped pickup probe wherein the pair of flat cable transmission lines are received between the legs of the U-shaped probe. The U-shaped probe is preferably formed of a flat coil conductor adhered to one side of a flexible substrate. On the other side of the flexible substrate there is a copper foil shield. The copper foil shield is connected to one end of the flat conductor coil and connected to one leg of the pickup loop which passes through the current transformer. The other end of the flat conductor coil is connected to the other leg of the pickup loop.

Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

DOEpatents

Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

1992-01-01

An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

DOEpatents

Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

1993-01-01

An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

Heat transfer measurements on biconics at incidence in hypersonic high enthalpy air and nitrogen flows

NASA Technical Reports Server (NTRS)

Gai, S. L.; Cain, T.; Joe, W. S.; Sandeman, R. J.; Miller, C. G.

1988-01-01

Heat transfer rate measurements have been obtained at 0, 5, 15, and 21 deg angles-of-attack for a straight biconic scale model of an aeroassisted orbital vehicle proposed for planetary probe missions. Heat-transfer distributions were measured using palladium thin-film resistance gauges deposited on a glass-ceramic substrate. The windward heat transfer correlations were based on equilibrium flow in the shock layer of the model, although the flow may depart from equilibrium in the flow-field.

An algorithm for temperature correcting substrate moisture measurements: aligning substrate moisture responses with environmental drivers in polytunnel-grown strawberry plants

NASA Astrophysics Data System (ADS)

Goodchild, Martin; Janes, Stuart; Jenkins, Malcolm; Nicholl, Chris; Kühn, Karl

2015-04-01

The aim of this work is to assess the use of temperature corrected substrate moisture data to improve the relationship between environmental drivers and the measurement of substrate moisture content in high porosity soil-free growing environments such as coir. Substrate moisture sensor data collected from strawberry plants grown in coir bags installed in a table-top system under a polytunnel illustrates the impact of temperature on capacitance-based moisture measurements. Substrate moisture measurements made in our coir arrangement possess the negative temperature coefficient of the permittivity of water where diurnal changes in moisture content oppose those of substrate temperature. The diurnal substrate temperature variation was seen to range from 7° C to 25° C resulting in a clearly observable temperature effect in substrate moisture content measurements during the 23 day test period. In the laboratory we measured the ML3 soil moisture sensor (ThetaProbe) response to temperature in Air, dry glass beads and water saturated glass beads and used a three-phase alpha (α) mixing model, also known as the Complex Refractive Index Model (CRIM), to derive the permittivity temperature coefficients for glass and water. We derived the α value and estimated the temperature coefficient for water - for sensors operating at 100MHz. Both results are good agreement with published data. By applying the CRIM equation with the temperature coefficients of glass and water the moisture temperature coefficient of saturated glass beads has been reduced by more than an order of magnitude to a moisture temperature coefficient of

Scanning MWCNT-Nanopipette and Probe Microscopy: Li Patterning and Transport Studies.

PubMed

Larson, Jonathan M; Bharath, Satyaveda C; Cullen, William G; Reutt-Robey, Janice E

2015-10-07

A carbon-nanotube-enabling scanning probe technique/nanotechnology for manipulating and measuring lithium at the nano/mesoscale is introduced. Scanning Li-nanopipette and probe microscopy (SLi-NPM) is based on a conductive atomic force microscope (AFM) cantilever with an open-ended multi-walled carbon nanotube (MWCNT) affixed to its apex. SLi-NPM operation is demonstrated with a model system consisting of a Li thin film on a Si(111) substrate. By control of bias, separation distance, and contact time, attograms of Li can be controllably pipetted to or from the MWCNT tip. Patterned surface Li features are then directly probed via noncontact AFM measurements with the MWCNT tip. The subsequent decay of Li features is simulated with a mesoscale continuum model, developed here. The Li surface diffusion coefficient for a four (two) Li layer thick film is measured as D=8(±1.2)×10(-15) cm(2) s(-1) (D=1.75(±0.15)×10(-15) cm(2) s(-1)). Dual-Li pipetting/measuring with SLi-NPM enables a broad range of time-dependent Li and nanoelectrode characterization studies of fundamental importance to energy-storage research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling linear and cyclic PKS intermediates through atom replacement.

PubMed

Shakya, Gaurav; Rivera, Heriberto; Lee, D John; Jaremko, Matt J; La Clair, James J; Fox, Daniel T; Haushalter, Robert W; Schaub, Andrew J; Bruegger, Joel; Barajas, Jesus F; White, Alexander R; Kaur, Parminder; Gwozdziowski, Emily R; Wong, Fiona; Tsai, Shiou-Chuan; Burkart, Michael D

2014-12-03

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs.

Vacuum Head Checks Foam/Substrate Bonds

NASA Technical Reports Server (NTRS)

Lloyd, James F.

1989-01-01

Electromechanical inspection system quickly gives measurements indicating adhesion, or lack thereof, between rigid polyurethane foam and aluminum substrate. Does not damage inspected article, easy to operate, and used to perform "go/no-go" evaluations or as supplement to conventional destructive pull-plug testing. Applies vacuum to small area of foam panel and measures distance through which foam pulled into vacuum. Probe head applied to specimen and evacuated through hose to controller/monitor unit. Digital voltmeter in unit reads deflection of LVDT probe head.

Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

DOEpatents

Ramsey, J. Michael; Foote, Robert S.

2003-12-09

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

DOEpatents

Ramsey, J. Michael; Foote, Robert S.

2002-01-01

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

Piezo-thermal Probe Array for High Throughput Applications

PubMed Central

Gaitas, Angelo; French, Paddy

2012-01-01

Microcantilevers are used in a number of applications including atomic-force microscopy (AFM). In this work, deflection-sensing elements along with heating elements are integrated onto micromachined cantilever arrays to increase sensitivity, and reduce complexity and cost. An array of probes with 5–10 nm gold ultrathin film sensors on silicon substrates for high throughput scanning probe microscopy is developed. The deflection sensitivity is 0.2 ppm/nm. Plots of the change in resistance of the sensing element with displacement are used to calibrate the probes and determine probe contact with the substrate. Topographical scans demonstrate high throughput and nanometer resolution. The heating elements are calibrated and the thermal coefficient of resistance (TCR) is 655 ppm/K. The melting temperature of a material is measured by locally heating the material with the heating element of the cantilever while monitoring the bending with the deflection sensing element. The melting point value measured with this method is in close agreement with the reported value in literature. PMID:23641125

Method and apparatus for synthesis of arrays of DNA probes

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

Cerrina, Francesco; Sussman, Michael R.; Blattner, Frederick R.

The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing an image former that includes a light source that provides light to a micromirror device comprising an array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two positions. Projection optics receives the light reflected from the micromirrors along an optical axis and precisely images the micromirrors onto the active surface of the substrate, whichmore » may be used to activate the surface of the substrate. The first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different pattern of micromirrors, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto. The micromirror array can be controlled in conjunction with a DNA synthesizer supplying appropriate reagents to a flow cell containing the active substrate to control the sequencing of images presented by the micromirror array in coordination of the reagents provided to the substrate.« less

Design, fabrication and experimental validation of a novel dry-contact sensor for measuring electroencephalography signals without skin preparation.

PubMed

Liao, Lun-De; Wang, I-Jan; Chen, Sheng-Fu; Chang, Jyh-Yeong; Lin, Chin-Teng

2011-01-01

In the present study, novel dry-contact sensors for measuring electro-encephalography (EEG) signals without any skin preparation are designed, fabricated by an injection molding manufacturing process and experimentally validated. Conventional wet electrodes are commonly used to measure EEG signals; they provide excellent EEG signals subject to proper skin preparation and conductive gel application. However, a series of skin preparation procedures for applying the wet electrodes is always required and usually creates trouble for users. To overcome these drawbacks, novel dry-contact EEG sensors were proposed for potential operation in the presence or absence of hair and without any skin preparation or conductive gel usage. The dry EEG sensors were designed to contact the scalp surface with 17 spring contact probes. Each probe was designed to include a probe head, plunger, spring, and barrel. The 17 probes were inserted into a flexible substrate using a one-time forming process via an established injection molding procedure. With these 17 spring contact probes, the flexible substrate allows for high geometric conformity between the sensor and the irregular scalp surface to maintain low skin-sensor interface impedance. Additionally, the flexible substrate also initiates a sensor buffer effect, eliminating pain when force is applied. The proposed dry EEG sensor was reliable in measuring EEG signals without any skin preparation or conductive gel usage, as compared with the conventional wet electrodes.

Design, Fabrication and Experimental Validation of a Novel Dry-Contact Sensor for Measuring Electroencephalography Signals without Skin Preparation

PubMed Central

Liao, Lun-De; Wang, I-Jan; Chen, Sheng-Fu; Chang, Jyh-Yeong; Lin, Chin-Teng

2011-01-01

In the present study, novel dry-contact sensors for measuring electro-encephalography (EEG) signals without any skin preparation are designed, fabricated by an injection molding manufacturing process and experimentally validated. Conventional wet electrodes are commonly used to measure EEG signals; they provide excellent EEG signals subject to proper skin preparation and conductive gel application. However, a series of skin preparation procedures for applying the wet electrodes is always required and usually creates trouble for users. To overcome these drawbacks, novel dry-contact EEG sensors were proposed for potential operation in the presence or absence of hair and without any skin preparation or conductive gel usage. The dry EEG sensors were designed to contact the scalp surface with 17 spring contact probes. Each probe was designed to include a probe head, plunger, spring, and barrel. The 17 probes were inserted into a flexible substrate using a one-time forming process via an established injection molding procedure. With these 17 spring contact probes, the flexible substrate allows for high geometric conformity between the sensor and the irregular scalp surface to maintain low skin-sensor interface impedance. Additionally, the flexible substrate also initiates a sensor buffer effect, eliminating pain when force is applied. The proposed dry EEG sensor was reliable in measuring EEG signals without any skin preparation or conductive gel usage, as compared with the conventional wet electrodes. PMID:22163929

Bridging of a substrate between cyclodextrin and an enzyme’s active site pocket triggers a unique mode of inhibition

PubMed Central

Sule, Nitesh V; Ugrinov, Angel; Mallik, Sanku; Srivastava, D. K.

2014-01-01

Background Methionyl-7-amino-4-methylcoumarin (MetAMC) serves as a substrate for the E. coli Methionine aminopeptidase (MetAP) catalyzed reaction, and is routinely used for screening compounds to identify potential antibiotic agents. In pursuit of screening the enzyme’s inhibitors, we observed that 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), utilized to solubilize hydrophobic inhibitors, inhibited the catalytic activity of the enzyme, and such inhibition was not solely due to sequestration of the substrate by HP-β-CD. Methods The mechanistic path for the HP-β-CD mediated inhibition of MetAP was probed by performing a detailed account of steady-state kinetics, ligand binding, X-ray crystallographic, and molecular modeling studies. Results X-ray crystallographic data of the β-cyclodextrin—substrate (β-CD—MetAMC) complex reveal that while the AMC moiety of the substrate is confined within the CD cavity, the methionine moiety protrudes outward. The steady-state kinetic data for inhibition of MetAP by HP-β-CD—MetAMC conform to a model mechanism in which the substrate is “bridged” between HP-β-CD and the enzyme’s active-site pocket, forming HP-β-CD—MetAMC—MetAP as the catalytically inactive ternary complex. Molecular modeling shows that the scissile bond of HP-β-CD-bound MetAMC substrate does not reach within the proximity of the enzyme’s catalytic metal center, and thus the substrate fails to undergo cleavage. Conclusions The data presented herein suggests that the bridging of the substrate between the enzyme and HP-β-CD cavities is facilitated by interaction of their surfaces, and the resulting complex inhibits the enzyme activity. General Significance Due to its potential interaction with physiological proteins via sequestered substrates, caution must be exercised in HP-β-CD mediated delivery of drugs under pathophysiological conditions. PMID:25450177

Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

PubMed

Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

2014-04-01

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

[Effect of Gegen Qinlian decoction on hepatic cytochrome CYP450 isozymes in rats by HPLC-MS/MS].

PubMed

Liu, Zi-hua; An, Rui; Zhang, Yi-zhu; Gu, Qing-qing; You, Li-sha; Wang, Xin-hong

2015-08-01

To study the effect of Gegen Qinlian decoction and its major effective components on five hepatic microsomal CYP450 isozymes in rats. The in vitro hepatic microsomal incubation technique was used to co-culture Gegen Qinlian decoction and its major effective components together with each probe substrate. HPLC-MS/MS was used to establish the analytical method for metabolites of the five isoform probe substrates of CYP450 isozymes, detect the linearity among micoromal protein concentration, incubation time and metabolite formation amount. And HPLC-MS/MS was applied to determine the formation rate (V) of corresponding metabolites (acetaminophen, 4-OH-chlorzoxazone, dextrophan, 6-OH-chlorzoxazone and 6β-hydroxytestosterone) specific probe substrates of the five isoform probe substrates of CYP450 isozymes (phenacetin, polbutamide, dextromethorphan, chlorzoxazone, testosterone), in order to determine the activity of each isozyme. The result showed good linearity among acetaminophen, 4-OH-tolbutamide, dextrophan, 6-OH-chlorzoxazone and 6β-hydroxytestosterone, satisfactory precision, stability and average recovery, suggesting the method was feasible. The optimized in vitro microsomal incubation conditions conformed to the requirements in the guideline of drug-drug interaction. Gegen Qinlian decoction showed different degrees of inhibitor effect on 5 CYP450 isoforms (CYP1A2, CYP2C11, CYP2D2, CYP2E1, CYP3A1/2). Its major effective component berberine could inhibit each CYP450 isoform at high concentrations (except for CYP1A2, CYP3A1/2).

Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

PubMed

Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

2015-12-18

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs.

Using phylogenetic probes for quantification of stable isotope labeling and microbial community analysis

DOEpatents

Brodie, Eoin L; DeSantis, Todd Z; Karaoz, Ulas; Andersen, Gary L

2014-12-09

Herein is described methods for a high-sensitivity means to measure the incorporation of stable isotope labeled substrates into RNA following stable isotope probing experiments (SIP). RNA is hybridized to a set of probes such as phylogenetic microarrays and isotope incorporation is quantified such as by secondary ion mass spectrometer imaging (NanoSIMS).

Interference of flavonoids with fluorescent intracellular probes: methodological implications in the evaluation of the oxidative burst by flow cytometry.

PubMed

Peluso, Ilaria; Manafikhi, Husseen; Reggi, Raffaella; Palmery, Maura

2014-08-01

The evaluation of oxidative burst is particularly relevant in many pathological and subclinical conditions. Flow cytometry provides quick and accurate measures of the reactive oxygen species production by leukocytes in most situations. However, spurious results, related to probes' efflux may be observed in several instances. Many factors affect the evaluation of the oxidative burst with fluorescent probes that require intracellular deacetylation and could be substrate of the multidrug resistance proteins (MDR). After discussing the implications of the efflux of fluorophores in the normalization strategies in flow cytometry assays, we have pointed out the possible interference of flavonoids with fluorescet probes' staining and signal. We have also reviewed the results from human intervention studies regarding the evaluation of oxidative burst with these probes. In vitro, at concentrations close to post-ingestion circulating levels, some flavonoids and their metabolites could interfere with probes' staining and fluorescence signal through different mechanisms, such as the inhibition of esterases, the modulation of the MDR-mediate efflux of probe and the inhibition of the oxidation of probe. These effects may explain the contrasting results obtained by human intervention studies. Finally, also inflammatory state or the use of drugs substrate of MDR proteins could affect the evaluation of the oxidative burst with intracellular probes. © 2014 International Society for Advancement of Cytometry.

Deltamethrin is metabolized by CYP6FU1, a cytochrome P450 associated with pyrethroid resistance, in Laodelphax striatellus.

PubMed

Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Peng, Yingchuan; Zhang, Haomiao; Jiang, Ling; Wu, Min; Han, Zhaojun

2018-06-01

Cytochrome P450s (CYPs) are known to play a major role in metabolizing a wide range compounds. CYP6FU1 has been found to be over-expressed in a deltamethrin-resistant strain of Laodelphax striatellus. This study was conducted to express CYP6FU1 in Sf9 cells as a recombinant protein, to confirm its ability to degrade deltamethrin, chlorpyrifos, imidacloprid and traditional P450 probing substrates. Carbon monoxide difference spectrum analysis indicated that the intact CYP6FU1 protein was expressed in insect Sf9 cells. Catalytic activity tests with four traditional P450 probing substrates revealed that the expressed CYP6FU1 preferentially metabolized p-nitroanisole and ethoxyresorufin, but not ethoxycoumarin and luciferin-HEGE. The enzyme kinetic parameters were tested using p-nitroanisole. The michaelis constant (K m ) and catalytic constant (K cat ) values were 17.51 ± 4.29 µm and 0.218 ± 0.001 pmol min -1 mg -1 protein, respectively. Furthermore, CYP6FU1 activity for degradation of insecticides was tested by measuring substrate depletion and metabolite formation. The chromatogram analysis showed obvious nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of deltamethrin, and formation of the unknown metabolite. Mass spectra and the molecular docking model showed that the metabolite was 4-hydroxy-deltamethrin. However, the recombinant CYP6FU1 could not metabolize imidacloprid and chlorpyrifos. These results confirmed that the over-expressed CYP6FU1 contributes to deltamethrin resistance in L. striatellus, and p-nitroanisole might be a potential diagnostic probe for deltamethrin metabolic resistance detection and monitoring. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Probing the substrate specificity of the bacterial Pnkp/Hen1 RNA repair system using synthetic RNAs

PubMed Central

Zhang, Can; Chan, Chio Mui; Wang, Pei; Huang, Raven H.

2012-01-01

Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed. PMID:22190744

Lanthanide tris(β-diketonates) as useful probes for chirality determination of biological amino alcohols in vibrational circular dichroism: ligand to ligand chirality transfer in lanthanide coordination sphere.

PubMed

Miyake, Hiroyuki; Terada, Keiko; Tsukube, Hiroshi

2014-06-01

A series of lanthanide tris(β-diketonates) functioned as useful chirality probes in the vibrational circular dichroism (VCD) characterization of biological amino alcohols. Various chiral amino alcohols induced intense VCD signals upon ternary complexation with racemic lanthanide tris(β-diketonates). The VCD signals observed around 1500 cm(-1) (β-diketonate IR absorption region) correlated well with the stereochemistry and enantiomeric purity of the targeted amino alcohol, while the corresponding monoalcohol, monoamine, and diol substrates induced very weak VCD signals. The high-coordination number and dynamic property of the lanthanide complex offer an effective chirality VCD probing of biological substrates. © 2014 Wiley Periodicals, Inc.

Probe-Substrate Distance Control in Desorption Electrospray Ionization

NASA Astrophysics Data System (ADS)

Yarger, Tyler J.; Yuill, Elizabeth M.; Baker, Lane A.

2018-03-01

We introduce probe-substrate distance (Dps)-control to desorption electrospray ionization (DESI) and report a systematic investigation of key experimental parameters. Examination of voltage, flow rate, and nebulizing gas pressure suggests as Dps decreases, the distance-dependent spray current increases, until a critical point. At the critical point the relationship inverts, and the spray current decreases as the probe moves closer to the surface due to constriction of solution flow by the nebulizing gas. Dps control was used to explore the use of spray current as a signal for feedback positioning, while mass spectrometry imaging was performed simultaneously. Further development of this technique is expected to find application in study of structure-function relationships for clinical diagnostics, biological investigation, and materials characterization. [Figure not available: see fulltext.

NASA Tech Briefs, June 2003

NASA Technical Reports Server (NTRS)

2003-01-01

Topics covered include: Nulling Infrared Radiometer for Measuring Temperature; The Ames Power Monitoring System; Hot Films on Ceramic Substrates for Measuring Skin Friction; Probe Without Moving Parts Measures Flow Angle; Detecting Conductive Liquid Leaking from Nonconductive Pipe; Adaptive Suppression of Noise in Voice Communications; High-Performance Solid-State W-Band Power Amplifiers; Microbatteries for Combinatorial Studies of Conventional Lithium-Ion Batteries; Correcting for Beam Aberrations in a Beam-Waveguide Antenna; Advanced Rainbow Solar Photovoltaic Arrays; Metal Side Reflectors for Trapping Light in QWIPs; Software for Collaborative Engineering of Launch Rockets; Software Assists in Extensive Environmental Auditing; Software Supports Distributed Operations via the Internet; Software Estimates Costs of Testing Rocket Engines; yourSky: Custom Sky-Image Mosaics via the Internet; Software for Managing Inventory of Flight Hardware; Lower-Conductivity Thermal-Barrier Coatings; Process for Smoothing an Si Substrate after Etching of SiO2; Flexible Composite-Material Pressure Vessel; Treatment to Destroy Chlorohydrocarbon Liquids in the Ground; Noncircular Cross Sections Could Enhance Mixing in Sprays; Small, Untethered, Mobile Roots for Inspecting Gas Pipes; Paint-Overspray Catcher; Preparation of Regular Specimens for Atom Probes; Inverse Tomo-Lithography for Making Microscopic 3D Parts; Predicting and Preventing Incipient Flameout in Combustors; MEMS-Based Piezoelectric/Electrostatic Inchworm Actuator; Metallized Capillaries as Probes for Raman Spectroscopy; Adaptation of Mesoscale Weather Models to Local Forecasting; Aerodynamic Design using Neural Networks; Combining Multiple Gyroscope Outputs for Increased Accuracy; and Improved Collision-Detection Method for Robotic Manipulator.

Soft material adhesion characterization for in vivo locomotion of robotic capsule endoscopes: Experimental and modeling results.

PubMed

Kern, Madalyn D; Ortega Alcaide, Joan; Rentschler, Mark E

2014-11-01

The objective of this work is to validate an experimental method and nondimensional model for characterizing the normal adhesive response between a polyvinyl chloride based synthetic biological tissue substrate and a flat, cylindrical probe with a smooth polydimethylsiloxane (PDMS) surface. The adhesion response is a critical mobility design parameter of a Robotic Capsule Endoscope (RCE) using PDMS treads to provide mobility to travel through the gastrointestinal tract for diagnostic purposes. Three RCE design characteristics were chosen as input parameters for the normal adhesion testing: pre-load, dwell time and separation rate. These parameters relate to the RCE׳s cross sectional dimension, tread length, and tread speed, respectively. An inscribed central composite design (CCD) prescribed 34 different parameter configurations to be tested. The experimental adhesion response curves were nondimensionalized by the maximum stress and total displacement values for each test configuration and a mean nondimensional curve was defined with a maximum relative error of 5.6%. A mathematical model describing the adhesion behavior as a function of the maximum stress and total displacement was developed and verified. A nonlinear regression analysis was done on the maximum stress and total displacement parameters and equations were defined as a function of the RCE design parameters. The nondimensional adhesion model is able to predict the adhesion curve response of any test configuration with a mean R(2) value of 0.995. Eight additional CCD studies were performed to obtain a qualitative understanding of the impact of tread contact area and synthetic material substrate stiffness on the adhesion response. These results suggest that the nondimensionalization technique for analyzing the adhesion data is sufficient for all values of probe radius and substrate stiffness within the bounds tested. This method can now be used for RCE tread design optimization given a set of environmental conditions for device operation. Copyright © 2014 Elsevier Ltd. All rights reserved.

A TEMPO-conjugated fluorescent probe for monitoring mitochondrial redox reactions.

PubMed

Hirosawa, Shota; Arai, Satoshi; Takeoka, Shinji

2012-05-18

We report a mitochondrial targeted redox probe (MitoRP) that comprises a nitroxide radical (TEMPO) moiety and coumarin 343. Using isolated mitochondria in the presence/absence of substrates and inhibitors of oxidative phosphorylation, we demonstrated that MitoRP is a useful probe to monitor the electron flow associated with complex I. This journal is © The Royal Society of Chemistry 2012

Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

PubMed

Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

2016-06-17

Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

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

Jana, Dipankar, E-mail: dip2602@gmail.com; Porwal, S.; Sharma, T. K., E-mail: tarun@rrcat.gov.in

Pump-probe Surface Photovoltage Spectroscopy (SPS) measurements are performed on semiconductor epitaxial layers. Here, an additional sub-bandgap cw pump laser beam is used in a conventional chopped light geometry SPS setup under the pump-probe configuration. The main role of pump laser beam is to saturate the sub-bandgap localized states whose contribution otherwise swamp the information related to the bandgap of material. It also affects the magnitude of Dember voltage in case of semi-insulating (SI) semiconductor substrates. Pump-probe SPS technique enables an accurate determination of the bandgap of semiconductor epitaxial layers even under the strong influence of localized sub-bandgap states. The pumpmore » beam is found to be very effective in suppressing the effect of surface/interface and bulk trap states. The overall magnitude of SPV signal is decided by the dependence of charge separation mechanisms on the intensity of the pump beam. On the contrary, an above bandgap cw pump laser can be used to distinguish the signatures of sub-bandgap states by suppressing the band edge related feature. Usefulness of the pump-probe SPS technique is established by unambiguously determining the bandgap of p-GaAs epitaxial layers grown on SI-GaAs substrates, SI-InP wafers, and p-GaN epilayers grown on Sapphire substrates.« less

Classification of Phase Transitions by Microcanonical Inflection-Point Analysis

NASA Astrophysics Data System (ADS)

Qi, Kai; Bachmann, Michael

2018-05-01

By means of the principle of minimal sensitivity we generalize the microcanonical inflection-point analysis method by probing derivatives of the microcanonical entropy for signals of transitions in complex systems. A strategy of systematically identifying and locating independent and dependent phase transitions of any order is proposed. The power of the generalized method is demonstrated in applications to the ferromagnetic Ising model and a coarse-grained model for polymer adsorption onto a substrate. The results shed new light on the intrinsic phase structure of systems with cooperative behavior.

Gap-mode enhancement on MoS2 probed by functionalized tip-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Alajlan, Abdulrahman M.; Voronine, Dmitri V.; Sinyukov, Alexander M.; Zhang, Zhenrong; Sokolov, Alexei V.; Scully, Marlan O.

2016-09-01

Surface enhancement of molecular spectroscopic signals has been widely used for sensing and nanoscale imaging. Because of the weak electromagnetic enhancement of Raman signals on semiconductors, it is motivating but challenging to study the electromagnetic effect separately from the chemical effects. We report tip-enhanced Raman scattering measurements on Au and bulk MoS2 substrates using a metallic tip functionalized with copper phthalocyanine molecules and demonstrate similar gap-mode enhancement on both substrates. We compare the experimental results with theoretical calculations to confirm the gap-mode enhancement on MoS2 using a well-established electrostatic model. The functionalized tip approach allows for suppressing the background and is ideal for separating electromagnetic and chemical enhancement mechanisms on various substrates. Our results may find a wide range of applications in MoS2-based devices, sensors, and metal-free nanoscale bio-imaging.

Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.

PubMed

Jones, Krysten A; Porterfield, William B; Rathbun, Colin M; McCutcheon, David C; Paley, Miranda A; Prescher, Jennifer A

2017-02-15

Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

Modeling Linear and Cyclic PKS Intermediates through Atom Replacement

PubMed Central

2015-01-01

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs. PMID:25406716

High-throughput, 384-well, LC-MS/MS CYP inhibition assay using automation, cassette-analysis technique, and streamlined data analysis.

PubMed

Halladay, Jason S; Delarosa, Erlie Marie; Tran, Daniel; Wang, Leslie; Wong, Susan; Khojasteh, S Cyrus

2011-08-01

Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

Determining Confounding Sensitivities In Eddy Current Thin Film Measurements

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

Gros, Ethan; Udpa, Lalita; Smith, James A.

Determining Confounding Sensitivities In Eddy Current Thin Film Measurements Ethan Gros, Lalita Udpa, Electrical Engineering, Michigan State University, East Lansing MI 48824 James A. Smith, Experiment Analysis, Idaho National Laboratory, Idaho Falls ID 83415 Eddy current (EC) techniques are widely used in industry to measure the thickness of non-conductive films on a metal substrate. This is done using a system whereby a coil carrying a high-frequency alternating current is used to create an alternating magnetic field at the surface of the instrument's probe. When the probe is brought near a conductive surface, the alternating magnetic field will induce ECs inmore » the conductor. The substrate characteristics and the distance of the probe from the substrate (the coating thickness) affect the magnitude of the ECs. The induced currents load the probe coil affecting the terminal impedance of the coil. The measured probe impedance is related to the lift off between coil and conductor as well as conductivity of the test sample. For a known conductivity sample, the probe impedance can be converted into an equivalent film thickness value. The EC measurement can be confounded by a number of measurement parameters. It is the goal of this research to determine which physical properties of the measurement set-up and sample can adversely affect the thickness measurement. The eddy current testing is performed using a commercially available, hand held eddy current probe (ETA3.3H spring loaded eddy probe running at 8 MHz) that comes with a stand to hold the probe. The stand holds the probe and adjusts the probe on the z-axis to help position the probe in the correct area as well as make precise measurements. The signal from the probe is sent to a hand held readout, where the results are recorded directly in terms of liftoff or film thickness. Understanding the effect of certain factors on the measurements of film thickness, will help to evaluate how accurate the ETA3.3H spring loaded eddy probe is at measuring film thickness under varying experimental conditions. This research will study the effects of a number of factors such as i) calibration, ii) conductivity, iii) edge effect, iv) surface finish of base material and v) cable condition and compare with the long term reproducibility of a standard measurement. This work was performed with support from the Department of Energy under the United States National Nuclear Security Administration (NNSA) at the Idaho National Laboratory.« less

Interaction model between a liquid film and a spherical probe

NASA Astrophysics Data System (ADS)

Ledesma Alonso, Rene; Legendre, Dominique; Tordjeman, Philippe

2012-11-01

To find a liquid surface profile, when performing AFM measurements, probe interaction effects should be identified. Herein, the behavior of a liquid film free surface (thickness E, surface tension γ and density difference Δρ), disposed over a flat surface and in the presence of a spherical probe (radius R) is forecast. A bump-like surface shape is observed, due to the probe/film interaction (characterized by the Hamaker constant Hpl). In addition, the attraction between the film and the substrate (depicted by Hsl) opposes the axial and radial deformation ranges. Several parameters portray the equilibrium shape: Bond Bo = (ΔρgR2) / γ and modified Hamaker Ha = 4Hpl / (3 πγR2) numbers, Hamaker ratio A =Hls /Hpl , separation distance D / R and film thickness E / R . We focus on the effect of geometry, nevertheless special attention is given to the role of physical parameters. Employing an augmented Young-Laplace equation, the equilibrium profile is described by a strongly non-linear ODE. A critical distance, below which the irreversible wetting process of the spherical probe occurs, is predicted. Our results provide simple relationships between parameters, which determine the optimal scanning conditions over liquid films.

Cyclic Voltammetry Probe Approach Curves with Alkali Amalgams at Mercury Sphere-Cap Scanning Electrochemical Microscopy Probes.

PubMed

Barton, Zachary J; Rodríguez-López, Joaquín

2017-03-07

We report a method of precisely positioning a Hg-based ultramicroelectrode (UME) for scanning electrochemical microscopy (SECM) investigations of any substrate. Hg-based probes are capable of performing amalgamation reactions with metal cations, which avoid unwanted side reactions and positive feedback mechanisms that can prove problematic for traditional probe positioning methods. However, prolonged collection of ions eventually leads to saturation of the amalgam accompanied by irreversible loss of Hg. In order to obtain negative feedback positioning control without risking damage to the SECM probe, we implement cyclic voltammetry probe approach surfaces (CV-PASs), consisting of CVs performed between incremental motor movements. The amalgamation current, peak stripping current, and integrated stripping charge extracted from a shared CV-PAS give three distinct probe approach curves (CV-PACs), which can be used to determine the tip-substrate gap to within 1% of the probe radius. Using finite element simulations, we establish a new protocol for fitting any CV-PAC and demonstrate its validity with experimental results for sodium and potassium ions in propylene carbonate by obtaining over 3 orders of magnitude greater accuracy and more than 20-fold greater precision than existing methods. Considering the timescales of diffusion and amalgam saturation, we also present limiting conditions for obtaining and fitting CV-PAC data. The ion-specific signals isolated in CV-PACs allow precise and accurate positioning of Hg-based SECM probes over any sample and enable the deployment of CV-PAS SECM as an analytical tool for traditionally challenging conditions.

Mechanistic Insights into the Specificity of Human Cytosolic Sulfotransferase 2A1 (hSULT2A1) for Hydroxylated Polychlorinated Biphenyls Through the Use of Fluoro-tagged Probes

PubMed Central

Ekuase, E.J.; van ’t Erve, T.J.; Rahaman, A.; Robertson, L.W.; Duffel, M.W.; Luthe, G.

2015-01-01

Determining the relationships between the structures of substrates and inhibitors and their interactions with drug-metabolizing enzymes is of prime importance in predicting the toxic potential of new and legacy xenobiotics. Traditionally, quantitative structure activity relationship (QSAR) studies are performed with many distinct compounds. Based on the chemical properties of the tested compounds, complex relationships can be established so that models can be developed to predict toxicity of novel compounds. In this study, the use of fluorinated analogues as supplemental QSAR compounds was investigated. Substituting fluorine induces changes in electronic and steric properties of the substrate without substantially changing the chemical backbone of the substrate. In vitro assays were performed using purified human cytosolic sulfotransferase hSULT2A1 as a model enzyme. A mono-hydroxylated polychlorinated biphenyl (4-OH PCB 14) and its four possible mono-fluoro analogues were used as test compounds. Remarkable similarities were found between this approach and previously published QSAR studies for hSULT2A1. Both studies implicate the importance of dipole moment and dihedral angle as being important to PCB structure in respect to being substrates for hSULT2A1. We conclude that mono-fluorinated analogues of a target substrate can be a useful tool to study the structure activity relationships for enzyme specificity. PMID:26165989

Development of a physical and electronic model for RuO 2 nanorod rectenna devices

NASA Astrophysics Data System (ADS)

Dao, Justin

Ruthenium oxide (RuO2) nanorods are an emergent technology in nanostructure devices. As the physical size of electronics approaches a critical lower limit, alternative solutions to further device miniaturization are currently under investigation. Thin-film nanorod growth is an interesting technology, being investigated for use in wireless communications, sensor systems, and alternative energy applications. In this investigation, self-assembled RuO2 nanorods are grown on a variety of substrates via a high density plasma, reactive sputtering process. Nanorods have been found to grow on substrates that form native oxide layers when exposed to air, namely silicon, aluminum, and titanium. Samples were analyzed with Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) techniques. Conductive Atomic Force Microscopy (C-AFM) measurements were performed on single nanorods to characterize structure and electrical conductivity. The C-AFM probe tip is placed on a single nanorod and I-V characteristics are measured, potentially exhibiting rectifying capabilities. An analysis of these results using fundamental semiconductor physics principles is presented. Experimental data for silicon substrates was most closely approximated by the Simmons model for direct electron tunneling, whereas that of aluminum substrates was well approximated by Fowler-Nordheim tunneling. The native oxide of titanium is regarded as a semiconductor rather than an insulator and its ability to function as a rectifier is not strong. An electronic model for these nanorods is described herein.

Single Molecule Electrochemical Detection in Aqueous Solutions and Ionic Liquids.

PubMed

Byers, Joshua C; Paulose Nadappuram, Binoy; Perry, David; McKelvey, Kim; Colburn, Alex W; Unwin, Patrick R

2015-10-20

Single molecule electrochemical detection (SMED) is an extremely challenging aspect of electroanalytical chemistry, requiring unconventional electrochemical cells and measurements. Here, SMED is reported using a "quad-probe" (four-channel probe) pipet cell, fabricated by depositing carbon pyrolytically into two diagonally opposite barrels of a laser-pulled quartz quadruple-barreled pipet and filling the open channels with electrolyte solution, and quasi-reference counter electrodes. A meniscus forms at the end of the probe covering the two working electrodes and is brought into contact with a substrate working electrode surface. In this way, a nanogap cell is produced whereby the two carbon electrodes in the pipet can be used to promote redox cycling of an individual molecule with the substrate. Anticorrelated currents generated at the substrate and tip electrodes, at particular distances (typically tens of nanometers), are consistent with the detection of single molecules. The low background noise realized in this droplet format opens up new opportunities in single molecule electrochemistry, including the use of ionic liquids, as well as aqueous solution, and the quantitative assessment and analysis of factors influencing redox cycling currents, due to a precisely known gap size.

Silica coating of PbS quantum dots and their position control using a nanohole on Si substrate

NASA Astrophysics Data System (ADS)

Mukai, Kohki; Okumura, Isao; Nishizaki, Yuta; Yamashita, Shuzo; Niwa, Keisuke

2018-04-01

We succeeded in controlling the apparent size of a colloidal PbS quantum dot (QD) in the range of 20 to 140 nm by coating with silica and trapping the coated QDs in a nanohole prepared by scanning probe microscope lithography. Photoluminescence intensity was improved by controlling the process of adding the silica source material of tetraethoxysilane for the coating. Nanoholes of different sizes were formed on a single substrate by scanning probe oxidation with the combination of SF6 dry etching and KOH wet etching. QDs having an arbitrary energy structure can be arranged at an arbitrary position on the semiconductor substrate using this technique, which will aid in the fabrication of future nanosize solid devices such as quantum information circuits.

All-optical in-depth detection of the acoustic wave emitted by a single gold nanorod

NASA Astrophysics Data System (ADS)

Xu, Feng; Guillet, Yannick; Ravaine, Serge; Audoin, Bertrand

2018-04-01

A single gold nanorod dropped on the surface of a silica substrate is used as a transient optoacoustic source of gigahertz hypersounds. We demonstrate the all-optical detection of the as-generated acoustic wave front propagating in the silica substrate. For this purpose, time-resolved femtosecond pump-probe experiments are performed in a reflection configuration. The fundamental breathing mode of the nanorod is detected at 23 GHz by interferometry, and the longitudinal acoustic wave radiated in the silica substrate is detected by time-resolved Brillouin scattering. By tuning the optical probe wavelength from 750 to 900 nm, hypersounds with wavelengths of 260-315 nm are detected in the silica substrate, with corresponding acoustic frequencies in the range of 19-23 GHz. To confirm the origin of these hypersounds, we theoretically analyze the influence of the acoustic excitation spectrum on the temporal envelope of the transient reflectivity. This analysis proves that the acoustic wave detected in the silica substrate results from the excitation of the breathing mode of the nanorod. These results pave the way for performing local in-depth elastic nanoscopy.

Impact of morphology on diffusive dynamics on curved surfaces

NASA Astrophysics Data System (ADS)

Kusters, Remy; Storm, Cornelis

2014-03-01

Diffusive processes on nonplanar substrates are deeply relevant for cellular function and transport and increasingly used to probe and characterize the behavior of proteins in membranes. We present analytical and numerical analyses of in-plane diffusion of discrete particles on curved geometries reflecting various generic motifs in biology and explore, in particular, the effect that the shape of the substrate has on the characteristic time scales of diffusive processes. To this end, we consider both collective measures (the relaxation of concentration profiles towards equilibrium) and single-particle measures (escape rates and first passage times of individual diffusing molecules): the first relevant for the correct interpretation of FRAP experiments in curved environments; the second, for single-particle tracking probes. Each of these measures is sensitively affected by the morphology of the substrate, and we find that the exit rate out of a domain is not uniquely set by the size of its boundary, illustrating the general principle we reveal: By varying the shape of a substrate, Nature can control the diffusive time scales in a microenvironment without changing the bare substrate properties.

Method and apparatus for staining immobilized nucleic acids

DOEpatents

Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

2000-01-01

A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

A new technique to transfer metallic nanoscale patterns to small and non-planar surfaces: Application to a fiber optic device for surface enhanced Raman scattering detection

NASA Astrophysics Data System (ADS)

Smythe, Elizabeth Jennings

This thesis focuses on the development of a bidirectional fiber optic probe for the detection of surface enhanced Raman scattering (SERS). One facet of this fiber-based probe featured an array of coupled optical antennas, which we designed to enhance the Raman signal of nearby analytes. When this array interacted with an analyte, it generated SERS signals specific to the chemical composition of the sample; some of these SERS signals coupled back into the fiber. We used the other facet of the probe to input light into the fiber and collect the SERS signals that coupled into the probe. In this dissertation, the development of the probe is broken into three sections: (i) characterization of antenna arrays, (ii) fabrication of the probe, and (iii) device measurements. In the first section we present a comprehensive study of metallic antenna arrays. We carried out this study to determine the effects of antenna geometry, spacing, and composition on the surface plasmon resonance (SPR) of a coupled antenna array; the wavelength range and strength of the SPR are functions of the shape and interactions of the antennas. The SPR of the array ultimately amplified the Raman signal of analytes and produced a measurable SERS signal, thus determination of the optimal array geometries for SERS generation was an important first step in the development of the SERS fiber probe. We then introduce a new technique developed to fabricate the SERS fiber probes. This technique involves transferring antenna arrays (created by standard lithographic methods) from a large silicon substrate to a fiber facet. We developed this fabrication technique to bypass many of the limitations presented by previously developed methods for patterning unconventional substrates (i.e. small and/or non-planar substrates), such as focused ion-beam milling and soft lithography. In the third section of this thesis, we present SERS measurements taken with the fiber probe. We constructed a measurement system to couple light into the probe and filter out background noise; this allowed simultaneous detection of multiple chemicals. Antenna array enhancement factor (EF) calculations are shown; these allowed us to determine that the probe efficiently collected SERS signals.

RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro

PubMed Central

Herrmann, Elena; Young, Wayne; Rosendale, Douglas; Reichert-Grimm, Verena; Conrad, Ralf

2017-01-01

RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that “cross-feeding” occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system. PMID:28299315

Breast imaging technology: Recent advances in imaging endogenous or transferred gene expression utilizing radionuclide technologies in living subjects - applications to breast cancer

PubMed Central

Berger, Frank; Sam Gambhir, Sanjiv

2001-01-01

A variety of imaging technologies is being investigated as tools for studying gene expression in living subjects. Two technologies that use radiolabeled isotopes are single photon emission computed tomography (SPECT) and positron emission tomography (PET). A relatively high sensitivity, a full quantitative tomographic capability, and the ability to extend small animal imaging assays directly into human applications characterize radionuclide approaches. Various radiolabeled probes (tracers) can be synthesized to target specific molecules present in breast cancer cells. These include antibodies or ligands to target cell surface receptors, substrates for intracellular enzymes, antisense oligodeoxynucleotide probes for targeting mRNA, probes for targeting intracellular receptors, and probes for genes transferred into the cell. We briefly discuss each of these imaging approaches and focus in detail on imaging reporter genes. In a PET reporter gene system for in vivo reporter gene imaging, the protein products of the reporter genes sequester positron emitting reporter probes. PET subsequently measures the PET reporter gene dependent sequestration of the PET reporter probe in living animals. We describe and review reporter gene approaches using the herpes simplex type 1 virus thymidine kinase and the dopamine type 2 receptor genes. Application of the reporter gene approach to animal models for breast cancer is discussed. Prospects for future applications of the transgene imaging technology in human gene therapy are also discussed. Both SPECT and PET provide unique opportunities to study animal models of breast cancer with direct application to human imaging. Continued development of new technology, probes and assays should help in the better understanding of basic breast cancer biology and in the improved management of breast cancer patients. PMID:11250742

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2016-08-22

Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric coupling between the allosteric pocket and the substrate binding region. The impact of novolactone on the conformational dynamics and allosteric communications in the HSPA1A complex was comparable to the substrate effect, which is consistent with the experimental evidence that PET-16, but not novolactone binding, can significantly decrease substrate affinity. We argue that the unique dynamic and network signatures of PET-16 and novolactone may be linked with the experimentally observed functional effects of these inhibitors on allosteric regulation and substrate binding.

RF Properties of Epitaxial Lift-Off HEMT Devices

NASA Technical Reports Server (NTRS)

Young, Paul G.; Alterovitz, Samuel A.; Mena, Rafael A.; Smith, Edwyn D.

1993-01-01

Epitaxial layers containing GaAs HEMT and P-HEMT structures have been lifted-off the GaAs substrate and attached to other host substrates using an AlAs parting layer. The devices were on-wafer RF probed before and after the lift-off step showing no degradation in the measured S-parameters. The maximum stable gain indicates a low frequency enhancement of the gain of 1-2 dB with some devices showing an enhancement of F(sub max)F(sub T) consistently shows an increase of 12-20% for all lifted-off HEMT structures. Comparison of the Hall measurements and small signal models show that the gain is improved and this is most probably associated with an enhanced carrier concentration.

Giant Polarization Rotation in BiFeO3/SrTiO3 Thin Films.

NASA Astrophysics Data System (ADS)

Langner, M. C.; Chu, Y. H.; Martin, L. M.; Gajek, M.; Ramesh, R.; Orenstein, J.

2008-03-01

We use optical second harmonic generation to probe dynamics of the ferroelectric polarization in (111) oriented BiFeO3 thin films grown on SrTiO3 substrates. The second harmonic response indicates 3m point group symmetry and is consistent with a spontaneous polarization normal to the surface of the film. We measure large changes in amplitude and lowering of symmetry, consistent with polarization rotation, when modest electric fields are applied in the plane of the film. At room temperature the rotation is an order of magnitude larger than expected from reported values of the dielectric constant and increases further (as 1/T) as temperature is lowered. We propose a substrate interaction model to explain these results.

The Accuracy of Al and Cu Film Thickness Determinations and the Implications for Electron Probe Microanalysis.

PubMed

Matthews, Mike B; Kearns, Stuart L; Buse, Ben

2018-04-01

The accuracy to which Cu and Al coatings can be determined, and the effect this has on the quantification of the substrate, is investigated. Cu and Al coatings of nominally 5, 10, 15, and 20 nm were sputter coated onto polished Bi using two configurations of coater: One with the film thickness monitor (FTM) sensor colocated with the samples, and one where the sensor is located to one side. The FTM thicknesses are compared against those calculated from measured Cu Lα and Al Kα k-ratios using PENEPMA, GMRFilm, and DTSA-II. Selected samples were also cross-sectioned using focused ion beam. Both systems produced repeatable coatings, the thickest coating being approximately four times the thinnest coating. The side-located FTM sensor indicated thicknesses less than half those of the software modeled results, propagating on to 70% errors in substrate quantification at 5 kV. The colocated FTM sensor produced errors in film thickness and substrate quantification of 10-20%. Over the range of film thicknesses and accelerating voltages modeled both the substrate and coating k-ratios can be approximated by linear trends as functions of film thickness. The Al films were found to have a reduced density of ~2 g/cm2.

Atom probe tomography of a Ti-Si-Al-C-N coating grown on a cemented carbide substrate.

PubMed

Thuvander, M; Östberg, G; Ahlgren, M; Falk, L K L

2015-12-01

The elemental distribution within a Ti-Si-Al-C-N coating grown by physical vapour deposition on a Cr-doped WC-Co cemented carbide substrate has been investigated by atom probe tomography. Special attention was paid to the coating/substrate interface region. The results indicated a diffusion of substrate binder phase elements into the Ti-N adhesion layer. The composition of this layer, and the Ti-Al-N interlayer present between the adhesion layer and the main Ti-Si-Al-C-N layer, appeared to be sub-stoichiometric. The analysis of the interlayer showed the presence of internal surfaces, possibly grain boundaries, depleted in Al. The composition of the main Ti-Al-Si-C-N layer varied periodically in the growth direction; layers enriched in Ti appeared with a periodicity of around 30 nm. Laser pulsing resulted in a good mass resolution that made it possible to distinguish between N(+) and Si(2+) at 14 Da. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein-based stable isotope probing.

PubMed

Jehmlich, Nico; Schmidt, Frank; Taubert, Martin; Seifert, Jana; Bastida, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Vogt, Carsten

2010-12-01

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.

Surface enhanced Raman gene probe and methods thereof

DOEpatents

Vo-Dinh, T.

1998-02-24

The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

Layered graphene-mica substrates induce melting of DNA origami

NASA Astrophysics Data System (ADS)

Green, Nathaniel S.; Pham, Phi H. Q.; Crow, Daniel T.; Burke, Peter J.; Norton, Michael L.

2018-04-01

Monolayer graphene supported on mica substrates induce melting of cross-shaped DNA origami. This behavior can be contrasted with the case of origami on graphene on graphite, where an expansion or partially re-organized structure is observed. On mica, only well-formed structures are observed. Comparison of the morphological differences observed for these probes after adsorption on these substrates provides insights into the sensitivity of DNA based nanostructures to the properties of the graphene monolayer, as modified by its substrate.

In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication

PubMed Central

Moorcroft, Matthew J.; Meuleman, Wouter R. A.; Latham, Steven G.; Nicholls, Thomas J.; Egeland, Ryan D.; Southern, Edwin M.

2005-01-01

In this paper, we demonstrate in situ synthesis of oligonucleotide probes on poly(dimethylsiloxane) (PDMS) microchannels through use of conventional phosphoramidite chemistry. PDMS polymer was moulded into a series of microchannels using standard soft lithography (micro-moulding), with dimensions <100 μm. The surface of the PDMS was derivatized by exposure to ultraviolet/ozone followed by vapour phase deposition of glycidoxypropyltrimethoxysilane and reaction with poly(ethylene glycol) spacer, resulting in a reactive surface for oligonucleotide coupling. High, reproducible yields were achieved for both 6mer and 21mer probes as assessed by hybridization to fluorescent oligonucleotides. Oligonucleotide surface density was comparable with that obtained on glass substrates. These results suggest PDMS as a stable and flexible alternative to glass as a suitable substrate in the fabrication and synthesis of DNA microarrays. PMID:15870385

Selective imaging of cathepsin L in breast cancer by fluorescent activity-based probes† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04303a

PubMed Central

Rut, Wioletta; Vizovisek, Matej; Groborz, Katarzyna; Kasperkiewicz, Paulina; Finlay, Darren; Vuori, Kristiina; Turk, Dusan; Turk, Boris; Salvesen, Guy S.

2018-01-01

Cysteine cathepsins normally function in the lysosomal degradation system where they are critical for the maintenance of cellular homeostasis and the MHC II immune response, and have been found to have major roles in several diseases and in tumor progression. Selective visualization of individual protease activity within a complex proteome is of major importance to establish their roles in both normal and tumor cells, thereby facilitating our understanding of the regulation of proteolytic networks. A generally accepted means to monitor protease activity is the use of small molecule substrates and activity-based probes. However, there are eleven human cysteine cathepsins, with a few of them displaying overlapping substrate specificity, making the development of small molecules that selectively target a single cathepsin very challenging. Here, we utilized HyCoSuL, a positional scanning substrate approach, to develop a highly-selective fluorogenic substrate and activity-based probe for monitoring cathepsin L activity in the breast cancer cell line MDA-MB-231. Use of this probe enabled us to distinguish the activity of cathepsin L from that of other cathepsins, particularly cathepsin B, which is abundant and ubiquitously expressed in normal and transformed cell types. We found that cathepsin L localization in MDA-MB-231 cells greatly overlaps with that of cathepsin B, however, several cathepsin L-rich lysosomes lacked cathepsin B activity. Overall, these studies demonstrate that HyCoSuL-derived small molecule probes are valuable tools to image cathepsin L activity in living cells. This approach thus enables evaluation of cathepsin L function in tumorigenesis and is applicable to other cysteine cathepsins. PMID:29719685

Imaging Spatial Variations in the Dissipation and Transport of Thermal Energy within Individual Silicon Nanowires Using Ultrafast Microscopy.

PubMed

Cating, Emma E M; Pinion, Christopher W; Van Goethem, Erika M; Gabriel, Michelle M; Cahoon, James F; Papanikolas, John M

2016-01-13

Thermal management is an important consideration for most nanoelectronic devices, and an understanding of the thermal conductivity of individual device components is critical for the design of thermally efficient systems. However, it can be difficult to directly probe local changes in thermal conductivity within a nanoscale system. Here, we utilize the time-resolved and diffraction-limited imaging capabilities of ultrafast pump-probe microscopy to determine, in a contact-free configuration, the local thermal conductivity in individual Si nanowires (NWs). By suspending single NWs across microfabricated trenches in a quartz substrate, the properties of the same NW both on and off the substrate are directly compared. We find the substrate has no effect on the recombination lifetime or diffusion length of photogenerated charge carriers; however, it significantly impacts the thermal relaxation properties of the NW. In substrate-supported regions, thermal energy deposited into the lattice by the ultrafast laser pulse dissipates within ∼10 ns through thermal diffusion and coupling to the substrate. In suspended regions, the thermal energy persists for over 100 ns, and we directly image the time-resolved spatial motion of the thermal signal. Quantitative analysis of the transient images permits direct determination of the NW's local thermal conductivity, which we find to be a factor of ∼4 smaller than in bulk Si. Our results point to the strong potential of pump-probe microscopy to be used as an all-optical method to quantify the effects of localized environment and morphology on the thermal transport characteristics of individual nanostructured components.

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

PubMed Central

Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

2008-01-01

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

Sensing the facet orientation in silver nano-plates using scanning Kelvin probe microscopy in air

NASA Astrophysics Data System (ADS)

Abdellatif, M. H.; Salerno, M.; Polovitsyn, Anatolii; Marras, Sergio; De Angelis, Francesco

2017-05-01

The work function of nano-materials is important for a full characterization of their electronic properties. Because the band alignment, band bending and electronic noise are very sensitive to work function fluctuations, the dependence of the work function of nano-scale crystals on facet orientation can be a critical issue in optimizing optoelectronic devices based on these materials. We used scanning Kelvin probe microscopy to assess the local work function on samples of silver nano-plates at sub-micrometric spatial resolution. With the appropriate choice of the substrate and based on statistical analysis, it was possible to distinguish the surface potential of the different facets of silver nano-plates even if the measurements were done in ambient conditions without the use of vacuum. A phenomenological model was used to calculate the differences of facet work function of the silver nano-plates and the corresponding shift in Fermi level. This theoretical prediction and the experimentally observed difference in surface potential on the silver nano-plates were in good agreement. Our results show the possibility to sense the nano-crystal facets by appropriate choice of the substrate in ambient conditions.

Glass transition in thin supported polystyrene films probed by temperature-modulated ellipsometry in vacuum.

PubMed

Efremov, Mikhail Yu; Kiyanova, Anna V; Last, Julie; Soofi, Shauheen S; Thode, Christopher; Nealey, Paul F

2012-08-01

Glass transition in thin (1-200 nm thick) spin-cast polystyrene films on silicon surfaces is probed by ellipsometry in a controlled vacuum environment. A temperature-modulated modification of the method is used alongside a traditional linear temperature scan. A clear glass transition is detected in films with thicknesses as low as 1-2 nm. The glass transition temperature (T(g)) shows no substantial dependence on thickness for coatings greater than 20 nm. Thinner films demonstrate moderate T(g) depression achieving 18 K for thicknesses 4-7 nm. Less than 4 nm thick samples are excluded from the T(g) comparison due to significant thickness nonuniformity (surface roughness). The transition in 10-20 nm thick films demonstrates excessive broadening. For some samples, the broadened transition is clearly resolved into two separate transitions. The thickness dependence of the glass transition can be well described by a simple 2-layer model. It is also shown that T(g) depression in 5 nm thick films is not sensitive to a wide range of experimental factors including molecular weight characteristics of the polymer, specifications of solvent used for spin casting, substrate composition, and pretreatment of the substrate surface.

GaN-based micro-LED arrays on flexible substrates for optical cochlear implants

NASA Astrophysics Data System (ADS)

Goßler, Christian; Bierbrauer, Colin; Moser, Rüdiger; Kunzer, Michael; Holc, Katarzyna; Pletschen, Wilfried; Köhler, Klaus; Wagner, Joachim; Schwaerzle, Michael; Ruther, Patrick; Paul, Oliver; Neef, Jakob; Keppeler, Daniel; Hoch, Gerhard; Moser, Tobias; Schwarz, Ulrich T.

2014-05-01

Currently available cochlear implants are based on electrical stimulation of the spiral ganglion neurons. Optical stimulation with arrays of micro-sized light-emitting diodes (µLEDs) promises to increase the number of distinguishable frequencies. Here, the development of a flexible GaN-based micro-LED array as an optical cochlear implant is reported for application in a mouse model. The fabrication of 15 µm thin and highly flexible devices is enabled by a laser-based layer transfer process of the GaN-LEDs from sapphire to a polyimide-on-silicon carrier wafer. The fabricated 50 × 50 µm2 LEDs are contacted via conducting paths on both p- and n-sides of the LEDs. Up to three separate channels could be addressed. The probes, composed of a linear array of the said µLEDs bonded to the flexible polyimide substrate, are peeled off the carrier wafer and attached to flexible printed circuit boards. Probes with four µLEDs and a width of 230 µm are successfully implanted in the mouse cochlea both in vitro and in vivo. The LEDs emit 60 µW at 1 mA after peel-off, corresponding to a radiant emittance of 6 mW mm-2.

Vertically aligned nanostructure scanning probe microscope tips

DOEpatents

Guillorn, Michael A.; Ilic, Bojan; Melechko, Anatoli V.; Merkulov, Vladimir I.; Lowndes, Douglas H.; Simpson, Michael L.

2006-12-19

Methods and apparatus are described for cantilever structures that include a vertically aligned nanostructure, especially vertically aligned carbon nanofiber scanning probe microscope tips. An apparatus includes a cantilever structure including a substrate including a cantilever body, that optionally includes a doped layer, and a vertically aligned nanostructure coupled to the cantilever body.

Experimental investigation and computational modeling of hot filament diamond chemical vapor deposition

NASA Astrophysics Data System (ADS)

Zumbach, Volker; Schäfer, Jörg; Tobai, Jens; Ridder, Michael; Dreier, Thomas; Schaich, Thomas; Wolfrum, Jürgen; Ruf, Bernhard; Behrendt, Frank; Deutschman, Olaf; Warnatz, Jürgen

1997-10-01

A joint investigation has been undertaken of the gas-phase chemistry taking place in a hot-filament chemical vapor-deposition (HFCVD) process for diamond synthesis on silica surfaces by a detailed comparison of numerical modeling and experimental results. Molecular beam sampling using quadrupole mass spectroscopy and resonance-enhanced multiphoton ionization time of flight mass spectroscopy (REMPI-TOF-MS) has been used to determine absolute concentrations of stable hydrocarbons and radicals. Resulting species of a CH4/H2, a CH4/D2 (both 0.5%/99.5%) and a C2H2/H2 (0.25%/99.75%) feedgas mixture were investigated for varying filament and substrate temperatures. Spatially resolved temperature profiles at various substrate temperatures, obtained from coherent anti-Stokes Raman spectroscopy (CARS) of hydrogen, are used as input parameters for the numerical code to reproduce hydrogen atom, methyl radical, methane, acetylene, and ethylene concentration profiles in the boundary layer of the substrate. In addition, the concentration of vibrationally excited hydrogen is determined by CARS. Results reveal only qualitative agreement between measured data and simulations, concerning concentrations of stable species and radicals probed near the surface, on filament and substrate temperature dependence, respectively. Hydrogen and deuterium experiments show similar behaviour for all species. In the case of CH4 as feedgas the model describes measured concentration profiles of CH3, CH4, and C2H2 qualitatively well. Large differences between model and experiment occur for hydrogen atoms (factor of 2) and C2H4 (factor of 3). For acetylene as feedgas the model is not able to give any predictions because no conversion of C2H2 is seen in the model in contrast to the experiment.

Monolayer Contact Doping of Silicon Surfaces and Nanowires Using Organophosphorus Compounds

PubMed Central

Hazut, Ori; Agarwala, Arunava; Subramani, Thangavel; Waichman, Sharon; Yerushalmi, Roie

2013-01-01

Monolayer Contact Doping (MLCD) is a simple method for doping of surfaces and nanostructures1. MLCD results in the formation of highly controlled, ultra shallow and sharp doping profiles at the nanometer scale. In MLCD process the dopant source is a monolayer containing dopant atoms. In this article a detailed procedure for surface doping of silicon substrate as well as silicon nanowires is demonstrated. Phosphorus dopant source was formed using tetraethyl methylenediphosphonate monolayer on a silicon substrate. This monolayer containing substrate was brought to contact with a pristine intrinsic silicon target substrate and annealed while in contact. Sheet resistance of the target substrate was measured using 4 point probe. Intrinsic silicon nanowires were synthesized by chemical vapor deposition (CVD) process using a vapor-liquid-solid (VLS) mechanism; gold nanoparticles were used as catalyst for nanowire growth. The nanowires were suspended in ethanol by mild sonication. This suspension was used to dropcast the nanowires on silicon substrate with a silicon nitride dielectric top layer. These nanowires were doped with phosphorus in similar manner as used for the intrinsic silicon wafer. Standard photolithography process was used to fabricate metal electrodes for the formation of nanowire based field effect transistor (NW-FET). The electrical properties of a representative nanowire device were measured by a semiconductor device analyzer and a probe station. PMID:24326774

Frictional properties of the end-grafted polymer layer in presence of salt solution

NASA Astrophysics Data System (ADS)

Raftari, Maryam; Zhang, Zhenyu; Leggett, Graham J.; Geoghegan, Mark

2012-02-01

We have studied the frictional behaviour of grafted poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) films using friction force microscopy (FFM). The films were prepared on native oxide-terminated silicon substrates using the technique of atom transfer radical polymerization (ATRP). These brushes had constant grafting density (1.18 nm2), and of a thickness of ˜66 nm, as measured by ellipsometry. We show that single asperity contact mechanics (Johnson-Kendall-Roberts (JKR) and Derjaguin-M"uller-Toporov (DMT) models) as well as a linear (Amontons) relation between applied load and frictional load all apply to these systems depending on the concentration of salt and the nature of the FFM probe. Measurements were made using gold-coating and polymer functionalized silicon nitride triangular probes. Polymer functionalized probe included growth the PDMAEMA with same method on tips. The frictional behaviour are investigated between PDMAEMA and gold coated and PDMAEMA tips immersed in different concentrations of KCl, KBr and KI.

Scanning Probe Platform | Materials Science | NREL

Science.gov Websites

level; this image obtained using a scanning tunneling microscope shows gray and white clusters of produce high-resolution color images or maps like this one obtained using scanning tunneling luminescence gray clusters. Gold substrate: (Left) STM image reveals the terraces of the H2 flamed substrate. (Right

Measuring the Thickness and Potential Profiles of the Space-Charge Layer at Organic/Organic Interfaces under Illumination and in the Dark by Scanning Kelvin Probe Microscopy.

PubMed

Rojas, Geoffrey A; Wu, Yanfei; Haugstad, Greg; Frisbie, C Daniel

2016-03-09

Scanning Kelvin probe microscopy was used to measure band-bending at the model donor/acceptor heterojunction poly(3-hexylthiophene) (P3HT)/fullerene (C60). Specifically, we measured the variation in the surface potential of C60 films with increasing thicknesses grown on P3HT to produce a surface potential profile normal to the substrate both in the dark and under illumination. The results confirm a space-charge carrier region with a thickness of 10 nm, consistent with previous observations. We discuss the possibility that the domain size in bulk heterojunction organic solar cells, which is comparable to the space-charge layer thickness, is actually partly responsible for less than expected electron/hole recombination rates.

In situ elasticity modulation with dynamic substrates to direct cell phenotype

PubMed Central

Kloxin, April M.; Benton, Julie A.; Anseth, Kristi S.

2009-01-01

Microenvironment elasticity influences critical cell functions such as differentiation, cytoskeletal organization, and process extension. Unfortunately, few materials allow elasticity modulation in real-time to probe its direct effect on these dynamic cellular processes. Here, a new approach is presented for the photochemical modulation of elasticity within the cell's microenvironment at any point in time. A photodegradable hydrogel was irradiated and degraded under cytocompatible conditions to generate a wide range of elastic moduli similar to soft tissues and characterized using rheometry and atomic force microscopy (AFM). The effect of the elastic modulus on valvular interstitial cell (VIC) activation into myofibroblasts was explored. In these studies, gradient samples were used to identify moduli that either promote or suppress VIC myofibroblastic activation. With this knowledge, VICs were cultured on a high modulus, activating hydrogel substrate, and uniquely, results show that decreasing the substrate modulus with irradiation reverses this activation, demonstrating that myofibroblasts can be de-activated solely by changing the modulus of the underlying substrate. This finding is important for the rational design of biomaterials for tissue regeneration and offers insight into fibrotic disease progression. These photodegradable hydrogels demonstrate the capability to both probe and direct cell function through dynamic changes in substrate elasticity. PMID:19788947

Therapeutic protein-drug interaction assessment for daclizumab high-yield process in patients with multiple sclerosis using a cocktail approach.

PubMed

Tran, Jonathan Q; Othman, Ahmed A; Wolstencroft, Paul; Elkins, Jacob

2016-07-01

To characterize the potential effect of daclizumab high-yield process (DAC HYP), a monoclonal antibody that blocks the high-affinity interleukin-2 receptors for treatment of multiple sclerosis, on activity of cytochrome P450 (CYP) enzymes. Twenty patients with multiple sclerosis received an oral cocktail of probe substrates of CYP1A2 (caffeine 200 mg), CYP2C9 (warfarin 10 mg/vitamin K 10 mg), CYP2C19 (omeprazole 40 mg), CYP2D6 (dextromethorphan 30 mg) and CYP3A (midazolam 5 mg) on two sequential occasions: 7 days before and 7 days after subcutaneous administration of DAC HYP 150 mg every 4 weeks for three doses. Serial pharmacokinetic blood samples up to 96 h post dose and 12-h urine samples were collected on both occasions. Area under the curve (AUC) for caffeine, S-warfarin, omeprazole and midazolam, and urine dextromethorphan to dextrorphan ratio were calculated. Statistical analyses were conducted on log-transformed parameters using a linear mixed-effects model. The 90% confidence intervals (CIs) for the geometric mean ratio (probe substrate with DAC HYP/probe substrate alone) for caffeine AUC from 0-12 h (0.93-1.15), S-warfarin AUC from 0 to infinity (AUC[0-inf]) (0.95-1.06), omeprazole AUC(0-inf) (0.88-1.13) and midazolam AUC(0-inf) (0.89-1.15) were within the no-effect boundary of 0.80-1.25. The geometric mean ratio for urine dextromethorphan to dextrorphan ratio was 1.01, with the 90% CI (0.76-1.34) extending slightly outside the no-effect boundary, likely due to high variability with urine collections and CYP2D6 activity. DAC HYP treatment in patients with multiple sclerosis had no effect on CYP 1A2, 2C9, 2C19, 2D6 and 3A activity. © 2016 The British Pharmacological Society.

Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity.

PubMed

Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G; Achilefu, Samuel

2013-01-01

Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

PubMed Central

Aubin-Tam, Marie-Eve; Olivares, Adrian O.; Sauer, Robert T.; Baker, Tania A.; Lang, Matthew J.

2011-01-01

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP. PMID:21496645

The Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA Binding†

PubMed Central

Clift, Michael; Ji, Haitao; Deniau, Gildas P.; O’Hagan, David; Silverman, Richard B.

2008-01-01

γ-Aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5’-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5’-phosphate (PLP) to pyridoxamine 5’-phosphate (PMP). The enzyme then catalyzes the conversion of α-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred based on the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches. PMID:17988152

Use of microdose phenotyping to individualise dosing of patients.

PubMed

Hohmann, Nicolas; Haefeli, Walter E; Mikus, Gerd

2015-09-01

Administering the right amount of the right drug at the right time is a key mission of clinical medicine. This comprises dose adaptation according to a patient's intrinsic and extrinsic factors influencing drug disposition. Several biomarkers are available for dose adaptation; still, prediction of individual drug disposition may be improved. Phenotyping is the quantification of drug metabolism with probe substrates specific to drug-metabolising enzymes. This allows measurement of baseline metabolism and changes after modulation of drug metabolism. This article explores the concept of phenotyping using pharmacologically ineffective microdoses of probe substrates to obtain information on drug metabolism. Several probe drugs such as midazolam for cytochrome P450 3A have already been used, but validation of other microdosed probe drugs, analytical procedures and drug formulations still face some challenges that have to be overcome. Since microdosed probe drugs have no risk of adverse drug reactions or interference with therapy, more widespread use is possible. This allows drug-drug interaction data to be safely obtained during first-in-man studies, enhancing the clinical safety of human healthy volunteers and patients in clinical trials, and, most importantly, allows determination of the drug-metabolising phenotype in severely ill patients. With harmless probe drugs at hand quantifying drug metabolism and adapting the dose accordingly, a phenotyping-based dosing strategy could become reality, offering the possibility of individualised drug therapy with reduced adverse effects and fewer therapeutic failures.

A hot-spot-active magnetic graphene oxide substrate for microRNA detection based on cascaded chemiluminescence resonance energy transfer.

PubMed

Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying

2015-02-28

Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal "off" state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an "open" configuration of the hairpin probe and a CRET signal "on" state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing.

Accessing the dynamics of end-grafted flexible polymer chains by atomic force-electrochemical microscopy. Theoretical modeling of the approach curves by the elastic bounded diffusion model and Monte Carlo simulations. Evidence for compression-induced lateral chain escape.

PubMed

Abbou, Jeremy; Anne, Agnès; Demaille, Christophe

2006-11-16

The dynamics of a molecular layer of linear poly(ethylene glycol) (PEG) chains of molecular weight 3400, bearing at one end a ferrocene (Fc) label and thiol end-grafted at a low surface coverage onto a gold substrate, is probed using combined atomic force-electrochemical microscopy (AFM-SECM), at the scale of approximately 100 molecules. Force and current approach curves are simultaneously recorded as a force-sensing microelectrode (tip) is inserted within the approximately 10 nm thick, redox labeled, PEG chain layer. Whereas the force approach curve gives access to the structure of the compressed PEG layer, the tip-current, resulting from tip-to-substrate redox cycling of the Fc head of the chain, is controlled by chain dynamics. The elastic bounded diffusion model, which considers the motion of the Fc head as diffusion in a conformational field, complemented by Monte Carlo (MC) simulations, from which the chain conformation can be derived for any degree of confinement, allows the theoretical tip-current approach curve to be calculated. The experimental current approach curve can then be very satisfyingly reproduced by theory, down to a tip-substrate separation of approximately 2 nm, using only one adjustable parameter characterizing the chain dynamics: the effective diffusion coefficient of the chain head. At closer tip-substrate separations, an unpredicted peak is observed in the experimental current approach curve, which is shown to find its origin in a compression-induced escape of the chain from within the narrowing tip-substrate gap. MC simulations provide quantitative support for lateral chain elongation as the escape mechanism.

Evaluation of software sensors for on-line estimation of culture conditions in an Escherichia coli cultivation expressing a recombinant protein.

PubMed

Warth, Benedikt; Rajkai, György; Mandenius, Carl-Fredrik

2010-05-03

Software sensors for monitoring and on-line estimation of critical bioprocess variables have mainly been used with standard bioreactor sensors, such as electrodes and gas analyzers, where algorithms in the software model have generated the desired state variables. In this article we propose that other on-line instruments, such as NIR probes and on-line HPLC, should be used to make more reliable and flexible software sensors. Five software sensor architectures were compared and evaluated: (1) biomass concentration from an on-line NIR probe, (2) biomass concentration from titrant addition, (3) specific growth rate from titrant addition, (4) specific growth rate from the NIR probe, and (5) specific substrate uptake rate and by-product rate from on-line HPLC and NIR probe signals. The software sensors were demonstrated on an Escherichia coli cultivation expressing a recombinant protein, green fluorescent protein (GFP), but the results could be extrapolated to other production organisms and product proteins. We conclude that well-maintained on-line instrumentation (hardware sensors) can increase the potential of software sensors. This would also strongly support the intentions with process analytical technology and quality-by-design concepts. 2010 Elsevier B.V. All rights reserved.

A hot-spot-active magnetic graphene oxide substrate for microRNA detection based on cascaded chemiluminescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying

2015-02-01

Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal ``off'' state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an ``open'' configuration of the hairpin probe and a CRET signal ``on'' state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing.Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal ``off'' state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an ``open'' configuration of the hairpin probe and a CRET signal ``on'' state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing. Electronic supplementary information (ESI) available: Sequences of RNA and DNA used in this study, relationship of the proposed three modes, CRET mechanism of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein system, calculation of the surface coverage of hairpin probe I-1 on MGO, control experiment, comparison between different modes for microRNA detection, and advantages of the proposed strategy. See DOI: 10.1039/c4nr06603k

Activity, specificity, and probe design for the smallpox virus protease K7L.

PubMed

Aleshin, Alexander E; Drag, Marcin; Gombosuren, Naran; Wei, Ge; Mikolajczyk, Jowita; Satterthwait, Arnold C; Strongin, Alex Y; Liddington, Robert C; Salvesen, Guy S

2012-11-16

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

Combination of inverted pyramidal nanovoid with silver nanoparticles to obtain further enhancement and its detection for ricin

NASA Astrophysics Data System (ADS)

Wang, Meng; Wang, Bin; Wu, Shixuan; Guo, Tingke; Li, Haoyu; Guo, Zhaoqing; Wu, Junhua; Jia, Peiyuan; Wang, Yuxia; Xu, Xiaoxuan; Wang, Yufang; Zhang, Cunzhou

2015-02-01

We have obtained the surface-enhanced Raman scattering substrate by depositing silver nanoparticles on the surface of the inverted pyramidal nanovoid in order to improve the enhance effects. Experimental results showed that the combined substrate exhibited greater enhancement than the nanovoid substrate or nanoparticles. In order to test the SERS activity of the combined substrates, Rh6G and ricin toxin were used as Raman probes. Finite element method was employed to simulate electric field and induced charge distribution of the substrates, which have been used to explore the interaction between nanoparticles and nanovoid as well as mechanism of the great enhancement.

Smooth perfluorinated surfaces with different chemical and physical natures: their unusual dynamic dewetting behavior toward polar and nonpolar liquids.

PubMed

Cheng, Dalton F; Masheder, Benjamin; Urata, Chihiro; Hozumi, Atsushi

2013-09-10

The effects of surface chemistry and the mobility of surface-tethered functional groups of various perfluorinated surfaces on their dewetting behavior toward polar (water) and nonpolar (n-hexadecane, n-dodecane, and n-decane) liquids were investigated. In this study, three types of common smooth perfluorinated surfaces, that is, a perfluoroalkylsilane (heptadecafluoro-1,1,2,2-tetrahydrooctyl-dimethylchlorosilane, FAS17) monomeric layer, an amorphous fluoropolymer film (Teflon AF 1600), and a perfluorinated polyether (PFPE)-terminated polymer brush film (Optool DSX), were prepared and their static/dynamic dewetting characteristics were compared. Although the apparent static contact angles (CAs) of these surfaces with all probe liquids were almost identical to each other, the ease of movement of liquid drops critically depended on the physical (solidlike or liquidlike) natures of the substrate surface. CA hysteresis and substrate tilt angles (TAs) of all probe liquids on the Optool DSX surface were found to be much lower than those of Teflon AF1600 and FAS17 surfaces due to its physical polymer chain mobility at room temperature and the resulting liquidlike nature. Only 6.0° of substrate incline was required to initiate movement for a small drop (5 μL) of n-decane, which was comparable to the reported substrate TA value (5.3°) for a superoleophobic surface (θ(S) > 160°, textured perfluorinated surface). Such unusual dynamic dewetting behavior of the Optool DSX surface was also markedly enhanced due to the significant increase in the chain mobility of PFPE by moderate heating (70 °C) of the surface, with substrate TA reducing to 3.0°. CA hysteresis and substrate TAs rather than static CAs were therefore determined to be of greater consequence for the estimation of the actual dynamic dewetting behavior of alkane probe liquids on these smooth perfluorinated surfaces. Their dynamic dewettability toward alkane liquids is in the order of Optool DSX > Teflon AF1600 ≈ FAS17.

Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts

PubMed Central

Deu, Edgar; Yang, Zhimou; Wang, Flora; Klemba, Michael; Bogyo, Matthew

2010-01-01

Background High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic. PMID:20700487

Na+/substrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-labeled Substrate

PubMed Central

Steed, P. Ryan; Stein, Richard A.; Mishra, Smriti; Goodman, Michael C.; Mchaourab, Hassane S.

2013-01-01

NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na+ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal to characterize mechanistically relevant binding interactions with NorM and to directly address the coupling of ion and drug binding. Fluorescence and EPR experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na+. Using double electron-electron resonance (DEER) spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na+ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na+ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na+ gradient across the native membrane, govern Na+/drug coupling during antiport. PMID:23902581

Bacterial plaque retention on oral hard materials: effect of surface roughness, surface composition, and physisorbed polycarboxylate.

PubMed

McConnell, Marla D; Liu, Yu; Nowak, Andrew P; Pilch, Shira; Masters, James G; Composto, Russell J

2010-03-15

Bacterial adhesion to oral hard materials is dependent on various factors, for example, surface roughness and surface composition. In this study, bacteria retention on three oral hard substrates, hydroxyapatite (HAP), enamel, and polished enamel (p-enamel) were investigated. The surface morphology and roughness of the three substrates were measured by scanning probe microscopy. HAP had the roughest surface, followed by enamel and polished enamel. For each individual substrate type, the roughness was shown to increase with scan size up to 50 microm x 50 microm. For HAP and enamel, roughness decreased considerably after formation of a pellicle, while addition of polymer coating to the pellicle layer reduced roughness much less in comparison. Bacterial surface coverage was measured at 30 min, 3 h, and 24 h on both native and surface-modified substrates, which were coated with two different polycarboxylate-based polymers, Gantrez S97 and Carbopol 940. As a result, the polymer coated surfaces had reduced bacteria coverage compared with the native surfaces over all time points and substrates measured. The reduction is the combined effect of electrostatic repulsion and sequestering of Ca(2+) ions at the surface, which plays a key role in the initial adhesion of bacteria to enamel surfaces in models of plaque formation. (c) 2009 Wiley Periodicals, Inc.

Polarity Control of Heteroepitaxial GaN Nanowires on Diamond.

PubMed

Hetzl, Martin; Kraut, Max; Hoffmann, Theresa; Stutzmann, Martin

2017-06-14

Group III-nitride materials such as GaN nanowires are characterized by a spontaneous polarization within the crystal. The sign of the resulting sheet charge at the top and bottom facet of a GaN nanowire is determined by the orientation of the wurtzite bilayer of the different atomic species, called N and Ga polarity. We investigate the polarity distribution of heteroepitaxial GaN nanowires on different substrates and demonstrate polarity control of GaN nanowires on diamond. Kelvin Probe Force Microscopy is used to determine the polarity of individual selective area-grown and self-assembled nanowires over a large scale. At standard growth conditions, mixed polarity occurs for selective GaN nanowires on various substrates, namely on silicon, on sapphire and on diamond. To obtain control over the growth orientation on diamond, the substrate surface is modified by nitrogen and oxygen plasma exposure prior to growth, and the growth parameters are adjusted simultaneously. We find that the surface chemistry and the substrate temperature are the decisive factors for obtaining control of up to 93% for both polarity types, whereas the growth mode, namely selective area or self-assembled growth, does not influence the polarity distribution significantly. The experimental results are discussed by a model based on the interfacial bonds between the GaN nanowires, the termination layer, and the substrate.

Disulphide linkage: To get cleaved or not? Bulk and nano copper based SERS of cystine

NASA Astrophysics Data System (ADS)

P. J., Arathi; Seemesh, Bhaskar; Rajendra Kumar Reddy, G.; Suresh Kumar, P.; Ramanathan, V.

2018-05-01

Different nano-structures of noble metals have been the conventional substrates for carrying out Surface Enhanced Raman Spectroscopy (SERS). In this paper we examine electrodeposited copper (Cu) nano-structures on pencil graphite as novel substrate to carry out SERS measurements by considering L-cystine (Cys-Cys) (dimer of the amino acid cysteine) as the probe. The formation of monolayer of the probe molecule on the substrates was confirmed using cyclic voltammetric measurements. Mode of adsorption of Cys-Cys was observed to be different on bulk Cu (taken in the wire form) and nano-structured Cu on pencil graphite. Whereas in the former the disulphide bond of Cys-Cys remained intact, it got cleaved when Cys-Cys was adsorbed on electrodeposited copper indicating the activated nature of the nano-structure compared to bulk copper. Csbnd S stretching mode of vibration underwent blue shift in Cys-Cys adsorbed on Cu on pencil graphite vis-à-vis Cys-Cys adsorbed on Cu wire. Further evidence on the cleavage of the Csbnd S bond on an activated substrate was obtained by considering a bimetallic substrate comprising of silver on copper which was electrodeposited on pencil graphite. Our studies have demonstrated that nano-copper surface is an excellent substrate for SERS giving 200 μM as lower detection limit for Cys-Cys.

Molecular modeling studies of substrate binding by penicillin acylase.

PubMed

Chilov, G G; Stroganov, O V; Svedas, V K

2008-01-01

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.

High performance and reusable SERS substrates using Ag/ZnO heterostructure on periodic silicon nanotube substrate

NASA Astrophysics Data System (ADS)

Lai, Yi-Chen; Ho, Hsin-Chia; Shih, Bo-Wei; Tsai, Feng-Yu; Hsueh, Chun-Hway

2018-05-01

Surface-enhanced Raman scattering (SERS) substrate with a higher surface area, enhanced light harvesting, multiple hot spots and strong electromagnetic field enhancements would exhibit enhanced Raman signals. Herein, the Ag nanoparticle/ZnO nanowire heterostructure decorated periodic silicon nanotube (Ag@ZnO@SiNT) substrate was proposed and fabricated. The proposed structure employed as SERS-active substrate was examined, and the results showed both the high performance in terms of high sensitivity and good reproducibility. Furthermore, the Ag@ZnO@SiNT substrate demonstrated the self-cleaning performance through the photocatalytic degradation of probed molecules upon UV-irradiation. The results showed that the proposed nanostructure had high performance, good reproducibility and reusability, and it is a promising SERS-active substrate for molecular sensing and cleaning.

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

Stokes, Adam; Al-Jassim, Mowafak; Norman, Andrew

The effects of alkali diffusion and post-deposition treatment in three-stage processed Cu(In,Ga)Se 2 solar cells are examined by using atom probe tomography and electrical property measurements. Cells, for which the substrate was treated at 650 °C to induce alkali diffusion from the substrate prior to absorber deposition, exhibited high open-circuit voltage (758 mV) and efficiency (18.2%) and also exhibited a 50 to 100-nm-thick ordered vacancy compound layer at the metallurgical junction. Surprisingly, these high-temperature samples exhibited higher concentrations of K at the junction (1.8 at.%) than post-deposition treatment samples (0.4 at.%). A model that uses Ga/(Ga + In) and Cu/(Gamore » + In) profiles to predict bandgaps (+/-17.9 meV) of 22 Cu(In,Ga)Se2 solar cells reported in literature was discussed and ultimately used to predict band properties at the nanoscale by using atom probe tomography data. The high-temperature samples exhibited a greater drop in the valence band maximum (200 meV) due to a lower Cu/(Ga + In) ratio than the post-deposition treatment samples. There was an anticorrelation of K concentrations and Cu/(Ga + In) ratios for all samples, regardless of processing conditions. In conclusion, changes in elemental profiles at the active junctions correlate well with the electrical behaviour of these devices.« less

Triplet state dissolved organic matter in aquatic photochemistry: reaction mechanisms, substrate scope, and photophysical properties.

PubMed

McNeill, Kristopher; Canonica, Silvio

2016-11-09

Excited triplet states of chromophoric dissolved organic matter ( 3 CDOM*) play a major role among the reactive intermediates produced upon absorption of sunlight by surface waters. After more than two decades of research on the aquatic photochemistry of 3 CDOM*, the need for improving the knowledge about the photophysical and photochemical properties of these elusive reactive species remains considerable. This critical review examines the efforts to date to characterize 3 CDOM*. Information on 3 CDOM* relies mainly on the use of probe compounds because of the difficulties associated with directly observing 3 CDOM* using transient spectroscopic methods. Singlet molecular oxygen ( 1 O 2 ), which is a product of the reaction between 3 CDOM* and dissolved oxygen, is probably the simplest indicator that can be used to estimate steady-state concentrations of 3 CDOM*. There are two major modes of reaction of 3 CDOM* with substrates, namely triplet energy transfer or oxidation (via electron transfer, proton-coupled electron transfer or related mechanisms). Organic molecules, including several environmental contaminants, that are susceptible to degradation by these two different reaction modes are reviewed. It is proposed that through the use of appropriate sets of probe compounds and model photosensitizers an improved estimation of the distribution of triplet energies and one-electron reduction potentials of 3 CDOM* can be achieved.

Improving surface-enhanced Raman scattering effect using gold-coated hierarchical polystyrene bead substrates modified with postgrowth microwave treatment.

PubMed

Yuen, Clement; Zheng, Wei; Huang, Zhiwei

2008-01-01

We report a novel postgrowth microwave heating implementation by selectively modifying hierarchical polystyrene (PS) bead substrates coated with gold (Au) films to effectively improve the surface-enhanced Raman scattering (SERS) effect on the analytes. The SERS signal of probe molecule rhodamine 6G (Rh 6G) on the microwave-treated Au-PS substrates can be improved by 10-fold, while the detection limit of Rh 6G in concentration can be enhanced by two orders of magnitude compared to the as-growth substrates. The high-quality SERS spectrum of saliva can also be acquired using the modified substrates, demonstrating the potential for the realization of the high-performance SERS substrates for biomedical applications.

Influence of smectite suspension structure on sheet orientation in dry sediments: XRD and AFM applications.

PubMed

Zbik, Marek S; Frost, Ray L

2010-06-15

The structure-building phenomena within clay aggregates are governed by forces acting between clay particles. Measurements of such forces are important to understand in order to manipulate the aggregate structure for applications such as dewatering of mineral processing tailings. A parallel particle orientation is required when conducting XRD investigation on the oriented samples and conduct force measurements acting between basal planes of clay mineral platelets using atomic force microscopy (AFM). To investigate how smectite clay platelets were oriented on silicon wafer substrate when dried from suspension range of methods like SEM, XRD and AFM were employed. From these investigations, we conclude that high clay concentrations and larger particle diameters (up to 5 microm) in suspension result in random orientation of platelets in the substrate. The best possible laminar orientation in the clay dry film, represented in the XRD 001/020 intensity ratio of 47 was obtained by drying thin layers from 0.02 wt.% clay suspensions of the natural pH. Conducted AFM investigations show that smectite studied in water based electrolytes show very long-range repulsive forces lower in strength than electrostatic forces from double-layer repulsion. It was suggested that these forces may have structural nature. Smectite surface layers rehydrate in water environment forms surface gel with spongy and cellular texture which cushion approaching AFM probe. This structural effect can be measured in distances larger than 1000 nm from substrate surface and when probe penetrate this gel layer, structural linkages are forming between substrate and clay covered probe. These linkages prevent subsequently smooth detachments of AFM probe on way back when retrieval. This effect of tearing new formed structure apart involves larger adhesion-like forces measured in retrieval. It is also suggested that these effect may be enhanced by the nano-clay particles interaction. 2010 Elsevier Inc. All rights reserved.

Temperature Dependent Performance of Coplanar Waveguide (CPW) on Substrates of Various Materials

NASA Technical Reports Server (NTRS)

Taub, Susan R.; Young, Paul

1994-01-01

The attenuation (a) and effective dielectric constant (E(sub eff)) of Coplanar Waveguide (CPW) transmission lines on high-resistivity silicon and diamond substrates as a function of both temperature and frequency are presented. The technique used to obtain the values for a and E(sub eff) involves the use of a unique cryogenic probe station designed and built by NASA. Attenuation of gold CPW lines on diamond substrates is compared with that of superconducting CPW lines.

Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes, caffeine, warfarin, omeprazole, metoprolol and midazolam, in common marmosets (Callithrix jacchus).

PubMed

Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Toda, Akiko; Shimizu, Makiko; Uno, Yasuhiro; Sasaki, Erika; Yamazaki, Hiroshi

2016-01-01

1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) composite, after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg(-1), respectively, to four male common marmosets were investigated. 2. The plasma concentrations of caffeine and warfarin decreased slowly in a monophasic manner but those of omeprazole, metoprolol and midazolam decreased extensively after intravenous and oral administrations, in a manner that approximated those as reported for pharmacokinetics in humans. 3. Bioavailabilities were ∼100% for caffeine and warfarin, but <25% for omeprazole and metoprolol. Bioavailability of midazolam was 4% in marmosets, presumably because of contribution of marmoset P450 3A4 expressed in small intestine and liver, with a high catalytic efficiency for midazolam 1'-hydroxylation as evident in the recombinant system. 4. These results suggest that common marmosets, despite their rapid clearance of some human P450 probe substrates, could be an experimental model for humans and that marmoset P450s have functional characteristics that differ from those of human and/or cynomolgus monkey P450s in some aspects, indicating their importance in modeling in P450-dependent drug metabolism studies in marmosets and of further studies.

Nanoscale laminin coating modulates cortical scarring response around implanted silicon microelectrode arrays

NASA Astrophysics Data System (ADS)

He, Wei; McConnell, George C.; Bellamkonda, Ravi V.

2006-12-01

Neural electrodes could significantly enhance the quality of life for patients with sensory and/or motor deficits as well as improve our understanding of brain functions. However, long-term electrical connectivity between neural tissue and recording sites is compromised by the development of astroglial scar around the recording probes. In this study we investigate the effect of a nanoscale laminin (LN) coating on Si-based neural probes on chronic cortical tissue reaction in a rat model. Tissue reaction was evaluated after 1 day, 1 week, and 4 weeks post-implant for coated and uncoated probes using immunohistochemical techniques to evaluate activated microglia/macrophages (ED-1), astrocytes (GFAP) and neurons (NeuN). The coating did not have an observable effect on neuronal density or proximity to the electrode surface. However, the response of microglia/macrophages and astrocytes was altered by the coating. One day post-implant, we observed an ~60% increase in ED-1 expression near LN-coated probe sites compared with control uncoated probe sites. Four weeks post-implant, we observed an ~20% reduction in ED-1 expression along with an ~50% reduction in GFAP expression at coated relative to uncoated probe sites. These results suggest that LN has a stimulatory effect on early microglia activation, accelerating the phagocytic function of these cells. This hypothesis is further supported by the increased mRNA expression of several pro-inflammatory cytokines (TNF-α, IL-1 and IL-6) in cultured microglia on LN-bound Si substrates. LN immunostaining of coated probes immediately after insertion and retrieval demonstrates that the coating integrity is not compromised by the shear force during insertion. We speculate, based on these encouraging results, that LN coating of Si neural probes could potentially improve chronic neural recordings through dispersion of the astroglial scar.

An Introduction to Drug Discovery by Probing Protein-Substrate Interactions Using Saturation Transfer Difference-Nuclear Magnetic Resonance (STD-NMR)

ERIC Educational Resources Information Center

Guegan, Jean-Paul; Daniellou, Richard

2012-01-01

NMR spectroscopy is a powerful tool for characterizing and identifying molecules and nowadays is even used to characterize complex systems in biology. In the experiment presented here, students learned how to apply this modern technique to probe interactions between small molecules and proteins. With the use of simple organic synthesis, students…

A sensitive plasmonic copper(II) sensor based on gold nanoparticles deposited on ITO glass substrate.

PubMed

Ding, Lijun; Gao, Yan; Di, Junwei

2016-09-15

Gold nanoparticles (Au NPs) based plasmonic probe was developed for sensitive and selective detection of Cu(2+) ion. The Au NPs were self-assembled on transparent indium tin oxide (ITO) film coated glass substrate using poly dimethyl diallyl ammonium chloride (PDDA) as a linker and then calcined at 400°C to obtain pure Au NPs on ITO surface (ITO/Au NPs). The probe was fabricated by functionalizing l-cysteine (Cys) on to gold surface (ITO/Au NPs/Cys). The strong chelation of Cu(2+) with Cys formed a stable Cys-Cu complex, and resulted in the red-shift of localized surface plasmon resonance (LSPR) peak of the Au NPs. The introduction of bovine serum albumin (BSA) as the second complexant could form complex of Cys-Cu-BAS and further markedly enhanced the red-shift of the LSPR peak. This plasmonic probe provided a highly sensitive and selective detection towards Cu(2+) ions, with a wide linear detection range (10(-11)-10(-5)M) over 6 orders of magnitude. The simple and cost-effective probe was successfully applied to the determination of Cu(2+) in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Probing phospholipase a(2) with fluorescent phospholipid substrates.

PubMed

Wichmann, Oliver; Gelb, Michael H; Schultz, Carsten

2007-09-03

The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.

Fabrication and surface-modification of implantable microprobes for neuroscience studies

NASA Astrophysics Data System (ADS)

Cao, H.; Nguyen, C. M.; Chiao, J. C.

2012-06-01

In this work implantable micro-probes for central nervous system (CNS) studies were developed on silicon and polyimide substrates. The probes which contained micro-electrode arrays with different surface modifications were designed for implantation in the CNS. The electrode surfaces were modified with nano-scale structures that could greatly increase the active surface area in order to enhance the electrochemical current outputs while maintaining micro-scale dimensions of the electrodes and probes. The electrodes were made of gold or platinum, and designed with different sizes. The silicon probes were modified by silicon nanowires fabricated with the vapor-liquid-solid mechanism at high temperatures. With polyimide substrates, the nanostructure modification was carried out by applying concentrated gold or silver colloid solutions onto the micro-electrodes at room temperature. The surfaces of electrodes before and after modification were observed by scanning electron microscopy. The silicon nanowire-modified surface was characterized by cyclic voltammetry. Experiments were carried out to investigate the improvement in sensing performance. The modified electrodes were tested with H2O2, electrochemical L-glutamate and dopamine. Comparisons between electrodes with and without nanostructure modification were conducted showing that the modifications have enhanced the signal outputs of the electrochemical neurotransmitter sensors.

SU-8 microprobe with microelectrodes for monitoring electrical impedance in living tissues.

PubMed

Tijero, M; Gabriel, G; Caro, J; Altuna, A; Hernández, R; Villa, R; Berganzo, J; Blanco, F J; Salido, R; Fernández, L J

2009-04-15

This paper presents a minimally invasive needle-shaped probe capable of monitoring the electrical impedance of living tissues. This microprobe consists of a 160 microm thick SU-8 substrate containing four planar platinum (Pt) microelectrodes. We design the probe to minimize damage to the surrounding tissue and to be stiff enough to be inserted in living tissues. The proposed batch fabrication process is low cost and low time consuming. The microelectrodes obtained with this process are strongly adhered to the SU-8 substrate and their impedance does not depend on frequency variation. In vitro experiments are compared with previously developed Si and SiC based microprobes and results suggest that it is preferable to use the SU-8 based microprobes due to their flexibility and low cost. The microprobe is assembled on a flexible printed circuit FPC with a conductive glue, packaged with epoxy and wired to the external instrumentation. This flexible probe is inserted into a rat kidney without fracturing and succeeds in demonstrating the ischemia monitoring.

An oscillator based on a single Au nanocluster

NASA Astrophysics Data System (ADS)

Gorshkov, O. N.; Filatov, D. O.; Antonov, D. A.; Antonov, I. N.; Shenina, M. E.; Pavlov, D. A.

2017-01-01

Metal nanoclusters embedded into the ultrathin dielectric films attracted much attention in recent years due to their unusual electronic, optical, etc., properties differing from those of the bulk metals essentially and, hence, to the prospects of their applications in novel nanoelectronic, single electronic, non-volatile memory, etc., devices. Here, we report on the experimental observation of the electrical oscillations in an oscillating loop connected to a contact of a conductive probe of an Atomic Force Microscope to a tunnel-transparent ( ˜6.5 nm thick) yttria stabilized zirconia film with embedded Au nanoclusters on the Si substrate. The oscillations were attributed to the negative differential resistance of the probe-to-sample contact originating from the resonant electron tunnelling between the probe and the Si substrate via the quantum confined electron energy levels in small ( ≈2.5 nm in diameter) Au nanoclusters. This observation demonstrates the prospects of building an oscillator nanoelectronic device based on an individual nanometer-sized metal nanocluster.

Quantum dot-based microfluidic biosensor for cancer detection

NASA Astrophysics Data System (ADS)

Ghrera, Aditya Sharma; Pandey, Chandra Mouli; Ali, Md. Azahar; Malhotra, Bansi Dhar

2015-05-01

We report results of the studies relating to fabrication of an impedimetric microfluidic-based nucleic acid sensor for quantification of DNA sequences specific to chronic myelogenous leukemia (CML). The sensor chip is prepared by patterning an indium-tin-oxide (ITO) coated glass substrate via wet chemical etching method followed by sealing with polydimethylsiloxane (PDMS) microchannel for fluid control. The fabricated microfluidic chip comprising of a patterned ITO substrate is modified by depositing cadmium selenide quantum dots (QCdSe) via Langmuir-Blodgett technique. Further, the QCdSe surface has been functionalized with specific DNA probe for CML detection. The probe DNA functionalized QCdSe integrated miniaturized system has been used to monitor target complementary DNA concentration by measuring the interfacial charge transfer resistance via hybridization. The presence of complementary DNA in buffer solution significantly results in decreased electro-conductivity of the interface due to presence of a charge barrier for transport of the redox probe ions. The microfluidic DNA biosensor exhibits improved linearity in the concentration range of 10-15 M to 10-11 M.

Microstrip antenna study for Pioneer Saturn/Uranus atmosphere entry probe

NASA Technical Reports Server (NTRS)

Kuhlman, E. A.

1974-01-01

The design parameters of a microstrip antenna were studied to determine its performance characteristics as affected by an atmospheric entry probe environment. The technical literature was reviewed to identify the known design and performance characteristics. These data were used to evaluate the expected effects of mission environments on the microstrip antenna design proposed for the Saturn/Uranus Atmospheric Entry Probe (SAEP). Radiation patterns and VSWR measurements were made to evaluate the performance in the SAEP thermal environment. Results of the literature search and pattern tests confirm that the microstrip antenna is a good choice as a transmitting antenna on the SAEP. The microstrip antenna is efficient, compact, and well suited to a space environment. The pattern can be controlled with a minimum beamwidth of 60 degrees (air substrate; e.g., honeycomb structure) and a maximum on the order of 100 degrees with higher dielectric constant substrates. The power handling capacity is good and can be improved by covering the antenna with a dielectric cover.

Investigating Nanoscale Electrochemistry with Surface- and Tip-Enhanced Raman Spectroscopy.

PubMed

Zaleski, Stephanie; Wilson, Andrew J; Mattei, Michael; Chen, Xu; Goubert, Guillaume; Cardinal, M Fernanda; Willets, Katherine A; Van Duyne, Richard P

2016-09-20

The chemical sensitivity of surface-enhanced Raman spectroscopy (SERS) methodologies allows for the investigation of heterogeneous chemical reactions with high sensitivity. Specifically, SERS methodologies are well-suited to study electron transfer (ET) reactions, which lie at the heart of numerous fundamental processes: electrocatalysis, solar energy conversion, energy storage in batteries, and biological events such as photosynthesis. Heterogeneous ET reactions are commonly monitored by electrochemical methods such as cyclic voltammetry, observing billions of electrochemical events per second. Since the first proof of detecting single molecules by redox cycling, there has been growing interest in examining electrochemistry at the nanoscale and single-molecule levels. Doing so unravels details that would otherwise be obscured by an ensemble experiment. The use of optical spectroscopies, such as SERS, to elucidate nanoscale electrochemical behavior is an attractive alternative to traditional approaches such as scanning electrochemical microscopy (SECM). While techniques such as single-molecule fluorescence or electrogenerated chemiluminescence have been used to optically monitor electrochemical events, SERS methodologies, in particular, have shown great promise for exploring electrochemistry at the nanoscale. SERS is ideally suited to study nanoscale electrochemistry because the Raman-enhancing metallic, nanoscale substrate duly serves as the working electrode material. Moreover, SERS has the ability to directly probe single molecules without redox cycling and can achieve nanoscale spatial resolution in combination with super-resolution or scanning probe microscopies. This Account summarizes the latest progress from the Van Duyne and Willets groups toward understanding nanoelectrochemistry using Raman spectroscopic methodologies. The first half of this Account highlights three techniques that have been recently used to probe few- or single-molecule electrochemical events: single-molecule SERS (SMSERS), superlocalization SERS imaging, and tip-enhanced Raman spectroscopy (TERS). While all of the studies we discuss probe model redox dye systems, the experiments described herein push the study of nanoscale electrochemistry toward the fundamental limit, in terms of both chemical sensitivity and spatial resolution. The second half of this Account discusses current experimental strategies for studying nanoelectrochemistry with SERS techniques, which includes relevant electrochemically and optically active molecules, substrates, and substrate functionalization methods. In particular, we highlight the wide variety of SERS-active substrates and optically active molecules that can be implemented for EC-SERS, as well as the need to carefully characterize both the electrochemistry and resultant EC-SERS response of each new redox-active molecule studied. Finally, we conclude this Account with our perspective on the future directions of studying nanoscale electrochemistry with SERS/TERS, which includes the integration of SECM with TERS and the use of theoretical methods to further describe the fundamental intricacies of single-molecule, single-site electrochemistry at the nanoscale.

LysoTracker and MitoTracker Red are transport substrates of P-glycoprotein: implications for anticancer drug design evading multidrug resistance.

PubMed

Zhitomirsky, Benny; Farber, Hodaya; Assaraf, Yehuda G

2018-04-01

LysoTracker and MitoTracker Red are fluorescent probes widely used for viable cell staining of lysosomes and mitochondria, respectively. They are utilized to study organelle localization and their resident proteins, assess organelle functionality and quantification of organelle numbers. The ATP-driven efflux transporter P-glycoprotein (P-gp) is expressed in normal and malignant tissues and extrudes structurally distinct endogenous and exogenous cytotoxic compounds. Thus, once aromatic hydrophobic compounds such as the above-mentioned fluorescent probes are recognized as transport substrates, efflux pumps including P-gp may abolish their ability to reach their cellular target organelles. Herein, we show that LysoTracker and MitoTracker Red are expelled from P-gp-overexpressing cancer cells, thus hindering their ability to fluorescently mark target organelles. We further demonstrate that tariquidar, a potent P-gp transport inhibitor, restores LysoTracker and MitoTracker Red cell entry. We conclude that LysoTracker and MitoTracker Red are P-gp transport substrates, and therefore, P-gp expression must be taken into consideration prior to cellular applications using these probes. Importantly, as MitoTracker was a superior P-gp substrate than LysoTracker Red, we discuss the implications for the future design of chemotherapeutics evading cancer multidrug resistance. Furthermore, restoration of MitoTracker Red fluorescence in P-gp-overexpressing cells may facilitate the identification of potent P-gp transport inhibitors (i.e. chemosensitizers). © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Laser interferometry force-feedback sensor for an interfacial force microscope

DOEpatents

Houston, Jack E.; Smith, William L.

2004-04-13

A scanning force microscope is provided with a force-feedback sensor to increase sensitivity and stability in determining interfacial forces between a probe and a sample. The sensor utilizes an interferometry technique that uses a collimated light beam directed onto a deflecting member, comprising a common plate suspended above capacitor electrodes situated on a substrate forming an interference cavity with a probe on the side of the common plate opposite the side suspended above capacitor electrodes. The probe interacts with the surface of the sample and the intensity of the reflected beam is measured and used to determine the change in displacement of the probe to the sample and to control the probe distance relative to the surface of the sample.

Anisotropic ultrafast response of MoS2 on rippled substrates

NASA Astrophysics Data System (ADS)

Cinquanta, Eugenio; Camellini, Andrea; Martella, Christian; Mennucci, Carlo; Lamperti, Alessio; Della Valle, Giuseppe; Zavelani Rossi, Margherita; Buatier de Mongeot, Francesco; Molle, Alessandro; Stagira, Salvatore

TMDs represent one of the most promising option for new devices characterized by high performances for opto- and nanoelectronics applications. Top-down schemes have been fruitfully exploited for the tuning of TMDs physics by stain engineering in exfoliated flakes. We propose an original bottom-up strategy based on the CVD growth of MoS2 on anisotropic substrates and its characterization by means of pump-probe spectroscopy. The ultrafast response of the rippled MoS2 reveals strongly anisotropic. While the transient absorption emerges as independent from the orientation of the pump beam polarization, the angle between the probe beam polarization and the ripples induces remarkable effects. Within an orthogonal geometry, both the overall intensity of the transient spectrum and the el-ph scattering decay time are halved while the photo-bleaching at 450 nm is blueshifted with respect to the parallel orientation case. Our results demonstrate that the coupling of TMDs with anisotropic substrates is a promising way for the integration of TMDs photonics devices.

Evanescent Microwave Probes on High-Resistivity Silicon and its Application in Characterization of Semiconductors

NASA Technical Reports Server (NTRS)

Tabib-Azar, M.; Akinwande, D.; Ponchak, George E.; LeClair, S. R.

1999-01-01

In this article we report the design, fabrication, and characterization of very high quality factor 10 GHz microstrip resonators on high-resistivity (high-rho) silicon substrates. Our experiments show that an external quality factor of over 13 000 can be achieved on microstripline resonators on high-rho silicon substrates. Such a high Q factor enables integration of arrays of previously reported evanescent microwave probe (EMP) on silicon cantilever beams. We also demonstrate that electron-hole pair recombination and generation lifetimes of silicon can be conveniently measured by illuminating the resonator using a pulsed light. Alternatively, the EMP was also used to nondestructively monitor excess carrier generation and recombination process in a semiconductor placed near the two-dimensional resonator.

Quantitative characterization of semiconductor structures with a scanning microwave microscope.

PubMed

Korolyov, S A; Reznik, A N

2018-02-01

In this work, our earlier method for measuring resistance R sh of semiconductor films with a near-field scanning microwave microscope [A. N. Reznik and S. A. Korolyov, J. Appl. Phys. 119, 094504 (2016)] is studied in a 0.1 kΩ/sq < R sh < 15 kΩ/sq range. The method is based on a microscope model in the form of a monopole or dipole antenna interacting with an arbitrary layered structure. The model fitting parameters are determined from the data yielded by calibration measurements on a system of etalon samples. The performance of the method was analyzed experimentally, using strip-probe and coaxial-probe microscopes in the frequency range of 1-3 GHz. For test structures, we used doped GaN films on the Al 2 O 3 substrate and also transistor structures based on the AlGaN/GaN heterojunction and AlGaAs/GaAs/InGaAs/GaAs/AlGaAs quantum well with a conducting channel. The obtained microwave microscope data were compared with the results of measurements by the van der Pauw method. At the first stage of the experiment, the calibration etalons were bulk homogeneous samples with different permittivity/conductivity values. In this case, satisfactory agreement between the microscope and the van der Pauw data was obtained with a strip probe on all tested samples in the entire range of R sh . With a coaxial probe, such accordance was observed only in high-ohmic samples with R sh > 1 kΩ/sq. The use of GaN film structures as a calibration system helped to increase the accuracy of the coaxial-probe-aided measurement of R sh to a level of ∼10%.

Quantitative characterization of semiconductor structures with a scanning microwave microscope

NASA Astrophysics Data System (ADS)

Korolyov, S. A.; Reznik, A. N.

2018-02-01

In this work, our earlier method for measuring resistance Rsh of semiconductor films with a near-field scanning microwave microscope [A. N. Reznik and S. A. Korolyov, J. Appl. Phys. 119, 094504 (2016)] is studied in a 0.1 kΩ/sq < Rsh < 15 kΩ/sq range. The method is based on a microscope model in the form of a monopole or dipole antenna interacting with an arbitrary layered structure. The model fitting parameters are determined from the data yielded by calibration measurements on a system of etalon samples. The performance of the method was analyzed experimentally, using strip-probe and coaxial-probe microscopes in the frequency range of 1-3 GHz. For test structures, we used doped GaN films on the Al2O3 substrate and also transistor structures based on the AlGaN/GaN heterojunction and AlGaAs/GaAs/InGaAs/GaAs/AlGaAs quantum well with a conducting channel. The obtained microwave microscope data were compared with the results of measurements by the van der Pauw method. At the first stage of the experiment, the calibration etalons were bulk homogeneous samples with different permittivity/conductivity values. In this case, satisfactory agreement between the microscope and the van der Pauw data was obtained with a strip probe on all tested samples in the entire range of Rsh. With a coaxial probe, such accordance was observed only in high-ohmic samples with Rsh > 1 kΩ/sq. The use of GaN film structures as a calibration system helped to increase the accuracy of the coaxial-probe-aided measurement of Rsh to a level of ˜10%.

Insights into the O-Acetylation Reaction of Hydroxylated Heterocyclic Amines by Human Arylamine N-Acetyltransferases: A Computational Study

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

Lau, E Y; Felton, J S; Lightstone, F C

2006-06-06

A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclicmore » amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.« less

Probing ligand binding modes of Mycobacterium tuberculosis MurC ligase by molecular modeling, dynamics simulation and docking.

PubMed

Anuradha, C M; Mulakayala, Chaitanya; Babajan, Banaganapalli; Naveen, M; Rajasekhar, Chikati; Kumar, Chitta Suresh

2010-01-01

Multi drug resistance capacity for Mycobacterium tuberculosis (MDR-Mtb) demands the profound need for developing new anti-tuberculosis drugs. The present work is on Mtb-MurC ligase, which is an enzyme involved in biosynthesis of peptidoglycan, a component of Mtb cell wall. In this paper the 3-D structure of Mtb-MurC has been constructed using the templates 1GQQ and 1P31. Structural refinement and energy minimization of the predicted Mtb-MurC ligase model has been carried out by molecular dynamics. The streochemical check failures in the energy minimized model have been evaluated through Procheck, Whatif ProSA, and Verify 3D. Further torsion angles for the side chains of amino acid residues of the developed model were determined using Predictor. Docking analysis of Mtb-MurC model with ligands and natural substrates enabled us to identify specific residues viz. Gly125, Lys126, Arg331, and Arg332, within the Mtb-MurC binding pocket to play an important role in ligand and substrate binding affinity and selectivity. The availability of Mtb-MurC ligase built model, together with insights gained from docking analysis will promote the rational design of potent and selective Mtb-MurC ligase inhibitors as antituberculosis therapeutics.

Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

PubMed

Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

2015-08-10

The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

Monolithically Integrated, Mechanically Resilient Carbon-Based Probes for Scanning Probe Microscopy

NASA Technical Reports Server (NTRS)

Kaul, Anupama B.; Megerian, Krikor G.; Jennings, Andrew T.; Greer, Julia R.

2010-01-01

Scanning probe microscopy (SPM) is an important tool for performing measurements at the nanoscale in imaging bacteria or proteins in biology, as well as in the electronics industry. An essential element of SPM is a sharp, stable tip that possesses a small radius of curvature to enhance spatial resolution. Existing techniques for forming such tips are not ideal. High-aspect-ratio, monolithically integrated, as-grown carbon nanofibers (CNFs) have been formed that show promise for SPM applications by overcoming the limitations present in wet chemical and separate substrate etching processes.

Real-Time Tracking the Synthesis and Degradation of Albumin in Complex Biological Systems with a near-Infrared Fluorescent Probe.

PubMed

Jin, Qiang; Feng, Lei; Zhang, Shui-Jun; Wang, Dan-Dan; Wang, Fang-Jun; Zhang, Yi; Cui, Jing-Nan; Guo, Wen-Zhi; Ge, Guang-Bo; Yang, Ling

2017-09-19

In this study, a novel fluorescent detection system for biological sensing of human albumin (HA) was developed on the basis of the pseudoesterase activity and substrate preference of HA. The designed near-infrared (NIR) fluorescent probe (DDAP) could be effectively hydrolyzed by HA, accompanied by significant changes in both color and fluorescence spectrum. The sensing mechanism was fully investigated by fluorescence spectroscopy, NMR, and mass spectra. DDAP exhibited excellent selectivity and sensitivity toward HA over a variety of human plasma proteins, hydrolases, and abundant biomolecules found in human body. The probe has been successfully applied to measure native HA in diluted plasma samples and the secreted HA in the hepatocyte culture supernatant. DDAP has also been used for fluorescence imaging of HA reabsorption in living renal cells, and the results show that the probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. Furthermore, DDAP has been successfully used for real-time tracking the uptaking and degradation of albumin in ex vivo mouse kidney models for the first time. All these results clearly demonstrated that DDAP-based assay held great promise for real-time sensing and tracking HA in complex biological systems, which would be very useful for basic researches and clinical diagnosis of HA-associated diseases.

Local Anodic Oxidation of Thin GeO Films and Formation of Nanostructures Based on Them

NASA Astrophysics Data System (ADS)

Astankova, K. N.; Kozhukhov, A. S.; Azarov, I. A.; Gorokhov, E. B.; Sheglov, D. V.; Latyshev, A. V.

2018-04-01

The process of local anodic oxidation of thin GeO films has been studied using an atomic force microscope. The electron-probe microanalysis showed that oxidized areas of a GeO film were germanium dioxide. The effect of the voltage pulse duration applied to the probe-substrate system and the atmospheric humidity on the height of the oxide structures has been studied. The kinetics of the local anodic oxidation (LAO) in a semi-contact mode obeys the Cabrera-Mott model for large times. The initial growth rate of the oxide ( R 0) significantly increases and the time of starting the oxidation ( t 0) decreases as the atmospheric humidity increases by 20%, which is related to an increase in the concentration of oxygen-containing ions at the surface of the oxidized GeO film. It was shown that nanostructures in thin GeO layers can be formed by the LAO method.

Raman and SERS microspectroscopy on living cells: a promising tool toward cellular drug response and medical diagnosis

NASA Astrophysics Data System (ADS)

Beljebbar, Abdelilah; Sockalingum, Ganesh D.; Morjani, Hamid; Manfait, Michel

1999-04-01

Raman spectroscopy has been sued to differentiate between sensitive and MDR-resistant cells using Raman spectral imaging with a 632.8 nm excitation wavelength. The comparison between two spectral images allowed to quantify the differences between sensitive and resistant cell lines in term of proteins, lipids when MDR phenotype is expressed. SER spectroscopy has become a powerful and non-invasive probe for investigating the molecular and cellular interaction of drugs with their targets. The comparison between these models allow to elucidate the biological effect of the drugs. The development of new types of SERS- active substrates has extended the applicability of this technique to medical diagnosis. Two kinds of SERS active substrates, characterized as 'bio-compatible' systems, can be used for investigation on single living cells: colloid suspensions and microelectrodes and island films. This methodology is used for the study of cell membrane components in interaction with the SERS substrates with the aim to understand the resistance mechanism. The constitution of a data bank will allow the follow-up of cancer and future monitoring of therapeutic intervention.

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

PubMed Central

Harris, M E; Pace, N R

1995-01-01

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs. PMID:7585250

Facile one-step synthesis of Ag@Fe3O4 core-shell nanospheres for reproducible SERS substrates

NASA Astrophysics Data System (ADS)

Sun, Lijuan; He, Jiang; An, Songsong; Zhang, Junwei; Ren, Dong

2013-08-01

A facile approach has been developed to synthesize Ag@Fe3O4 core-shell nanospheres, in which the Ag nanoparticle core was well wrapped by a permeable Fe3O4 shell. An in situ reduction of AgNO3 and Fe(NO3)3 was the basis of this one-step method with ethylene glycol as the reducing agent. The as-obtained Ag@Fe3O4 nanospheres were a highly efficient surface-enhanced Raman scattering (SERS) substrate; high reproducibility, stability, and reusability were obtained by employing 4-aminothiophenol (4-ATP) and rhodamine 6G (R6G) as the Raman probe molecules. It was revealed that the SERS signals of 4-ATP and R6G on the Ag@Fe3O4 nanospheres were much stronger than those on the pure Ag nanoparticles, demonstrating that the magnetic enrichment procedures can improve SERS detection sensitivity efficiently. A highly efficient and recyclable SERS substrate was produced by the new model system that has potential applications in chemical and biomolecular assays.

Microstructural Analysis and Transport Properties of Thermally Sprayed Multiple-Layer Ceramic Coatings

NASA Astrophysics Data System (ADS)

Wang, Hsin; Muralidharan, Govindarajan; Leonard, Donovan N.; Haynes, J. Allen; Porter, Wallace D.; England, Roger D.; Hays, Michael; Dwivedi, Gopal; Sampath, Sanjay

2018-02-01

Multilayer, graded ceramic/metal coatings were prepared by an air plasma spray method on Ti-6Al-4V, 4140 steel and graphite substrates. The coatings were designed to provide thermal barriers for diesel engine pistons to operate at higher temperatures with improved thermal efficiency and cleaner emissions. A systematic, progressive variation in the mixture of yttria-stabilized zirconia and bondcoat alloys (NiCoCrAlYHfSi) was designed to provide better thermal expansion match with the substrate and to improve thermal shock resistance and cycle life. Heat transfer through the layers was evaluated by a flash diffusivity technique based on a model of one-dimensional heat flow. The aging effect of the as-sprayed coatings was captured during diffusivity measurements, which included one heating and cooling cycle. The hysteresis of thermal diffusivity due to aging was not observed after 100-h annealing at 800 °C. The measurements of coatings on substrate and freestanding coatings allowed the influence of interface resistance to be evaluated. The microstructure of the multilayer coating was examined using scanning electron microscope and electron probe microanalysis.

Design, Validation, and Testing of a Hot-Film Anemometer for Hypersonic Flow

NASA Astrophysics Data System (ADS)

Sheplak, Mark

The application of constant-temperature hot-film anemometry to hypersonic flow has been reviewed and extended in this thesis. The objective of this investigation was to develop a measurement tool capable of yielding continuous, high-bandwidth, quantitative, normal mass-flux and total -temperature measurements in moderate-enthalpy environments. This research has produced a probe design that represents a significant advancement over existing designs, offering the following improvements: (1) a five-fold increase in bandwidth; (2) true stagnation-line sensor placement; (3) a two order-of-magnitude decrease in sensor volume; and (4) over a 70% increase in maximum film temperature. These improvements were achieved through substrate design, sensor placement, the use of high-temperature materials, and state -of-the-art microphotolithographic fabrication techniques. The experimental study to characterize the probe was performed in four different hypersonic wind tunnels at NASA-Langley Research Center. The initial test consisted of traversing the hot film through a Mach 6, flat-plate, turbulent boundary layer in air. The detailed static-calibration measurements that followed were performed in two different hypersonic flows: a Mach 11 helium flow and Mach 6 air flow. The final test of this thesis consisted of traversing the probe through the Mach 6 wake of a 70^ circ blunt body. The goal of this test was to determine the state (i.e., laminar or turbulent) of the wake. These studies indicate that substrate conduction effects result in instrumentation characteristics that prevent the hot-film anemometer from being used as a quantitative tool. The extension of this technique to providing quantitative information is dependent upon the development of lower thermal-conductivity substrate materials. However, the probe durability, absence of strain gauging, and high bandwidth represent significant improvements over the hot-wire technique for making qualitative measurements. Potential uses for this probe are: frequency identification for resonant flows, transition studies, turbulence detection for quiet-tunnel development and reattaching turbulent shear flows, and qualitative turbulence studies of shock-wave/turbulent boundary layer interactions.

Material optimization of multi-layered enhanced nanostructures

NASA Astrophysics Data System (ADS)

Strobbia, Pietro

The employment of surface enhanced Raman scattering (SERS)-based sensing in real-world scenarios will offer numerous advantages over current optical sensors. Examples of these advantages are the intrinsic and simultaneous detection of multiple analytes, among many others. To achieve such a goal, SERS substrates with throughput and reproducibility comparable to commonly used fluorescence sensors have to be developed. To this end, our lab has discovered a multi-layer geometry, based on alternating films of a metal and a dielectric, that amplifies the SERS signal (multi-layer enhancement). The advantage of these multi-layered structures is to amplify the SERS signal exploiting layer-to-layer interactions in the volume of the structures, rather than on its surface. This strategy permits an amplification of the signal without modifying the surface characteristics of a substrate, and therefore conserving its reproducibility. Multi-layered structures can therefore be used to amplify the sensitivity and throughput of potentially any previously developed SERS sensor. In this thesis, these multi-layered structures were optimized and applied to different SERS substrates. The role of the dielectric spacer layer in the multi-layer enhancement was elucidated by fabricating spacers with different characteristics and studying their effect on the overall enhancement. Thickness, surface coverage and physical properties of the spacer were studied. Additionally, the multi-layered structures were applied to commercial SERS substrates and to isolated SERS probes. Studies on the dependence of the multi-layer enhancement on the thickness of the spacer demonstrated that the enhancement increases as a function of surface coverage at sub-monolayer thicknesses, due to the increasing multi-layer nature of the substrates. For fully coalescent spacers the enhancement decreases as a function of thickness, due to the loss of interaction between proximal metallic films. The influence of the physical properties of the spacer on the multi-layer enhancement were also studied. The trends in Schottky barrier height, interfacial potential and dielectric constant were isolated by using different materials as spacers (i.e., TiO2, HfO2, Ag 2O and Al2O3). The results show that the bulk dielectric constant of the material can be used to predict the relative magnitude of the multi-layer enhancement, with low dielectric constant materials performing more efficiently as spacers. Optimal spacer layers were found to be ultrathin coalescent films (ideally a monolayer) of low dielectric constant materials. Finally, multi-layered structures were observed to be employable to amplify SERS in drastically different substrate geometries. The multi-layered structures were applied to disposable commercial SERS substrates (i.e., Klarite). This project involved the regeneration of the used substrates, by stripping and redepositing the gold coating layer, and their amplification, by using the multi-layer geometry. The latter was observed to amplify the sensitivity of the substrates. Additionally, the multi-layered structures were applied to probes dispersed in solution. Such probes were observed to yield stronger SERS signal when optically trapped and to reduce the background signal. The application of the multi-layered structures on trapped probes, not only further amplified the SERS signal, but also increased the maximum number of applicable layers for the structures.

Evaluation of first-pass cytochrome P4503A (CYP3A) and P-glycoprotein activities using alfentanil and fexofenadine in combination.

PubMed

Kharasch, Evan D; Walker, Alysa; Hoffer, Christine; Sheffels, Pamela

2005-01-01

Cytochrome P4503A (CYP3A) and P-glycoprotein (P-gp) are major determinants of oral bioavailability. Development of in vivo probe(s), for both CYP3A and P-gp, which could be administered in combination, is a current goal. Nevertheless, there is considerable overlap in CYP3A and P-gp substrate selectivities; there are few discrete probes. Alfentanil is a selective CYP3A probe but not a P-gp substrate. Fexofenadine is a P-gp probe but not a CYP3A substrate. This investigation tested the hypothesis that alfentanil and fexofenadine could be administered in combination to probe first-pass CYP3A and P-gp activities in humans. Two 3-way crossover studies were conducted in healthy volunteers. In the first protocol, subjects received oral alfentanil alone, fexofenadine alone, or fexofenadine 1 hour after alfentanil. In the second protocol, subjects abstained from citrus and apple products for 5 days and received fexofenadine alone, fexofenadine 1 hour after alfentanil, or alfentanil 4 hours after fexofenadine. An assay using solid-phase extraction and electrospray liquid chromatography/mass spectrometry was developed for the simultaneous quantification of plasma alfentanil and fexofenadine. In both protocols, alfentanil plasma concentrations and area under the concentration versus time curve (AUC) were unaffected by fexofenadine or meal composition. Fexofenadine given 1 hour after alfentanil and followed 1 hour later by a meal containing orange or apple juice had a somewhat lower AUC compared with fexofenadine alone (geometric mean ratio with and without the interacting drug = 0.73, 90% confidence interval [CI] = 0.59-1.04). Fexofenadine given 1 hour after alfentanil and followed 2 hours later by a meal not containing citrus or apple products had an AUC that was unchanged compared with fexofenadine alone (ratio = 0.91, 90% CI = 0.70-1.35). These results show that alfentanil disposition was not affected by fexofenadine. A dosing regimen was identified in which fexofenadine disposition was not affected by alfentanil. The timing and content of meals after fexofenadine had a significant effect on fexofenadine disposition. Alfentanil and fexofenadine in combination appear to be a useful probe for evaluating both first-pass CYP3A and P-gp activities in humans.

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

PubMed

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

NASA Astrophysics Data System (ADS)

Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

2002-03-01

We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

In vivo detection of c-Met expression in a rat C6 glioma model.

PubMed

Towner, R A; Smith, N; Doblas, S; Tesiram, Y; Garteiser, P; Saunders, D; Cranford, R; Silasi-Mansat, R; Herlea, O; Ivanciu, L; Wu, D; Lupu, F

2008-01-01

The tyrosine kinase receptor, c-Met, and its substrate, the hepatocyte growth factor (HGF), are implicated in the malignant progression of glioblastomas. In vivo detection of c-Met expression may be helpful in the diagnosis of malignant tumours. The C6 rat glioma model is a widely used intracranial brain tumour model used to study gliomas experimentally. We used a magnetic resonance imaging (MRI) molecular targeting agent to specifically tag the cell surface receptor, c-Met, with an anti-c-Met antibody (Ab) linked to biotinylated Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-albumin in rat gliomas to detect overexpression of this antigen in vivo. The anti-c-Met probe (anti-c-Met-Gd-DTPA-albumin) was administered intravenously, and as determined by an increase in MRI signal intensity and a corresponding decrease in regional T(1) relaxation values, this probe was found to detect increased expression of c-Met protein levels in C6 gliomas. In addition, specificity for the binding of the anti-c-Met contrast agent was determined by using fluorescence microscopic imaging of the biotinylated portion of the targeting agent within neoplastic and 'normal'brain tissues following in vivo administration of the anti-c-Met probe. Controls with no Ab or with a normal rat IgG attached to the contrast agent component indicated no non-specific binding to glioma tissue. This is the first successful visualization of in vivo overexpression of c-Met in gliomas.

In vivo detection of c-Met expression in a rat C6 glioma model

PubMed Central

Towner, RA; Smith, N; Doblas, S; Tesiram, Y; Garteiser, P; Saunders, D; Cranford, R; Silasi-Mansat, R; Herlea, O; Ivanciu, L; Wu, D; Lupu, F

2008-01-01

Abstract The tyrosine kinase receptor, c-Met, and its substrate, the hepatocyte growth factor (HGF), are implicated in the malignant progression of glioblastomas. In vivo detection of c-Met expression may be helpful in the diagnosis of malignant tumours. The C6 rat glioma model is a widely used intracranial brain tumour model used to study gliomas experimentally. We used a magnetic resonance imaging (MRI) molecular targeting agent to specifically tag the cell surface receptor, c-Met, with an anti-c-Met antibody (Ab) linked to biotinylated Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-albumin in rat gliomas to detect overexpression of this antigen in vivo. The anti-c-Met probe (anti-c-Met-Gd-DTPA-albumin) was administered intravenously, and as determined by an increase in MRI signal intensity and a corresponding decrease in regional T1 relaxation values, this probe was found to detect increased expression of c-Met protein levels in C6 gliomas. In addition, specificity for the binding of the anti-c-Met contrast agent was determined by using fluorescence microscopic imaging of the biotinylated portion of the targeting agent within neoplastic and ‘normal’brain tissues following in vivo administration of the anti-c-Met probe. Controls with no Ab or with a normal rat IgG attached to the contrast agent component indicated no non-specific binding to glioma tissue. This is the first successful visualization of in vivo overexpression of c-Met in gliomas. PMID:18194445

SPM Investigation of Thiolated Gold Nanoparticle Patterns Deposited on Different Self-Assembled Substrates

NASA Astrophysics Data System (ADS)

Sbrana, F.; Parodi, M. T.; Ricci, D.; Di Zitti, E.

We present the results of a Scanning Probe Microscopy (SPM) investigation of ordered nanosized metallo-organic structures. Our aim is to investigate the organization and stability of thiolated gold nanoparticles in a compact pattern when deposited onto gold substrates functionalized with self-assembled monolayers made from two molecules that differ essentially in their terminating group: 1,4-benzenedimethanethiol and 4-methylbenzylthiol.

Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.

PubMed

Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric; Ploegh, Hidde L

2009-04-17

Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.

An atomic-force-microscopy study of the structure of surface layers of intact fibroblasts

NASA Astrophysics Data System (ADS)

Khalisov, M. M.; Ankudinov, A. V.; Penniyaynen, V. A.; Nyapshaev, I. A.; Kipenko, A. V.; Timoshchuk, K. I.; Podzorova, S. A.; Krylov, B. V.

2017-02-01

Intact embryonic fibroblasts on a collagen-treated substrate have been studied by atomic-force microscopy (AFM) using probes of two types: (i) standard probes with tip curvature radii of 2-10 nm and (ii) special probes with a calibrated 325-nm SiO2 ball radius at the tip apex. It is established that, irrespective of probe type, the average maximum fibroblast height is on a level of 1.7 μm and the average stiffness of the probe-cell contact amounts to 16.5 mN/m. The obtained AFM data reveal a peculiarity of the fibroblast structure, whereby its external layers move as a rigid shell relative to the interior and can be pressed inside to a depth dependent on the load only.

Self-assembled monolayers of alkyl-thiols on InAs: A Kelvin probe force microscopy study

NASA Astrophysics Data System (ADS)

Szwajca, A.; Wei, J.; Schukfeh, M. I.; Tornow, M.

2015-03-01

We report on the preparation and characterization of self-assembled monolayers from aliphatic thiols with different chain length and termination on InAs (100) planar surfaces. This included as first step the development and investigation of a thorough chemical InAs surface preparation step using a dedicated bromine/NH4OH-based etching process. Ellipsometry, contact angle measurements and atomic force microscopy (AFM) indicated the formation of smooth, surface conforming monolayers. The molecular tilt angles were obtained as 30 ± 10° with respect to the surface normal. Kelvin probe force microscopy (KPFM) measurements in hand with Parameterized Model number 5 (PM5) calculations of the involved molecular dipoles allowed for an estimation of the molecular packing densities on the surface. We obtained values of up to n = 1014 cm- 2 for the SAMs under study. These are close to what is predicted from a simple geometrical model that would calculate a maximum density of about n = 2.7 × 1014 cm- 2. We take this as additional conformation of the substrate smoothness and quality of our InAs-SAM hybrid layer systems.

Activity-Dependent Enzymatic Assay for the Detection of Toluene-Oxidizing Bacteria Capable of Trichloroethylene Degradation

NASA Astrophysics Data System (ADS)

Kauffman, M. E.; Kauffman, M. E.; Keener, W. K.; Watwood, M. E.; Lehman, R. M.

2001-12-01

Toluene-oxidizing bacteria produce enzymes that cometabolically degrade trichloroethylene (TCE). These inducible enzymes are produced only in the presence of certain aromatic substrates such as toluene or phenol. Recent laboratory studies have utilized analog chemical substrates to identify production of bacterial enzymes capable of degrading trichloroethylene. These analog substrates produce chromogenic and/or fluorescent products when biotransformed by the enzymes of interest. In this study, 3-hydroxyphenylacetylene (3-HPA) was identified as an activity-dependent enzymatic probe for the detection of three of the four known toluene oxygenase enzymes capable of TCE degradation. Laboratory studies were conducted using pure cultures of Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas putida F1. Cell cultures grown on lactate (non-enzyme inducing) or lactate and toluene (inducing) were trapped trapped on black polycarbonate filters, exposed to 3-HPA, and examined for fluorescence using an epifluorescent microscope. Additionally, B. cepacia G4 cells were grown under the same conditions, but in the presence of mineral and basalt specimens to allow for bacterial attachment. The specimens were then exposed to 3-HPA and examined under an epifluorescent microscope. Our results demonstrate that cells induced for the production of oxygenase enzymes, both unattached and attached, are able to transform 3-HPA to a fluorescent product, although cells attached to geologic materials, such as basalt, take substantially longer to transform the probe. Cells grown under non-inducing conditions do not transform the probe, regardless of their attachment status. Additionally, well water samples taken from a TCE-contaminated aquifer were successfully assayed using the 3-HPA enzymatic probe. The development of this enzyme activity-dependent enzymatic assay provides a fast and reliable method to assess the potential for TCE and aromatic contaminant bioremediation.

Quantitative fluorescent in-situ hybridization: a hypothesized competition mode between two dominant bacteria groups in hydrogen-producing anaerobic sludge processes.

PubMed

Huang, C-L; Chen, C-C; Lin, C-Y; Liu, W-T

2009-01-01

Two hydrogen-producing continuous flow stirred tank reactors (CSTRs) fed respectively with glucose and sucrose were investigated by polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) and fluorescent in-situ hybridization (FISH). The substrate was fed in a continuous mode decreased from hydraulic retention time (HRT) 10 hours to 6, 5, 4, 3, and 2 hours. Quantitative fluorescent in-situ hybridization (FISH) observations further demonstrated that two morphotypes of bacteria dominated both microbial communities. One was long rod bacteria which can be targeted either by Chis150 probe designed to hybridize the gram positive low G + C bacteria or the specific oligonucleotide probe Lg10-6. The probe Lg10-6, affiliated with Clostridium pasteurianum, was designed and then checked with other reference organisms. The other type, unknown group, which cannot be detected by Chis150 was curved rod bacteria. Notably, the population ratios of the two predominant groups reflected the different operational performance of the two reactors, such as hydrogen producing rates, substrate turnover rates and metabolites compositions. Therefore, a competition mode of the two dominant bacteria groups was hypothesized. In the study, 16S rRNA-based gene library of hydrogen-producing microbial communities was established. The efficiency of hydrogen yields was correlated with substrates (glucose or sucrose), HRT, metabolites compositions (acetate, propionate, butyrate and ethanol), thermal pre-treatment (seed biomass was heated at 100 degrees C for 45 minutes), and microbial communities in the bioreactor, not sludge sources (municipal sewage sludge, alcohol-processing sludge, or bean-processing sludge). The designed specific oligonucleotide probe Lg10-6 also provides us a useful and fast molecular tool to screen hydrogen-producing microbial communities in the future research.

Substrate preparation effects on defect density in molecular beam epitaxial growth of CdTe on CdTe (100) and (211)B

DOE PAGES

Burton, George L.; Diercks, David R.; Perkins, Craig L.; ...

2017-07-01

Recent studies have demonstrated that growth of CdTe on CdTe (100) and (211)B substrates via molecular beam epitaxy (MBE) results in planar defect densities 2 and 3 orders of magnitude higher than growth on InSb (100) substrates, respectively. To understand this shortcoming, MBE growth on CdTe substrates with a variety of substrate preparation methods is studied by scanning electron microscopy, secondary ion mass spectrometry, x-ray photoelectron spectroscopy, cross sectional transmission electron microscopy, and atom probe tomography (APT). Prior to growth, carbon is shown to remain on substrate surfaces even after atomic hydrogen cleaning. APT revealed that following the growth ofmore » films, trace amounts of carbon remained at the substrate/film interface. This residual carbon may lead to structural degradation, which was determined as the main cause of higher defect density.« less

Gold@silver bimetal nanoparticles/pyramidal silicon 3D substrate with high reproducibility for high-performance SERS

NASA Astrophysics Data System (ADS)

Zhang, Chao; Jiang, Shou Zhen; Yang, Cheng; Li, Chong Hui; Huo, Yan Yan; Liu, Xiao Yun; Liu, Ai Hua; Wei, Qin; Gao, Sai Sai; Gao, Xing Guo; Man, Bao Yuan

2016-05-01

A novel and efficient surface enhanced Raman scattering (SERS) substrate has been presented based on Gold@silver/pyramidal silicon 3D substrate (Au@Ag/3D-Si). By combining the SERS activity of Ag, the chemical stability of Au and the large field enhancement of 3D-Si, the Au@Ag/3D-Si substrate possesses perfect sensitivity, homogeneity, reproducibility and chemical stability. Using R6G as probe molecule, the SERS results imply that the Au@Ag/3D-Si substrate is superior to the 3D-Si, Ag/3D-Si and Au/3D-Si substrate. We also confirmed these excellent behaviors in theory via a commercial COMSOL software. The corresponding experimental and theoretical results indicate that our proposed Au@Ag/3D-Si substrate is expected to develop new opportunities for label-free SERS detections in biological sensors, biomedical diagnostics and food safety.

Performance-Enhancing Methods for Au Film over Nanosphere Surface-Enhanced Raman Scattering Substrate and Melamine Detection Application

PubMed Central

Wang, Jun Feng; Wu, Xue Zhong; Xiao, Rui; Dong, Pei Tao; Wang, Chao Guang

2014-01-01

A new high-performance surface-enhanced Raman scattering (SERS) substrate with extremely high SERS activity was produced. This SERS substrate combines the advantages of Au film over nanosphere (AuFON) substrate and Ag nanoparticles (AgNPs). A three order enhancement of SERS was observed when Rhodamine 6G (R6G) was used as a probe molecule to compare the SERS effects of the new substrate and commonly used AuFON substrate. These new SERS substrates can detect R6G down to 1 nM. The new substrate was also utilized to detect melamine, and the limit of detection (LOD) is 1 ppb. A linear relationship was also observed between the SERS intensity at Raman peak 682 cm−1 and the logarithm of melamine concentrations ranging from 10 ppm to 1 ppb. This ultrasensitive SERS substrate is a promising tool for detecting trace chemical molecules because of its simple and effective fabrication procedure, high sensitivity and high reproducibility of the SERS effect. PMID:24886913

Performance-enhancing methods for Au film over nanosphere surface-enhanced Raman scattering substrate and melamine detection application.

PubMed

Wang, Jun Feng; Wu, Xue Zhong; Xiao, Rui; Dong, Pei Tao; Wang, Chao Guang

2014-01-01

A new high-performance surface-enhanced Raman scattering (SERS) substrate with extremely high SERS activity was produced. This SERS substrate combines the advantages of Au film over nanosphere (AuFON) substrate and Ag nanoparticles (AgNPs). A three order enhancement of SERS was observed when Rhodamine 6G (R6G) was used as a probe molecule to compare the SERS effects of the new substrate and commonly used AuFON substrate. These new SERS substrates can detect R6G down to 1 nM. The new substrate was also utilized to detect melamine, and the limit of detection (LOD) is 1 ppb. A linear relationship was also observed between the SERS intensity at Raman peak 682 cm(-1) and the logarithm of melamine concentrations ranging from 10 ppm to 1 ppb. This ultrasensitive SERS substrate is a promising tool for detecting trace chemical molecules because of its simple and effective fabrication procedure, high sensitivity and high reproducibility of the SERS effect.

13C ENDOR Spectroscopy of Lipoxygenase-Substrate Complexes Reveals the Structural Basis for C-H Activation by Tunneling.

PubMed

Horitani, Masaki; Offenbacher, Adam R; Carr, Cody A Marcus; Yu, Tao; Hoeke, Veronika; Cutsail, George E; Hammes-Schiffer, Sharon; Klinman, Judith P; Hoffman, Brian M

2017-02-08

In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn 2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13 C ENDOR then reveals the locations of 13 C10 and reactive 13 C11 of linoleic acid relative to the metal; 1 H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

Probing cardiac metabolism by hyperpolarized 13C MR using an exclusively endogenous substrate mixture and photo-induced non-persistent radicals

PubMed Central

Bastiaansen, Jessica A. M.; Yoshihara, Hikari A. I.; Capozzi, Andrea; Schwitter, Juerg; Gruetter, Rolf; Merritt, Matthew E.; Comment, Arnaud

2018-01-01

Purpose To probe the cardiac metabolism of carbohydrates and short chain fatty acids simultaneously in vivo following the injection of a hyperpolarized 13C-labeled substrate mixture prepared using photo-induced non-persistent radicals. Methods Droplets of mixed [1-13C]pyruvic and [1-13C]butyric acids were frozen into glassy beads in liquid nitrogen. Ethanol addition was investigated as a means to increase the polarization level. The beads were irradiated with ultraviolet (UV) light and the radical concentration was measured by ESR spectroscopy. Following dynamic nuclear polarization (DNP) in a 7T polarizer, the beads were dissolved, and the radical-free hyperpolarized solution was rapidly transferred into an injection pump located inside a 9.4T scanner. The hyperpolarized solution was injected in healthy rats to measure cardiac metabolism in vivo. Results UV-irradiation created non-persistent radicals in a mixture containing 13C-labeled pyruvic and butyric acids and enabled the hyperpolarization of both substrates by DNP. Ethanol addition increased the radical concentration from 16 to 26 mM. Liquid-state 13C polarization was 3% inside the pump at the time of injection, and increased to 5% by addition of ethanol to the substrate mixture prior to UV irradiation. In the rat heart, the in vivo13C signals from lactate, alanine, bicarbonate and acetylcarnitine were detected following the metabolism of the injected substrate mixture. Conclusion Co-polarization of two 13C-labeled substrates and the detection of their myocardial metabolism in vivo was achieved without using persistent radicals. The absence of radicals in the solution containing the hyperpolarized 13C-substrates may simplify the translation to clinical use because no filtration is required prior to injection. PMID:29411415

NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

PubMed Central

Dunn, Michael F.

2013-01-01

Conspectus NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry – offers unprecedented insight into three-dimensional, chemically-detailed structure. From its initial role in refining diffraction data of organic and inorganic solids, NMR crystallography is now being developed for application to active sites in biomolecules, where it reveals chemically-rich detail concerning the interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. For example, typical X-ray crystal structures (1.5 to 2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate, but do not directly identify the protonation state of either. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them, only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but rely on chemical details that must be specified. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which models of the active site can be developed using computational chemistry; these models can be distinguished by comparison of their calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at highest resolution. In this Account, we detail our first steps in the development of NMR crystallography for application to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation. PMID:23537227

Tomography of a Probe Potential Using Atomic Sensors on Graphene.

PubMed

Wyrick, Jonathan; Natterer, Fabian D; Zhao, Yue; Watanabe, Kenji; Taniguchi, Takashi; Cullen, William G; Zhitenev, Nikolai B; Stroscio, Joseph A

2016-12-27

Our ability to access and explore the quantum world has been greatly advanced by the power of atomic manipulation and local spectroscopy with scanning tunneling and atomic force microscopes, where the key technique is the use of atomically sharp probe tips to interact with an underlying substrate. Here we employ atomic manipulation to modify and quantify the interaction between the probe and the system under study that can strongly affect any measurement in low charge density systems, such as graphene. We transfer Co atoms from a graphene surface onto a probe tip to change and control the probe's physical structure, enabling us to modify the induced potential at a graphene surface. We utilize single Co atoms on a graphene field-effect device as atomic scale sensors to quantitatively map the modified potential exerted by the scanning probe over the whole relevant spatial and energy range.

van der Waals interaction between a moving nano-cylinder and a liquid thin film.

PubMed

Ledesma-Alonso, René; Raphaël, Elie; Salez, Thomas; Tordjeman, Philippe; Legendre, Dominique

2017-05-24

We study the static and dynamic interaction between a horizontal cylindrical nano-probe and a thin liquid film. The effects of the physical and geometrical parameters, with a special focus on the film thickness, the probe speed, and the distance between the probe and the free surface are analyzed. Deformation profiles have been computed numerically from a Reynolds lubrication equation, coupled to a modified Young-Laplace equation, which takes into account the probe/liquid and the liquid/substrate non-retarded van der Waals interactions. We have found that the film thickness and the probe speed have a significant effect on the threshold separation distance below which the jump-to-contact instability is triggered. These results encourage the use of horizontal cylindrical nano-probes to scan thin liquid films, in order to determine either the physical or geometrical properties of the latter, through the measurement of interaction forces.

Effect of substrate roughness on the apparent surface free energy of sputter deposited superhydrophobic polytetrafluoroethylene coatings: A comparison of experimental data with different theoretical models

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

Selvakumar, N.; Barshilia, Harish C.; Rajam, K. S.

2010-07-15

We have studied the effect of substrate roughness on the wettability and the apparent surface free energy (SFE) of sputter deposited polytetrafluoroethylene (PTFE) coatings deposited on untreated glass (average roughness, R{sub a}=2.0 nm), plasma etched glass (R{sub a}=7.4 nm), and sandblasted glass (R{sub a}=4500 nm) substrates. The wettability of the PTFE coatings deposited on substrates with varying roughnesses was evaluated by measuring the apparent contact angle (CA) using a series of probe liquids from nonpolar aprotic to polar protic. The wettability measurements indicate that an apparent water CA of 152 deg. with a sliding angle of 8 deg. was achievedmore » for PTFE coatings deposited on a substrate with R{sub a}=4500 nm. The superhydrophobicity observed in these coatings is attributed to the presence of dual scale roughness, densely packed microstructure and the presence of CF{sub 3} groups. Unlike the bulk PTFE which is mainly dispersive, the sputter deposited PTFE coatings are expected to have some degree of polar component due to the plasma treatment. In order to calculate the dispersive SFE of PTFE coatings, we have used the Girifalco-Good-Fowkes (GGF) method and validated it with the Zisman model. Furthermore, the Owens-Wendt model has been used to calculate the dispersive and the polar components of the apparent SFE of the PTFE coatings. These results are further corroborated using the Fowkes method. Finally, an ''equation of state'' theory proposed by Neumann has been used to calculate the apparent SFE values of the PTFE coatings. The results indicate that the apparent SFE values of the PTFE coatings obtained from the Owens-Wendt and the Fowkes methods are comparable to those obtained from the Neumann's method. The analyses further demonstrate that the GGF and the Zisman methods underestimate the apparent SFE values of the sputter deposited PTFE coatings.« less

Control of the interaction strength of photonic molecules by nanometer precise 3D fabrication.

PubMed

Rawlings, Colin D; Zientek, Michal; Spieser, Martin; Urbonas, Darius; Stöferle, Thilo; Mahrt, Rainer F; Lisunova, Yuliya; Brugger, Juergen; Duerig, Urs; Knoll, Armin W

2017-11-28

Applications for high resolution 3D profiles, so-called grayscale lithography, exist in diverse fields such as optics, nanofluidics and tribology. All of them require the fabrication of patterns with reliable absolute patterning depth independent of the substrate location and target materials. Here we present a complete patterning and pattern-transfer solution based on thermal scanning probe lithography (t-SPL) and dry etching. We demonstrate the fabrication of 3D profiles in silicon and silicon oxide with nanometer scale accuracy of absolute depth levels. An accuracy of less than 1nm standard deviation in t-SPL is achieved by providing an accurate physical model of the writing process to a model-based implementation of a closed-loop lithography process. For transfering the pattern to a target substrate we optimized the etch process and demonstrate linear amplification of grayscale patterns into silicon and silicon oxide with amplification ratios of ∼6 and ∼1, respectively. The performance of the entire process is demonstrated by manufacturing photonic molecules of desired interaction strength. Excellent agreement of fabricated and simulated structures has been achieved.

Nanoscale insight into the p-n junction of alkali-incorporated Cu(In,Ga)Se 2 solar cells

DOE PAGES

Stokes, Adam; Al-Jassim, Mowafak; Norman, Andrew; ...

2017-04-05

The effects of alkali diffusion and post-deposition treatment in three-stage processed Cu(In,Ga)Se 2 solar cells are examined by using atom probe tomography and electrical property measurements. Cells, for which the substrate was treated at 650 °C to induce alkali diffusion from the substrate prior to absorber deposition, exhibited high open-circuit voltage (758 mV) and efficiency (18.2%) and also exhibited a 50 to 100-nm-thick ordered vacancy compound layer at the metallurgical junction. Surprisingly, these high-temperature samples exhibited higher concentrations of K at the junction (1.8 at.%) than post-deposition treatment samples (0.4 at.%). A model that uses Ga/(Ga + In) and Cu/(Gamore » + In) profiles to predict bandgaps (+/-17.9 meV) of 22 Cu(In,Ga)Se2 solar cells reported in literature was discussed and ultimately used to predict band properties at the nanoscale by using atom probe tomography data. The high-temperature samples exhibited a greater drop in the valence band maximum (200 meV) due to a lower Cu/(Ga + In) ratio than the post-deposition treatment samples. There was an anticorrelation of K concentrations and Cu/(Ga + In) ratios for all samples, regardless of processing conditions. In conclusion, changes in elemental profiles at the active junctions correlate well with the electrical behaviour of these devices.« less

Probing catalytic rate enhancement during intramembrane proteolysis.

PubMed

Arutyunova, Elena; Smithers, Cameron C; Corradi, Valentina; Espiritu, Adam C; Young, Howard S; Tieleman, D Peter; Lemieux, M Joanne

2016-09-01

Rhomboids are ubiquitous intramembrane serine proteases involved in various signaling pathways. While the high-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed an active site comprised of a serine-histidine dyad and an extensive oxyanion hole, the molecular details of rhomboid catalysis were unclear because substrates are unknown for most of the family members. Here we used the only known physiological pair of AarA rhomboid with its psTatA substrate to decipher the contribution of catalytically important residues to the reaction rate enhancement. An MD-refined homology model of AarA was used to identify residues important for catalysis. We demonstrated that the AarA active site geometry is strict and intolerant to alterations. We probed the roles of H83 and N87 oxyanion hole residues and determined that substitution of H83 either abolished AarA activity or reduced the transition state stabilization energy (ΔΔG‡) by 3.1 kcal/mol; substitution of N87 decreased ΔΔG‡ by 1.6-3.9 kcal/mol. Substitution M154, a residue conserved in most rhomboids that stabilizes the catalytic general base, to tyrosine, provided insight into the mechanism of nucleophile generation for the catalytic dyad. This study provides a quantitative evaluation of the role of several residues important for hydrolytic efficiency and oxyanion stabilization during intramembrane proteolysis.

In vitro and physiologically‐based pharmacokinetic based assessment of drug–drug interaction potential of canagliflozin

PubMed Central

Dallas, Shannon; Sensenhauser, Carlo; Lim, Heng Keang; Scheers, Ellen; Verboven, Peter; Cuyckens, Filip; Leclercq, Laurent; Evans, David C.; Kelley, Michael F.; Johnson, Mark D.; Snoeys, Jan

2016-01-01

Aims Canagliflozin is a recently approved drug for use in the treatment of type 2 diabetes. The potential for canagliflozin to cause clinical drug–drug interactions (DDIs) was assessed. Methods DDI potential of canagliflozin was investigated using in vitro test systems containing drug metabolizing enzymes or transporters. Basic predictive approaches were applied to determine potential interactions in vivo. A physiologically‐based pharmacokinetic (PBPK) model was developed and clinical DDI simulations were performed to determine the likelihood of cytochrome P450 (CYP) inhibition by canagliflozin. Results Canagliflozin was primarily metabolized by uridine 5′‐diphospho‐glucuronosyltransferase 1A9 and 2B4 enzymes. Canagliflozin was a substrate of efflux transporters (P‐glycoprotein, breast cancer resistance protein and multidrug resistance‐associated protein‐2) but was not a substrate of uptake transporters (organic anion transporter polypeptide isoforms OATP1B1, OATP1B3, organic anion transporters OAT1 and OAT3, and organic cationic transporters OCT1, and OCT2). In inhibition assays, canagliflozin was shown to be a weak in vitro inhibitor (IC50) of CYP3A4 (27 μmol l –1, standard error [SE] 4.9), CYP2C9 (80 μmol l –1, SE 8.1), CYP2B6 (16 μmol l–1, SE 2.1), CYP2C8 (75 μmol l –1, SE 6.4), P‐glycoprotein (19.3 μmol l –1, SE 7.2), and multidrug resistance‐associated protein‐2 (21.5 μmol l –1, SE 3.1). Basic models recommended in DDI guidelines (US Food & Drug Administration and European Medicines Agency) predicted moderate to low likelihood of interaction for these CYPs and efflux transporters. PBPK DDI simulations of canagliflozin with CYP probe substrates (simvastatin, S‐warfarin, bupropion, repaglinide) did not show relevant interaction in humans since mean areas under the concentration‐time curve and maximum plasma concentration ratios for probe substrates with and without canagliflozin and its 95% CIs were within 0.80–1.25. Conclusions In vitro DDI followed by a predictive or PBPK approach was applied to determine DDI potential of canagliflozin. Overall, canagliflozin is neither a perpetrator nor a victim of clinically important interactions. PMID:27862160

Mechanically adjustable single-molecule transistors and stencil mask nanofabrication of high-resolution scanning probes

NASA Astrophysics Data System (ADS)

Champagne, Alexandre

This dissertation presents the development of two original experimental techniques to probe nanoscale objects. The first one studies electronic transport in single organic molecule transistors in which the source-drain electrode spacing is mechanically adjustable. The second involves the fabrication of high-resolution scanning probe microscopy sensors using a stencil mask lithography technique. We describe the fabrication of transistors in which a single organic molecule can be incorporated. The source and drain leads of these transistors are freely suspended above a flexible substrate, and their spacing can be adjusted by bending the substrate. We detail the technology developed to carry out measurements on these samples. We study electronic transport in single C60 molecules at low temperature. We observe Coulomb blockaded transport and can resolve the discrete energy spectrum of the molecule. We are able to mechanically tune the spacing between the electrodes (over a range of 5 A) to modulate the lead-molecule coupling, and can electrostatically tune the energy levels on the molecule by up to 160 meV using a gate electrode. Initial progress in studying different transport regimes in other molecules is also discussed. We present a lithographic process that allows the deposition of metal nanostructures with a resolution down to 10 nm directly onto atomic force microscope (AFM) tips. We show that multiple layers of lithography can be deposited and aligned. We fabricate high-resolution magnetic force microscopy (MFM) probes using this method and discuss progress to fabricate other scanning probe microscopy (SPM) sensors.

Design and measurement technique of surface-enhanced Raman scattering for detection of bisphenol A

NASA Astrophysics Data System (ADS)

Abu Bakar, Norhayati; Mat Salleh, Muhamad; Umar, Akrajas Ali; Shapter, Joseph George

2017-06-01

Surface-enhanced Raman scattering (SERS) is a highly sensitive measurement technique that provides Raman peaks at different Raman shift for different molecule structures. The SERS sensor is potentially used to detect food contamination and monitor environmental pollutants. A self-developed SERS system for specific analysis with low development cost is a challenging issue. This study attempts to develop a simple SERS sensor system for detection of bisphenol A (BPA) molecule using SERS substrate of silver nanoplate film. A SERS sensor system was developed, consisting of a light source to excite analyte molecules, Inphotonic Raman probe, sensor chamber and spectrophotometer as an analyser system. A duplex fibre optic is used to transmit light from the source to the probe and from the probe to the spectrophotometer. For SERS measurement, BPA detection was done by comparing the Raman signal spectra of the BPA on the quartz substrate and BPA on the silver nanoplate film. This SERS sensor successfully sensed BPA with SERS enhancement factor (EF) 5.55  ×  103 and a detection limit of BPA concentration at 1 mM.

SERS Detection of Dopamine Using Label-Free Acridine Red as Molecular Probe in Reduced Graphene Oxide/Silver Nanotriangle Sol Substrate

NASA Astrophysics Data System (ADS)

Luo, Yanghe; Ma, Lu; Zhang, Xinghui; Liang, Aihui; Jiang, Zhiliang

2015-05-01

The reduced graphene oxide/silver nanotriangle (rGO/AgNT) composite sol was prepared by the reduction of silver ions with sodium borohydride in the presence of H2O2 and sodium citrate. In the nanosol substrate, the molecular probe of acridine red (AR) exhibited a weak surface-enhanced Raman scattering (SERS) peak at 1506 cm-1 due to its interaction with the rGO of rGO/AgNT. Upon addition of dopamine (DA), the competitive adsorption between DA and AR with the rGO took place, and the AR molecules were adsorbed on the AgNT aggregates with a strong SERS peak at 1506 cm-1 that caused the SERS peak increase. The increased SERS intensity is linear to the DA concentration in the range of 2.5-500 μmol/L. This new analytical system was investigated by SERS, fluorescence, absorption, transmission electron microscope (TEM), and scanning electron microscope (SEM) techniques, and a SERS quantitative analysis method for DA was established, using AR as a label-free molecular probe.

Quantum dot-based microfluidic biosensor for cancer detection

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

Ghrera, Aditya Sharma; School of Engineering and Technology, ITM University, Gurgaon-122017; Pandey, Chandra Mouli

2015-05-11

We report results of the studies relating to fabrication of an impedimetric microfluidic–based nucleic acid sensor for quantification of DNA sequences specific to chronic myelogenous leukemia (CML). The sensor chip is prepared by patterning an indium–tin–oxide (ITO) coated glass substrate via wet chemical etching method followed by sealing with polydimethylsiloxane (PDMS) microchannel for fluid control. The fabricated microfluidic chip comprising of a patterned ITO substrate is modified by depositing cadmium selenide quantum dots (QCdSe) via Langmuir–Blodgett technique. Further, the QCdSe surface has been functionalized with specific DNA probe for CML detection. The probe DNA functionalized QCdSe integrated miniaturized system hasmore » been used to monitor target complementary DNA concentration by measuring the interfacial charge transfer resistance via hybridization. The presence of complementary DNA in buffer solution significantly results in decreased electro-conductivity of the interface due to presence of a charge barrier for transport of the redox probe ions. The microfluidic DNA biosensor exhibits improved linearity in the concentration range of 10{sup −15} M to 10{sup −11} M.« less

Synergistic effects of semiconductor substrate and noble metal nano-particles on SERS effect both theoretical and experimental aspects

NASA Astrophysics Data System (ADS)

Yang, Chen; Liang, Pei; Tang, Lisha; Zhou, Yongfeng; Cao, Yanting; Wu, Yanxiong; Zhang, De; Dong, Qianmin; Huang, Jie; He, Peng

2018-04-01

As a means of chemical identification and analysis, Surface enhanced Raman spectroscopy (SERS), with the advantages of high sensitivity and selectivity, non-destructive, high repeatability and in situ detection etc., has important significance in the field of composition detection, environmental science, biological medicine etc. Physical model of coupling effect between different semiconductor substrates and noble metal particles were investigated by using 3D-FDTD method. Mechanism and the effects of excitation wavelength, particle spacing and semiconductor substrate types on the SERS effect were discussed. The results showed that the optimal excitation wavelengths of three noble metals of Ag, Au, Cu, were located at 510, 600 and 630 nm, respectively; SERS effect of Ag, Au, Cu increases with the decreasing of the inter distance of particles, while the distance of the NPs reaches the critical value of 3 nm, the strength of SERS effect will be greatly enhanced. For the four different types of substrate of Ge, Si, SiO2 (glass) and Al2O3, the SERS effect of Ag on SiO2 > Ge > Al2O3 > Si. For Au and Cu nanoparticles, the SERS effect of them on oxide substrate is stronger than that on non-oxide substrate. In order to verify FDTD simulations, taking silver nanoparticles as an example, and silver nanoparticles prepared by chemical method were spinning coating on the four different substrates with R6G as probe molecules. The results show that the experimental results are consistent with FDTD theoretical simulations, and the SERS enhancement effect of Ag-SiO2 substrate is best. The results of this study have important theoretical significance to explain the variations of SERS enhancement on different noble metals, which is also an important guide for the preparation of SERS substrates, especially for the microfluidics. The better Raman effect can be realized by choosing proper substrate type, particle spacing and excitation wavelength, result in expanding the depth and width of SERS application.

Intrinsic Charge Trapping Observed as Surface Potential Variations in diF-TES-ADT Films.

PubMed

Hoffman, Benjamin C; McAfee, Terry; Conrad, Brad R; Loth, Marsha A; Anthony, John E; Ade, Harald W; Dougherty, Daniel B

2016-08-24

Spatial variations in surface potential are measured with Kelvin probe force microscopy for thin films of 2,8-difluoro-5,11-bis(triethylsilylethynyl)anthradithiophenes (diF-TES-ADT) grown on SiO2 and silane-treated SiO2 substrates by organic molecular beam deposition. The variations are observed both between and within grains of the polycrystalline organic film and are quantitatively different than electrostatic variations on the substrate surfaces. The skewness of surface potential distributions is larger on SiO2 than on HMDS-treated substrates. This observation is attributed to the impact of substrate functionalization on minimizing intrinsic crystallographic defects in the organic film that can trap charge.

Surface-potential undulation of Alq3 thin films prepared on ITO, Au, and n-Si.

PubMed

Ozasa, Kazunari; Ito, Hiromi; Maeda, Mizuo; Hara, Masahiko

2012-01-01

The surface potential (SP) morphology on thin films of tris(8-hydroxyquinolinato) aluminum (Alq3) was investigated with Kelvin probe force microscopy. Thin Alq3 films of 100 nm were prepared on ITO/glass substrates, Au/mica substrates, and n-Si substrates. Cloud-like morphologies of the SP undulation with 200-400 nm in lateral size were observed for all three types of the substrates. New larger peaks were observed in the cloud-like morphologies when the surfaces were exposed shortly to a light, while the SP average was reduced monotonically. The nonuniform distribution of charged traps and mobility was deduced from the SP undulation morphology and its photoexposure dependences.

HA/Bioglass composite films deposited by pulsed laser with different substrate temperature

NASA Astrophysics Data System (ADS)

Wang, D. G.; Chen, C. Z.; Jin, Q. P.; Li, H. C.; Pan, Y. K.

2014-03-01

In this experiment, the HA/Bioglass composite films on Ti-6Al-4V were deposited by a pulsed laser at Ar atmosphere, and the influence of substrate temperature on the morphology, phase constitutions, bonding configurations and adhesive strength of the films was studied. The obtained films were characterized by an electron probe microanalyzer (EPMA), scanning electron microscope (SEM), X-ray diffractometer (XRD), Fourier transform infrared spectrometer (FTIR), scratch apparatus, and so on. The results show that the amount of the droplets, the crystallinity, and the critical load of the deposited films all increase with the increase of the substrate temperature; however, the substrate temperature has little influence on the functional groups of the films.

Synthesis and characterization of macromolecular rhodamine tethers and their interactions with P-glycoprotein.

PubMed

Crawford, Lindsey; Putnam, David

2014-08-20

Rhodamine dyes are well-known P-glycoprotein (P-gp) substrates that have played an important role in the detection of inhibitors and other substrates of P-gp, as well as in the understanding of P-gp function. Macromolecular conjugates of rhodamines could prove useful as tethers for further probing of P-gp structure and function. Two macromolecular derivatives of rhodamine, methoxypolyethylene glycol-rhodamine6G and methoxypolyethylene glycol-rhodamine123, were synthesized through the 2'-position of rhodamine6G and rhodamine123, thoroughly characterized, and then evaluated by inhibition with verapamil for their ability to interact with P-gp and to act as efflux substrates. To put the results into context, the P-gp interactions of the new conjugates were compared to the commercially available methoxypolyethylene glycol-rhodamineB. FACS analysis confirmed that macromolecular tethers of rhodamine6G, rhodamine123, and rhodamineB were accumulated in P-gp expressing cells 5.2 ± 0.3%, 26.2 ± 4%, and 64.2 ± 6%, respectively, compared to a sensitive cell line that does not overexpress P-gp. Along with confocal imaging, the efflux analysis confirmed that the macromolecular rhodamine tethers remain P-gp substrates. These results open potential avenues for new ways to probe the function of P-gp both in vitro and in vivo.

Structural identifiability analyses of candidate models for in vitro Pitavastatin hepatic uptake.

PubMed

Grandjean, Thomas R B; Chappell, Michael J; Yates, James W T; Evans, Neil D

2014-05-01

In this paper a review of the application of four different techniques (a version of the similarity transformation approach for autonomous uncontrolled systems, a non-differential input/output observable normal form approach, the characteristic set differential algebra and a recent algebraic input/output relationship approach) to determine the structural identifiability of certain in vitro nonlinear pharmacokinetic models is provided. The Organic Anion Transporting Polypeptide (OATP) substrate, Pitavastatin, is used as a probe on freshly isolated animal and human hepatocytes. Candidate pharmacokinetic non-linear compartmental models have been derived to characterise the uptake process of Pitavastatin. As a prerequisite to parameter estimation, structural identifiability analyses are performed to establish that all unknown parameters can be identified from the experimental observations available. Copyright © 2013. Published by Elsevier Ireland Ltd.

Stripline feed for a microstrip array of patch elements with teardrop shaped probes

NASA Technical Reports Server (NTRS)

Huang, John (Inventor)

1990-01-01

A circularly polarized microstrip array antenna utilizing a honeycomb substrate made of dielectric material to support on one side the microstrip patch elements in an array, and on the other side a stripline circuit for feeding the patch elements in subarray groups of four with angular orientation and phase for producing circularly polarized radiation, preferably at a 0.degree., 90.degree., 180.degree. and 270.degree. relationship. The probe used for coupling each feed point in the stripline circuit to a microstrip patch element is teardrop shaped in order to introduce capacitance between the coupling probe and the metal sheet of the stripline circuit that serves as an antenna ground plane. The capacitance thus introduced tunes out inductance of the probe. The shape of the teardrop probe is not critical. The probe capacitance required is controlled by the maximum diameter for the teardrop shaped probe, which can be empirically determined for the operating frequency. An aluminum baffle around each subarray blocks out surface waves between subarrays.

Carbon Nanofibers Synthesized on Selective Substrates for Nonvolatile Memory and 3D Electronics

NASA Technical Reports Server (NTRS)

Kaul, Anupama B.; Khan, Abdur R.

2011-01-01

A plasma-enhanced chemical vapor deposition (PECVD) growth technique has been developed where the choice of starting substrate was found to influence the electrical characteristics of the resulting carbon nanofiber (CNF) tubes. It has been determined that, if the tubes are grown on refractory metallic nitride substrates, then the resulting tubes formed with dc PECVD are also electrically conducting. Individual CNFs were formed by first patterning Ni catalyst islands using ebeam evaporation and liftoff. The CNFs were then synthesized using dc PECVD with C2H2:NH3 = [1:4] at 5 Torr and 700 C, and approximately equal to 200-W plasma power. Tubes were grown directly on degenerately doped silicon <100> substrates with resistivity rho approximately equal to 1-5 meterohm-centimeter, as well as NbTiN. The approximately equal to 200-nanometer thick refractory NbTiN deposited using magnetron sputtering had rho approximately equal to 113 microohm-centimeter and was also chemically compatible with CNF synthesis. The sample was then mounted on a 45 beveled Al holder, and placed inside a SEM (scanning electron microscope). A nanomanipulator probe stage was placed inside the SEM equipped with an electrical feed-through, where tungsten probes were used to make two-terminal electrical measurements with an HP 4156C parameter analyzer. The positive terminal nanoprobe was mechanically manipulated to physically contact an individual CNF grown directly on NbTiN as shown by the SEM image in the inset of figure (a), while the negative terminal was grounded to the substrate. This revealed the tube was electrically conductive, although measureable currents could not be detected until approximately equal to 6 V, after which point current increased sharply until compliance (approximately equal to 50 nA) was reached at approximately equal to 9.5 V. A native oxide on the tungsten probe tips may contribute to a tunnel barrier, which could be the reason for the suppressed transport at low biases. Currents up to approximately 100 nA could be cycled, which are likely to propagate via the tube surface, or sidewalls, rather than the body, which is shown by the I-V in figure (a). Electrical conduction via the sidewalls is a necessity for dc NEMS (nanoelectromechanical system) applications, more so than for the field emission applications of such tubes. During the tests, high conductivity was expected, because both probes were shorted to the substrate, as shown by curve 1 in the I-V characteristic in figure (b). When a tube grown on NbTiN was probed, the response was similar to the approximately equal to 100 nA and is represented by curve 2 in figure (b), which could be cycled and propagated via the tube surface or the sidewalls. However, no measureable currents for the tube grown directly on Si were observed as shown by curve 3 in figure (b), even after testing over a range of samples. This could arise from a dielectric coating on the sidewalls for tubes on Si. As a result of the directional nature of ion bombardment during dc PECVD, Si from the substrate is likely re-sputtered and possibly coats the sidewalls.

Photo-Attachment of Biomolecules for Miniaturization on Wicking Si-Nanowire Platform

PubMed Central

Cheng, He; Zheng, Han; Wu, Jia Xin; Xu, Wei; Zhou, Lihan; Leong, Kam Chew; Fitzgerald, Eugene; Rajagopalan, Raj; Too, Heng Phon; Choi, Wee Kiong

2015-01-01

We demonstrated the surface functionalization of a highly three-dimensional, superhydrophilic wicking substrate using light to immobilize functional biomolecules for sensor or microarray applications. We showed here that the three-dimensional substrate was compatible with photo-attachment and the performance of functionalization was greatly improved due to both increased surface capacity and reduced substrate reflectivity. In addition, photo-attachment circumvents the problems induced by wicking effect that was typically encountered on superhydrophilic three-dimensional substrates, thus reducing the difficulty of producing miniaturized sites on such substrate. We have investigated various aspects of photo-attachment process on the nanowire substrate, including the role of different buffers, the effect of wavelength as well as how changing probe structure may affect the functionalization process. We demonstrated that substrate fabrication and functionalization can be achieved with processes compatible with microelectronics processes, hence reducing the cost of array fabrication. Such functionalization method coupled with the high capacity surface makes the substrate an ideal candidate for sensor or microarray for sensitive detection of target analytes. PMID:25689680

Electrospun Nanofibers Made of Silver Nanoparticles, Cellulose Nanocrystals, and Polyacrylonitrile as Substrates for Surface-Enhanced Raman Scattering

PubMed Central

Ren, Suxia; Dong, Lili; Zhang, Xiuqiang; Lei, Tingzhou; Ehrenhauser, Franz; Song, Kunlin; Li, Meichun; Sun, Xiuxuan; Wu, Qinglin

2017-01-01

Nanofibers with excellent activities in surface-enhanced Raman scattering (SERS) were developed through electrospinning precursor suspensions consisting of polyacrylonitrile (PAN), silver nanoparticles (AgNPs), silicon nanoparticles (SiNPs), and cellulose nanocrystals (CNCs). Rheology of the precursor suspensions, and morphology, thermal properties, chemical structures, and SERS sensitivity of the nanofibers were investigated. The electrospun nanofibers showed uniform diameters with a smooth surface. Hydrofluoric (HF) acid treatment of the PAN/CNC/Ag composite nanofibers (defined as p-PAN/CNC/Ag) led to rougher fiber surfaces with certain pores and increased mean fiber diameters. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) results confirmed the existence of AgNPs that were formed during heat and HF acid treatment processes. In addition, thermal stability of the electrospun nanofibers increased due to the incorporation of CNCs and AgNPs. The p-PAN/CNC/Ag nanofibers were used as a SERS substrate to detect p-aminothiophenol (p-ATP) probe molecule. The results show that this substrate exhibited high sensitivity for the p-ATP probe detection. PMID:28772428

Nanoscale Electrostructural Characterization of Compositionally Graded Al(x)Ga(1-x)N Heterostructures on GaN/Sapphire (0001) Substrate.

PubMed

Kuchuk, Andrian V; Lytvyn, Petro M; Li, Chen; Stanchu, Hryhorii V; Mazur, Yuriy I; Ware, Morgan E; Benamara, Mourad; Ratajczak, Renata; Dorogan, Vitaliy; Kladko, Vasyl P; Belyaev, Alexander E; Salamo, Gregory G

2015-10-21

We report on AlxGa1-xN heterostructures resulting from the coherent growth of a positive then a negative gradient of the Al concentration on a [0001]-oriented GaN substrate. These polarization-doped p-n junction structures were characterized at the nanoscale by a combination of averaging as well as depth-resolved experimental techniques including: cross-sectional transmission electron microscopy, high-resolution X-ray diffraction, Rutherford backscattering spectrometry, and scanning probe microscopy. We observed that a small miscut in the substrate orientation along with the accumulated strain during growth led to a change in the mosaic structure of the AlxGa1-xN film, resulting in the formation of macrosteps on the surface. Moreover, we found a lateral modulation of charge carriers on the surface which were directly correlated with these steps. Finally, using nanoscale probes of the charge density in cross sections of the samples, we have directly measured, semiquantitatively, both n- and p-type polarization doping resulting from the gradient concentration of the AlxGa1-xN layers.

Extended substrate specificity and first potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease.

PubMed

Rut, Wioletta; Zhang, Linlin; Kasperkiewicz, Paulina; Poreba, Marcin; Hilgenfeld, Rolf; Drąg, Marcin

2017-03-01

Zika virus is spread by Aedes mosquitoes and is linked to acute neurological disorders, especially to microcephaly in newborn children and Guillan-Barré Syndrome. The NS2B-NS3 protease of this virus is responsible for polyprotein processing and therefore considered an attractive drug target. In this study, we have used the Hybrid Combinatorial Substrate Library (HyCoSuL) approach to determine the substrate specificity of ZIKV NS2B-NS3 protease in the P4-P1 positions using natural and a large spectrum of unnatural amino acids. Obtained data demonstrate a high level of specificity of the S3-S1 subsites, especially for basic amino acids. However, the S4 site exhibits a very broad preference toward natural and unnatural amino acids with selected D-amino acids being favored over L enantiomers. This information was used for the design of a very potent phosphonate inhibitor/activity-based probe of ZIKV NS2B-NS3 protease. Copyright © 2016 Elsevier B.V. All rights reserved.

Probes of the catalytic site of cysteine dioxygenase.

PubMed

Chai, Sergio C; Bruyere, John R; Maroney, Michael J

2006-06-09

The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.

Fluorescence X-ray absorption spectroscopy using a Ge pixel array detector: application to high-temperature superconducting thin-film single crystals.

PubMed

Oyanagi, H; Tsukada, A; Naito, M; Saini, N L; Lampert, M O; Gutknecht, D; Dressler, P; Ogawa, S; Kasai, K; Mohamed, S; Fukano, A

2006-07-01

A Ge pixel array detector with 100 segments was applied to fluorescence X-ray absorption spectroscopy, probing the local structure of high-temperature superconducting thin-film single crystals (100 nm in thickness). Independent monitoring of pixel signals allows real-time inspection of artifacts owing to substrate diffractions. By optimizing the grazing-incidence angle theta and adjusting the azimuthal angle phi, smooth extended X-ray absorption fine structure (EXAFS) oscillations were obtained for strained (La,Sr)2CuO4 thin-film single crystals grown by molecular beam epitaxy. The results of EXAFS data analysis show that the local structure (CuO6 octahedron) in (La,Sr)2CuO4 thin films grown on LaSrAlO4 and SrTiO3 substrates is uniaxially distorted changing the tetragonality by approximately 5 x 10(-3) in accordance with the crystallographic lattice mismatch. It is demonstrated that the local structure of thin-film single crystals can be probed with high accuracy at low temperature without interference from substrates.

Nanoindentation data analysis of loading curve performed on DLC thin films: Effect of residual stress on the elasto-plastic properties

NASA Astrophysics Data System (ADS)

Ouchabane, M.; Dublanche-Tixier, Ch.; Dergham, D.

2017-11-01

The present work is a contribution to the understanding of the mechanical behavior of DLC thin films through nanoindentation tests. DLC films of different thicknesses deposited by the PECVD process on a silicon substrate contain high residual compressive stresses when they are very thin and the stresses become relatively low and more relaxed as the film thickens. These different levels of residual stress influence the values of hardness (H) and Young's modulus (E) obtained when probing the film-substrate system by nanoindentation. It is observed that the DLC layers exhibit different mechanical behaviors even when they are deposited under the same conditions. It is proposed that the compressive stress induces structural modifications resulting in modifying the elasto-plastic properties of each thin film-substrate system. Data analysis of the loading curve can provide information on the elasto-plastic properties of DLC thin films, particularly the stiffness (S) and Er2/H, as a function of residual compressive stresses. The structural changes induced by residual stresses were probed by using Raman spectroscopy and correlated to the mechanical properties.

Surface-enhanced Raman spectroscopic monitor of triglyceride hydrolysis in a skin pore phantom

NASA Astrophysics Data System (ADS)

Weldon, Millicent K.; Morris, Michael D.

1999-04-01

Bacterial hydrolysis of triglycerides is followed in a sebum probe phantom by microprobe surface-enhanced Raman scattering (SERS) spectroscopy. The phantom consists of a purpose-built syringe pump operating at physiological flow rates connected to a 300 micron i.d. capillary. We employ silicon substrate SERS microprobes to monitor the hydrolysis products. The silicon support allows some tip flexibility that makes these probes ideal for insertion into small structures. Propionibacterium acnes are immobilized on the inner surface of the capillary. These bacteria hydrolyze the triglycerides in a model sebum emulsion flowing through the capillary. The transformation is followed in vitro as changes in the SERS caused by hydrolysis of triglyceride to fatty acid. The breakdown products consists of a mixture of mono- and diglycerides and their parent long chain fatty acids. The fatty acids adsorb as their carboxylates and can be readily identified by their characteristic spectra. The technique can also confirm the presence of bacteria by detection of short chain carboxylic acids released as products of glucose fermentation during the growth cycle of these cells. Co-adsorption of propionate is observed. Spatial localization of the bacteria is obtained by ex-situ line imaging of the probe.

Substrate-dependent cell elasticity measured by optical tweezers indentation

NASA Astrophysics Data System (ADS)

Yousafzai, Muhammad S.; Ndoye, Fatou; Coceano, Giovanna; Niemela, Joseph; Bonin, Serena; Scoles, Giacinto; Cojoc, Dan

2016-01-01

In the last decade, cell elasticity has been widely investigated as a potential label free indicator for cellular alteration in different diseases, cancer included. Cell elasticity can be locally measured by pulling membrane tethers, stretching or indenting the cell using optical tweezers. In this paper, we propose a simple approach to perform cell indentation at pN forces by axially moving the cell against a trapped microbead. The elastic modulus is calculated using the Hertz-model. Besides the axial component, the setup also allows us to examine the lateral cell-bead interaction. This technique has been applied to measure the local elasticity of HBL-100 cells, an immortalized human cell line, originally derived from the milk of a woman with no evidence of breast cancer lesions. In addition, we have studied the influence of substrate stiffness on cell elasticity by performing experiments on cells cultured on two substrates, bare and collagen-coated, having different stiffness. The mean value of the cell elastic modulus measured during indentation was 26±9 Pa for the bare substrate, while for the collagen-coated substrate it diminished to 19±7 Pa. The same trend was obtained for the elastic modulus measured during the retraction of the cell: 23±10 Pa and 13±7 Pa, respectively. These results show the cells adapt their stiffness to that of the substrate and demonstrate the potential of this setup for low-force probing of modifications to cell mechanics induced by the surrounding environment (e.g. extracellular matrix or other cells).

Cyclic Nucleotide Monophosphates in Plants and Plant Signaling.

PubMed

Marondedze, Claudius; Wong, Aloysius; Thomas, Ludivine; Irving, Helen; Gehring, Chris

2017-01-01

Cyclic nucleotide monophosphates (cNMPs) and the enzymes that can generate them are of increasing interest in the plant sciences. Arguably, the major recent advance came with the release of the complete Arabidopsis thaliana genome that has enabled the systematic search for adenylate (ACs) or guanylate cyclases (GCs) and did eventually lead to the discovery of a number of GCs in higher plants. Many of these proteins have complex domain architectures with AC or GC centers moonlighting within cytosolic kinase domains. Recent reports indicated the presence of not just the canonical cNMPs (i.e., cAMP and cGMP), but also the noncanonical cCMP, cUMP, cIMP, and cdTMP in plant tissues, and this raises several questions. Firstly, what are the functions of these cNMPs, and, secondly, which enzymes can convert the substrate triphosphates into the respective noncanonical cNMPs? The first question is addressed here by comparing the reactive oxygen species (ROS) response of cAMP and cGMP to that elicited by the noncanonical cCMP or cIMP. The results show that particularly cIMP can induce significant ROS production. To answer, at least in part, the second question, we have evaluated homology models of experimentally confirmed plant GCs probing the substrate specificity by molecular docking simulations to determine if they can conceivably catalytically convert substrates other than ATP or GTP. In summary, molecular modeling and substrate docking simulations can contribute to the evaluation of cyclases for noncanonical cyclic mononucleotides and thereby further our understanding of the molecular mechanism that underlie cNMP-dependent signaling in planta.

[Preparation of a kind of SERS-active substrates for spot fast analysis].

PubMed

Ji, Nan; Li, Zhi-Shi; Zhao, Bing; Zou, Bo

2013-02-01

A kind of SERS-active substrates was prepared using chemical self-assembly method, aiming at spot fast analysis using portable Raman spectrometer. PDDA was first absorbed on the inner wall of vials, and then Ag colloids were assembled on the inner wall. UV-Vis spectra and Raman spectra of two kinds of blank vials were investigated and the transparent vials were thought to be better for SERS-vials. UV-Vis spectra were used to monitor the assembly process of Ag colloids. SERS activity of our substrates was characterized using p-ATP as probing molecules.

Excess Substrate is a Spectator Ligand in a Rhodium-Catalyzed Asymmetric [2+2+2] Cycloaddition of Alkenyl Isocyanates with Tolanes

PubMed Central

Oinen, Mark Emil; Yu, Robert T.; Rovis, Tomislav

2009-01-01

Excess substrate has been identified as an unintended spectator ligand affecting enantioselectivity in the [2+2+2] cycloaddition of alkenyl isocyanates with tolanes. Replacement of excess substrate with an exogenous additive affords products with consistent and higher ee’s. The increase in enantioselectivity is the result of a change in composition of a proposed rhodium(III) intermediate on the catalytic cycle. The net result is a rational probe of a short-lived rhodium(III) intermediate, and gives insight that may have applications in many rhodium catalyzed reactions. PMID:19803471

Analysis of an infinite array of rectangular microstrip patches with idealized probe feeds

NASA Technical Reports Server (NTRS)

Pozar, D. M.; Schaubert, D. H.

1984-01-01

A solution is presented to the problem of an infinite array of microstrip patches fed by idealized current probes. The input reflection coefficient is calculated versus scan angle in an arbitrary scan plane, and the effects of substrate parameters and grid spacing are considered. It is pointed out that even when a Galerkin method is used the impedance matrix is not symmetric due to phasing through a unit cell, as required for scanning. The mechanism by which scan blindness can occur is discussed. Measurement results are presented for the reflection coefficient magnitude variation with angle for E-plane, H-plane, and D-plane scans, for various substrate parameters. Measured results from waveguide simulators are also presented, and the scan blindness phenomenon is observed and discussed in terms of forced surface waves and a modified grating lobe diagram.

High-voltage SPM oxidation of ZrN: materials for multiscale applications

NASA Astrophysics Data System (ADS)

Farkas, N.; Comer, J. R.; Zhang, G.; Evans, E. A.; Ramsier, R. D.; Dagata, J. A.

2005-02-01

Scanning probe microscope (SPM) oxidation was used to form zirconium oxide features on 200 nm thick ZrN films. The features exhibit rapid yet controlled growth kinetics, even in contact mode with 70 V dc applied between the probe tip and substrate. The features grown for times longer than 10 s are higher than 200 nm, and reach more than 1000 nm in height after 300 s. Long-time oxidation experiments and selective etching of the oxides and nitrides lead us to propose that as the oxidation reaches the silicon substrate, delamination occurs with the simultaneous formation of a thin layer of new material at the ZrN/Si interface. High-voltage oxide growth on ZrN is fast and sustainable, and the robust oxide features are promising candidates for multiscale (nanometre-to-micrometre) applications.

Recycle polymer characterization and adhesion modeling

NASA Astrophysics Data System (ADS)

Holbery, James David

Contaminants from paper product producers that adversely affect fiber yield have been collected from mills located in three North American geographic regions. Samples have been fractionated using a modified solvent extraction process and subsequently quantitatively characterized and it was found that agglomerates were comprised of the following: approximately 30% extractable polymeric material, 25--35% fiber, 12--15% inorganic material, 15% non-extractable high molecular-weight polyethylene or cross-linked polymers, and 2--4% starch residue. Three representative polymers, paraffin, low-molecular weight polyethylene, and a commercial hot-melt adhesive were selected for further analysis to model the attractive and repulsive behavior using Scanning Probe Microscopy in an aqueous cell. Scanning force probes were characterized using an original technique utilizing a nano-indentation apparatus that is non-destructive and is accurate to within 10% for probes with force constants as low as 1 N/m. Surface force measurements were performed between a Poly (Styrene/30% Butyl Methacrylate) sphere and substrates produced from paraffin, polyethylene, and a commercial hot-melt adhesive in solutions ranging in NaF ionic concentrations from 0.001M to 1M. Reasonable theoretical agreement with experimental data has been shown between a combined model applying van der Waals force contributions using the Derjaguin approximation and electrostatic contributions as predicted by a Debye-Huckel linearization of the Poisson-Boltzmann equation utilizing Hamaker constants derived from critical surface energies determined from Zisman and Lifshitz-van der Waals energy approaches. This model has been applied to measured data and indicates the strength of adhesion for the hot-melt to be 0.14 nN while that of paraffin is 1.9 nN and polyethylene 2.8 nN. Paraffin and polyethylene are 13.5 and 20 times greater in attraction than the hot-melt adhesive. Hot-melt adhesive repulsion is predicted to be 220 pN while for paraffin it is 9.1 nN and polyethylene 12.2 nN, a factor of 41 and 55 greater for paraffin and polyethylene, respectively. Decay lengths for repulsion is fit to be 2.3 nm for hotmelt indicating, approximately one-third that of paraffin and polyethylene. Johnson-Kendall-Roberts contact mechanic theory for viscoelastic materials has been applied with reasonable accuracy, particularly in experiments performed in solutions, to model the approach snap-in magnitude and detachment forces between sphere and substrate. Two representative commercial agglomeration formulations have been analyzed to determine the impact on adhesion and detachment forces although at room temperature, no measurable effect was identified.

Label-free liquid crystal biosensor based on specific oligonucleotide probes for heavy metal ions.

PubMed

Yang, Shengyuan; Wu, Chao; Tan, Hui; Wu, Yan; Liao, Shuzhen; Wu, Zhaoyang; Shen, Guoli; Yu, Ruqin

2013-01-02

In this study, to enhance the capability of metal ions disturbing the orientation of liquid crystals (LCs), we designed a new label-free LC biosensor for the highly selective and sensitive detection of heavy metal ions. This strategy makes use of the target-induced DNA conformational change to enhance the disruption of target molecules for the orientation of LC leading to an amplified optical signal. The Hg(2+) ion, which possesses a unique property to bind specifically to two DNA thymine (T) bases, is used as a model heavy metal ion. In the presence of Hg(2+), the specific oligonucleotide probes form a conformational reorganization of the oligonucleotide probes from hairpin structure to duplex-like complexes. The duplex-like complexes are then bound on the triethoxysilylbutyraldehyde/N,N-dimethyl-N-octadecyl (3-aminopropyl) trimethoxysilyl chloride (TEA/DMOAP)-coated substrate modified with capture probes, which can greatly distort the orientational profile of LC, making the optical image of LC cell birefringent as a result. The optical signal of LC sensor has a visible change at the Hg(2+) concentration of low to 0.1 nM, showing good detection sensitivity. The cost-effective LC sensing method can translate the concentration signal of heavy metal ions in solution into the presence of DNA duplexes and is expected to be a sensitive detection platform for heavy metal ions and other small molecule monitors.

Mode pumping experiments on biomolecules

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

Austin, R.H.; Erramilli, S.; Xie, A.

1995-12-31

We will explore several aspects of protein dynamics and energy transfer that can be explored by using the intense, picosecond, tunable mid-IR output of the FEL. In order of appearance they are: (1) Saturation recovery and inter-level coupling of the low temperature amide-I band in acetanilide. This is a continuation of earlier experiments to test soliton models in crystalline hydrogen bonded solids. In this experiment we utilize the sub-picosecond time resolution and low repetition rate of the Stanford SCLA FEL to do both T{sub 1} and T{sub 2} relaxation measurements at 1650 cm{sup -1}. (2) Probing the influence of collectivemore » dynamics in sensory rhodopsin. In this experiment we use the FIR output of the Stanford FIREFLY FEL to determine the lifetime of collective modes in the photo-active protein sensory rhodopsin, and begin experiments on the influence of collective modes on retinal reaction dynamics. (3) Probing the transition states of enzymes. This experiment, in the initial stages, attempts to use the intense IR output of the FEL to probe and influence the reaction path of a transition state analog for the protein nucleoside hydrolase. The transition state of the inosine substrate is believed to have critical modes softened by the protein so that bond-breaking paths show absorption at approximately 800 cm{sup -1}. A form of action spectrum using FEL excitation will be used to probe this state.« less

Kron-Branin modelling of ultra-short pulsed signal microelectrode

NASA Astrophysics Data System (ADS)

Xu, Zhifei; Ravelo, Blaise; Liu, Yang; Zhao, Lu; Delaroche, Fabien; Vurpillot, Francois

2018-06-01

An uncommon circuit modelling of microelectrode for ultra-short signal propagation is developed. The proposed model is based on the Tensorial Analysis of Network (TAN) using the Kron-Branin (KB) formalism. The systemic graph topology equivalent to the considered structure problem is established by assuming as unknown variables the branch currents. The TAN mathematical solution is determined after the KB characteristic matrix identification. The TAN can integrate various structure physical parameters. As proof of concept, via hole ended microelectrodes implemented on Kapton substrate were designed, fabricated and tested. The 0.1-MHz-to-6-GHz S-parameter KB model, simulation and measurement are in good agreement. In addition, time-domain analyses with nanosecond duration pulse signals were carried out to predict the microelectrode signal integrity. The modelled microstrip electrode is usually integrated in the atom probe tomography. The proposed unfamiliar KB method is particularly beneficial with respect to the computation speed and adaptability to various structures.

The study on the nanomachining property and cutting model of single-crystal sapphire by atomic force microscopy.

PubMed

Huang, Jen-Ching; Weng, Yung-Jin

2014-01-01

This study focused on the nanomachining property and cutting model of single-crystal sapphire during nanomachining. The coated diamond probe is used to as a tool, and the atomic force microscopy (AFM) is as an experimental platform for nanomachining. To understand the effect of normal force on single-crystal sapphire machining, this study tested nano-line machining and nano-rectangular pattern machining at different normal force. In nano-line machining test, the experimental results showed that the normal force increased, the groove depth from nano-line machining also increased. And the trend is logarithmic type. In nano-rectangular pattern machining test, it is found when the normal force increases, the groove depth also increased, but rather the accumulation of small chips. This paper combined the blew by air blower, the cleaning by ultrasonic cleaning machine and using contact mode probe to scan the surface topology after nanomaching, and proposed the "criterion of nanomachining cutting model," in order to determine the cutting model of single-crystal sapphire in the nanomachining is ductile regime cutting model or brittle regime cutting model. After analysis, the single-crystal sapphire substrate is processed in small normal force during nano-linear machining; its cutting modes are ductile regime cutting model. In the nano-rectangular pattern machining, due to the impact of machined zones overlap, the cutting mode is converted into a brittle regime cutting model. © 2014 Wiley Periodicals, Inc.

Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy

NASA Technical Reports Server (NTRS)

Mathur, A. B.; Collinsworth, A. M.; Reichert, W. M.; Kraus, W. E.; Truskey, G. A.

2001-01-01

This study evaluated the hypothesis that, due to functional and structural differences, the apparent elastic modulus and viscous behavior of cardiac and skeletal muscle and vascular endothelium would differ. To accurately determine the elastic modulus, the contribution of probe velocity, indentation depth, and the assumed shape of the probe were examined. Hysteresis was observed at high indentation velocities arising from viscous effects. Irreversible deformation was not observed for endothelial cells and hysteresis was negligible below 1 microm/s. For skeletal muscle and cardiac muscle cells, hysteresis was negligible below 0.25 microm/s. Viscous dissipation for endothelial and cardiac muscle cells was higher than for skeletal muscle cells. The calculated elastic modulus was most sensitive to the assumed probe geometry for the first 60 nm of indentation for the three cell types. Modeling the probe as a blunt cone-spherical cap resulted in variation in elastic modulus with indentation depth that was less than that calculated by treating the probe as a conical tip. Substrate contributions were negligible since the elastic modulus reached a steady value for indentations above 60 nm and the probe never indented more than 10% of the cell thickness. Cardiac cells were the stiffest (100.3+/-10.7 kPa), the skeletal muscle cells were intermediate (24.7+/-3.5 kPa), and the endothelial cells were the softest with a range of elastic moduli (1.4+/-0.1 to 6.8+/-0.4 kPa) depending on the location of the cell surface tested. Cardiac and skeletal muscle exhibited nonlinear elastic behavior. These passive mechanical properties are generally consistent with the function of these different cell types.

Substrate flexibility regulates growth and apoptosis of normal but not transformed cells

NASA Technical Reports Server (NTRS)

Wang, H. B.; Dembo, M.; Wang, Y. L.

2000-01-01

One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.

Self-Powered Wireless Affinity-Based Biosensor Based on Integration of Paper-Based Microfluidics and Self-Assembled RFID Antennas.

PubMed

Yuan, Mingquan; Alocilja, Evangelyn C; Chakrabartty, Shantanu

2016-08-01

This paper presents a wireless, self-powered, affinity-based biosensor based on the integration of paper-based microfluidics with our previously reported method for self-assembling radio-frequency (RF) antennas. At the core of the proposed approach is a silver-enhancement technique that grows portions of a RF antenna in regions where target antigens hybridize with target specific affinity probes. The hybridization regions are defined by a network of nitrocellulose based microfluidic channels which implement a self-powered approach to sample the reagent and control its flow and mixing. The integration substrate for the biosensor has been constructed using polyethylene and the patterning of the antenna on the substrate has been achieved using a low-cost ink-jet printing technique. The substrate has been integrated with passive radio-frequency identification (RFID) tags to demonstrate that the resulting sensor-tag can be used for continuous monitoring in a food supply-chain where direct measurement of analytes is typically considered to be impractical. We validate the proof-of-concept operation of the proposed sensor-tag using IgG as a model analyte and using a 915 MHz Ultra-high-frequency (UHF) RFID tagging technology.

Microstructural Analysis and Transport Properties of Thermally Sprayed Multiple-Layer Ceramic Coatings

DOE PAGES

Wang, Hsin; Muralidharan, Govindarajan; Leonard, Donovan N.; ...

2018-01-04

In this paper, multilayer, graded ceramic/metal coatings were prepared by an air plasma spray method on Ti-6Al-4V, 4140 steel and graphite substrates. The coatings were designed to provide thermal barriers for diesel engine pistons to operate at higher temperatures with improved thermal efficiency and cleaner emissions. A systematic, progressive variation in the mixture of yttria-stabilized zirconia and bondcoat alloys (NiCoCrAlYHfSi) was designed to provide better thermal expansion match with the substrate and to improve thermal shock resistance and cycle life. Heat transfer through the layers was evaluated by a flash diffusivity technique based on a model of one-dimensional heat flow.more » The aging effect of the as-sprayed coatings was captured during diffusivity measurements, which included one heating and cooling cycle. The hysteresis of thermal diffusivity due to aging was not observed after 100-h annealing at 800 °C. The measurements of coatings on substrate and freestanding coatings allowed the influence of interface resistance to be evaluated. Finally, the microstructure of the multilayer coating was examined using scanning electron microscope and electron probe microanalysis.« less

Microstructural Analysis and Transport Properties of Thermally Sprayed Multiple-Layer Ceramic Coatings

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

Wang, Hsin; Muralidharan, Govindarajan; Leonard, Donovan N.

In this paper, multilayer, graded ceramic/metal coatings were prepared by an air plasma spray method on Ti-6Al-4V, 4140 steel and graphite substrates. The coatings were designed to provide thermal barriers for diesel engine pistons to operate at higher temperatures with improved thermal efficiency and cleaner emissions. A systematic, progressive variation in the mixture of yttria-stabilized zirconia and bondcoat alloys (NiCoCrAlYHfSi) was designed to provide better thermal expansion match with the substrate and to improve thermal shock resistance and cycle life. Heat transfer through the layers was evaluated by a flash diffusivity technique based on a model of one-dimensional heat flow.more » The aging effect of the as-sprayed coatings was captured during diffusivity measurements, which included one heating and cooling cycle. The hysteresis of thermal diffusivity due to aging was not observed after 100-h annealing at 800 °C. The measurements of coatings on substrate and freestanding coatings allowed the influence of interface resistance to be evaluated. Finally, the microstructure of the multilayer coating was examined using scanning electron microscope and electron probe microanalysis.« less

A Ag synchronously deposited and doped TiO2 hybrid as an ultrasensitive SERS substrate: a multifunctional platform for SERS detection and photocatalytic degradation.

PubMed

Yang, Libin; Sang, Qinqin; Du, Juan; Yang, Ming; Li, Xiuling; Shen, Yu; Han, Xiaoxia; Jiang, Xin; Zhao, Bing

2018-06-06

Ag simultaneously deposited and doped TiO2 (Ag-TiO2) hybrid nanoparticles (NPs) were prepared via a sol-hydrothermal method, as both a sensitive surface-enhanced Raman scattering (SERS) substrate and a superior photocatalyst for the first time. Ag-TiO2 hybrid NPs exhibit excellent SERS performance for several probe molecules and the enhancement factor is calculated to be 1.86 × 105. The detection limit of the 4-mercaptobenzoic acid (4-MBA) probe on the Ag-TiO2 substrate is 1 × 10-9 mol L-1, which is four orders of magnitude lower than that on pure TiO2 as a consequence of the synergistic effects of TiO2 and Ag. This is the highest SERS sensitivity among the reported semiconductor substrates and even comparable to noble metal substrates, and a SERS enhancement mechanism from the synergistic contribution of the semiconductor and noble metal was proposed. And importantly, the Ag-TiO2 hybrid shows excellent photocatalytic degradation activity for the detected species under UV light irradiation at lower concentration conditions, even for the hard to degrade 4-MBA molecule. This makes the Ag-TiO2 hybrid promising as a dual-function platform for both highly sensitive SERS detection and photocatalytic degradation of a pollutant system. Moreover, it also proves that the Ag-TiO2 hybrid can serve as a promising recyclable SERS-active substrate by virtue of its photocatalytic self-cleaning properties for some specific applications, for instance comparative studies of different species on the same SERS platform, in addition to the economic benefit.

Strategies in the design of small-molecule fluorescent probes for peptidases.

PubMed

Chen, Laizhong; Li, Jing; Du, Lupei; Li, Minyong

2014-11-01

Peptidases, which can cleave specific peptide bonds in innumerable categories of substrates, usually present pivotal positions in protein activation, cell signaling and regulation as well as in the origination of amino acids for protein generation or application in other metabolic pathways. They are also involved in many pathological conditions, such as cancer, atherosclerosis, arthritis, and neurodegenerative disorders. This review article aims to conduct a wide-ranging survey on the development of small-molecule fluorescent probes for peptidases, as well as to realize the state of the art in the tailor-made probes for diverse types of peptidases. © 2014 Wiley Periodicals, Inc.

XPS study of graphene oxide reduction induced by (100) and (111)-oriented Si substrates

NASA Astrophysics Data System (ADS)

Priante, F.; Salim, M.; Ottaviano, L.; Perrozzi, F.

2018-02-01

The reduction of graphene oxide (GO) has been extensively studied in literature in order to let GO partially recover the properties of graphene. Most of the techniques proposed to reduce GO are based on high temperature annealing or chemical reduction. A new procedure, based on the direct reduction of GO by etched Si substrate, was recently proposed in literature. In the present work, we accurately investigated the Si-GO interaction with x-ray photoelectron spectroscopy. In order to avoid external substrate oxidation factors we used EtOH as the GO solvent instead of water, and thermal annealing was carried out in UHV. We investigated the effect of Si(100), Si(111) and Au substrates on GO, to probe the role played by both the substrate composition and substrate orientation during the reduction process. A similar degree of GO reduction was observed for all samples but only after thermal annealing, ruling out the direct reduction effect of the substrate.

Virtual substrate method for nanomaterials characterization

PubMed Central

Da, Bo; Liu, Jiangwei; Yamamoto, Mahito; Ueda, Yoshihiro; Watanabe, Kazuyuki; Cuong, Nguyen Thanh; Li, Songlin; Tsukagoshi, Kazuhito; Yoshikawa, Hideki; Iwai, Hideo; Tanuma, Shigeo; Guo, Hongxuan; Gao, Zhaoshun; Sun, Xia; Ding, Zejun

2017-01-01

Characterization techniques available for bulk or thin-film solid-state materials have been extended to substrate-supported nanomaterials, but generally non-quantitatively. This is because the nanomaterial signals are inevitably buried in the signals from the underlying substrate in common reflection-configuration techniques. Here, we propose a virtual substrate method, inspired by the four-point probe technique for resistance measurement as well as the chop-nod method in infrared astronomy, to characterize nanomaterials without the influence of underlying substrate signals from four interrelated measurements. By implementing this method in secondary electron (SE) microscopy, a SE spectrum (white electrons) associated with the reflectivity difference between two different substrates can be tracked and controlled. The SE spectrum is used to quantitatively investigate the covering nanomaterial based on subtle changes in the transmission of the nanomaterial with high efficiency rivalling that of conventional core-level electrons. The virtual substrate method represents a benchmark for surface analysis to provide ‘free-standing' information about supported nanomaterials. PMID:28548114

Investigation of dielectric substrates on electrical and optical performance of wafer-scale graphene using non-contact methods

NASA Astrophysics Data System (ADS)

Wang, Dong; Ning, Jing; Zhang, Jincheng; Guo, Lixin; Hao, Yue

2017-10-01

Here we systemically discussed the influence of dielectric substrates on the surface morphology, electrical and optical performance of transferred graphene. The electrical properties were investigated using a microwave-probing technique without metal-graphene contact. We found that a complex mechanism governed the influence of the surface properties of the dielectric substrates, such as morphology, hydrophilicity, crystallinity, and polarization, on the performance of the graphene. We also found that graphene on r-Al2O3 was more effective for graphene-based devices with a high carrier mobility of ˜5000 cm2 V-1 s-1. This provides a new method to choose the most suitable substrate for fabricating graphene-based devices.

Identifying suitable substrates for high-quality graphene-based heterostructures

NASA Astrophysics Data System (ADS)

Banszerus, L.; Janssen, H.; Otto, M.; Epping, A.; Taniguchi, T.; Watanabe, K.; Beschoten, B.; Neumaier, D.; Stampfer, C.

2017-06-01

We report on a scanning confocal Raman spectroscopy study investigating the strain-uniformity and the overall strain and doping of high-quality chemical vapour deposited (CVD) graphene-based heterostuctures on a large number of different substrate materials, including hexagonal boron nitride (hBN), transition metal dichalcogenides, silicon, different oxides and nitrides, as well as polymers. By applying a hBN-assisted, contamination free, dry transfer process for CVD graphene, high-quality heterostructures with low doping densities and low strain variations are assembled. The Raman spectra of these pristine heterostructures are sensitive to substrate-induced doping and strain variations and are thus used to probe the suitability of the substrate material for potential high-quality graphene devices. We find that the flatness of the substrate material is a key figure for gaining, or preserving high-quality graphene.

Colloidal silica films for high-capacity DNA arrays

NASA Astrophysics Data System (ADS)

Glazer, Marc Irving

The human genome project has greatly expanded the amount of genetic information available to researchers, but before this vast new source of data can be fully utilized, techniques for rapid, large-scale analysis of DNA and RNA must continue to develop. DNA arrays have emerged as a powerful new technology for analyzing genomic samples in a highly parallel format. The detection sensitivity of these arrays is dependent on the quantity and density of immobilized probe molecules. We have investigated substrates with a porous, "three-dimensional" surface layer as a means of increasing the surface area available for the synthesis of oligonucleotide probes, thereby increasing the number of available probes and the amount of detectable bound target. Porous colloidal silica films were created by two techniques. In the first approach, films were deposited by spin-coating silica colloid suspensions onto flat glass substrates, with the pores being formed by the natural voids between the solid particles (typically 23nm pores, 35% porosity). In the second approach, latex particles were co-deposited with the silica and then pyrolyzed, creating films with larger pores (36 nm), higher porosity (65%), and higher surface area. For 0.3 mum films, enhancements of eight to ten-fold and 12- to 14-fold were achieved with the pure silica films and the films "templated" with polymer latex, respectively. In gene expression assays for up to 7,000 genes using complex biological samples, the high-capacity films provided enhanced signals and performed equivalently or better than planar glass on all other functional measures, confirming that colloidal silica films are a promising platform for high-capacity DNA arrays. We have also investigated the kinetics of hybridization on planar glass and high-capacity substrates. Adsorption on planar arrays is similar to ideal Langmuir-type adsorption, although with an "overshoot" at high solution concentration. Hybridization on high-capacity films is controlled by traditional adsorption (ka) and desorption (kd) coefficients, as well as morphology factors and transient binding interactions between the target and probes. The strength of the transient probe/target binding interactions are on the order of 5--7 DNA base pairs, which suggests the formation of nucleation or other metastable complexes, rather than fully-zippered duplexes.

Contrasting visual working memory for verbal and non-verbal material with multivariate analysis of fMRI

PubMed Central

Habeck, Christian; Rakitin, Brian; Steffener, Jason; Stern, Yaakov

2012-01-01

We performed a delayed-item-recognition task to investigate the neural substrates of non-verbal visual working memory with event-related fMRI (‘Shape task’). 25 young subjects (mean age: 24.0 years; STD=3.8 years) were instructed to study a list of either 1,2 or 3 unnamable nonsense line drawings for 3 seconds (‘stimulus phase’ or STIM). Subsequently, the screen went blank for 7 seconds (‘retention phase’ or RET), and then displayed a probe stimulus for 3 seconds in which subject indicated with a differential button press whether the probe was contained in the studied shape-array or not (‘probe phase’ or PROBE). Ordinal Trend Canonical Variates Analysis (Habeck et al., 2005a) was performed to identify spatial covariance patterns that showed a monotonic increase in expression with memory load during all task phases. Reliable load-related patterns were identified in the stimulus and retention phase (p<0.01), while no significant pattern could be discerned during the probe phase. Spatial covariance patterns that were obtained from an earlier version of this task (Habeck et al., 2005b) using 1, 3, or 6 letters (‘Letter task’) were also prospectively applied to their corresponding task phases in the current non-verbal task version. Interestingly, subject expression of covariance patterns from both verbal and non-verbal retention phases correlated positively in the non-verbal task for all memory loads (p<0.0001). Both patterns also involved similar frontoparietal brain regions that were increasing in activity with memory load, and mediofrontal and temporal regions that were decreasing. Mean subject expression of both patterns across memory load during retention also correlated positively with recognition accuracy (dL) in the Shape task (p<0.005). These findings point to similarities in the neural substrates of verbal and non-verbal rehearsal processes. Encoding processes, on the other hand, are critically dependent on the to-be-remembered material, and seem to necessitate material-specific neural substrates. PMID:22652306

Physics and Applications of Defects in Advanced Semiconductors. Materials Research Society Symposium Proceedings. Volume 325

DTIC Science & Technology

1994-01-01

MAGNETOOPTICAL STUDIES OF ACCEPTORS CONFINED IN GaAs/AMGaAs QUANTUM WELLS ............................................... 73 P.O. Holtz, Q.X. Zhao, B. Momar...PROBE-PROBE TRANSMISSION STUDIES OF LT-GROWN GaAs NEAR THE BAND EDGE ...................................... 389 H.B. Radousky, A.F. Bello, DJ. Erskine...SUBSTRATE ...................... 449 M. Shah, M.O. Manareh, R. Kaspi, M.Y. Yen, B.A. Philips, M. Skowronki, and J. Shi•rm A TEM STUDY OF DEFECT STRUCTURE IN

Construction of promoter-probe shuttle vectors for Escherichia coli and corynebacteria on the basis of promoterless alpha-amylase gene.

PubMed

Ugorcáková, J; Bukovská, G; Timko, J

2000-01-01

We constructed new promoter-probe vectors for E. coli and corynebacteria based on the promoterless alpha-amylase gene originating from Bacillus subtilis. Vectors pJUPAE1 and pJUPAE2 are suitable for isolation of transcriptionally active fragments from plasmids, phages or genomic DNA. alpha-Amylase activity can be easily visually detected on agar plates containing a chromogenic substrate, or by direct measurement of alpha-amylase activity.

From Lab to Fab: Developing a Nanoscale Delivery Tool for Scalable Nanomanufacturing

NASA Astrophysics Data System (ADS)

Safi, Asmahan A.

The emergence of nanomaterials with unique properties at the nanoscale over the past two decades carries a capacity to impact society and transform or create new industries ranging from nanoelectronics to nanomedicine. However, a gap in nanomanufacturing technologies has prevented the translation of nanomaterial into real-world commercialized products. Bridging this gap requires a paradigm shift in methods for fabricating structured devices with a nanoscale resolution in a repeatable fashion. This thesis explores the new paradigms for fabricating nanoscale structures devices and systems for high throughput high registration applications. We present a robust and scalable nanoscale delivery platform, the Nanofountain Probe (NFP), for parallel direct-write of functional materials. The design and microfabrication of NFP is presented. The new generation addresses the challenges of throughput, resolution and ink replenishment characterizing tip-based nanomanufacturing. To achieve these goals, optimized probe geometry is integrated to the process along with channel sealing and cantilever bending. The capabilities of the newly fabricated probes are demonstrated through two type of delivery: protein nanopatterning and single cell nanoinjection. The broad applications of the NFP for single cell delivery are investigated. An external microfluidic packaging is developed to enable delivery in liquid environment. The system is integrated to a combined atomic force microscope and inverted fluorescence microscope. Intracellular delivery is demonstrated by injecting a fluorescent dextran into Hela cells in vitro while monitoring the injection forces. Such developments enable in vitro cellular delivery for single cell studies and high throughput gene expression. The nanomanufacturing capabilities of NFPs are explored. Nanofabrication of carbon nanotube-based electronics presents all the manufacturing challenges characterizing of assembling nanomaterials precisely onto devices. The presented study combines top-down and bottom-approaches by integrating the catalyst patterning and carbon nanotube growth directly on structures. Large array of iron-rich catalyst are patterned on an substrate for subsequent carbon nanotubes synthesis. The dependence of probe geometry and substrate wetting is assessed by modeling and experimental studies. Finally preliminary results on synthesis of carbon nanotube by catalyst assisted chemical vapor deposition suggest increasing the catalyst yield is critical. Such work will enable high throughput nanomanufacturing of carbon nanotube based devices.

Comparison of inkjet-printed silver conductors on different microsystem substrates

NASA Astrophysics Data System (ADS)

Kruger, Jené; Bezuidenhout, Petroné H.; Joubert, Trudi-Heleen

2016-02-01

Applications for diagnostic and environmental point-of-need require processes and building blocks to add smart features to disposable biosensors on low-cost substrates. A novel method for producing such biosensors is printing electronics using additive technologies. This work contributes to the toolbox of processes, materials and components for printed electronics manufacturing - as well as rapid prototyping - of circuits. Printing protocols were developed to facilitate successful inkjet printing of nanosilver ink (Harima NPS-JL) onto different microsystem substrates using a functional printer (Dimatix DMP-3281). Photo paper is a standard inkjet substrate, which were compared with glass, polycarbonate (PC), plastic projector transparency foil, and polydimethylsiloxane (PDMS). Comparison attributes include physical and electrical properties. The layout design comprised dogbone elements of 8 mm length, and widths varying between 100 μm and 2 mm. All printed features were thermally cured for 1 hour at 120 °C. The physical characteristics were measured with a laser scanning microscope (Zeiss LSM-5) to determine the width, thickness and surface roughness of the printed features. An LCR meter (GW-Instek 8110) was used to measure the printed structures' electrical characteristics (resistance, capacitance and inductance). A lumped element model and layout design rules were extracted to assist in standardized design procedures. The model incorporates prediction of the bandwidth attainable with these structures. The layer thickness on all substrates is larger than the 1 μm on photo paper, and varies between 1.6 μm (PC) and 7 μm (PDMS). The spreading for PDMS is similar to photo paper, but since for the other substrates it is between 5 (glass) and 10 (PC) times larger than for photo paper, the layout design rules require large spacing, leading to larger area networks. Electrical probing on the PDMS is not consistent and results are inconclusive. For the other substrates, the comparative dogbone resistance (100 μm width) is significantly larger than the 2 Ω standard, varying from 12.6 Ω (PC) to 19.3 Ω (glass). The bandwidth relative to photo paper is smaller by a factor of between 6 (PC) and 9.5 (glass).

Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues.

PubMed

Liu, Xianjun; Xiang, Meihao; Tong, Zongxuan; Luo, Fengyan; Chen, Wen; Liu, Feng; Wang, Fenglin; Yu, Ru-Qin; Jiang, Jian-Hui

2018-05-01

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 μm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

Development of PEGylated peptide probes conjugated with (18)F-labeled BODIPY for PET/optical imaging of MT1-MMP activity.

PubMed

Kondo, Naoya; Temma, Takashi; Deguchi, Jun; Sano, Kohei; Ono, Masahiro; Saji, Hideo

2015-12-28

Since the processing activity of the matrix metalloproteinase MT1-MMP regulates various cellular functions such as motility, invasion, growth, differentiation and apoptosis, precise in vivo evaluation of MT1-MMP activity in cancers can provide beneficial information for both basic and clinical studies. For this purpose, we designed a cleavable Positron Emission Tomography (PET)/optical imaging probe consisting of BODIPY650/665 and polyethylene glycol (PEG) conjugated to opposite ends of MT1-MMP substrate peptides. We used in vitro and in vivo fluorescence experiments to select suitable substrate peptide sequences and PEG sizes for the MT1-MMP probes and obtained an optimized structure referred to here as MBP-2k. Radiofluorinated MBP-2k ([(18)F]MBP-2k) was then successfully synthesized via an (18)F-(19)F isotopic exchange reaction in BODIPY650/665. After intravenous injection into mice with xenografted tumors, [(18)F]MBP-2k showed significantly higher accumulation in HT1080 tumors with high MT1-MMP activity than in A549 tumors that have low MT1-MMP activity. Moreover, PET images showed better contrast in HT1080 tumors. These results show that [(18)F]MBP-2k can be used as a hybrid PET/optical imaging agent and is a promising probe for non-invasive monitoring of MT1-MMP activity in cancers. This probe may also efficiently combine targeted tumor imaging with image-guided surgery that could be beneficial for patients in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

Nondestructive Superresolution Imaging of Defects and Nonuniformities in Metals, Semiconductors, Dielectrics, Composites, and Plants Using Evanescent Microwaves

NASA Technical Reports Server (NTRS)

Tabib-Azar, M.; Pathak, P. S.; Ponchak, G.; LeClair, S.

1999-01-01

We have imaged and mapped material nonuniformities and defects using microwaves generated at the end of a microstripline resonator with 0.4 micrometer lateral spatial resolution at 1 GHz. Here we experimentally examine the effect of microstripline substrate permittivity, the feedline-to-resonator coupling strength, and probe tip geometry on the spatial resolution of the probe. Carbon composites, dielectrics, semiconductors, metals, and botanical samples were scanned for defects, residual stresses, subsurface features, areas of different film thickness, and moisture content. The resulting evanescent microwave probe (EMP) images are discussed. The main objective of this work is to demonstrate the overall capabilities of the EMP imaging technique as well as to discuss various probe parameters that can be used to design EMPs for different applications.

A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics.

PubMed

Ma, Y; Dai, X; Hong, T; Munk, G B; Libera, M

2016-12-19

Despite their many advantages and successes, molecular beacon (MB) hybridization probes have not been extensively used in microarray formats because of the complicating probe-substrate interactions that increase the background intensity. We have previously shown that tethering to surface-patterned microgels is an effective means for localizing MB probes to specific surface locations in a microarray format while simultaneously maintaining them in as water-like an environment as possible and minimizing probe-surface interactions. Here we extend this approach to include both real-time detection together with integrated NASBA amplification. We fabricate small (∼250 μm × 250 μm) simplex, duplex, and five-plex assays with microarray spots of controllable size (∼20 μm diameter), position, and shape to detect bacteria and fungi in a bloodstream-infection model. The targets, primers, and microgel-tethered probes can be combined in a single isothermal reaction chamber with no post-amplification labelling. We extract total RNA from clinical blood samples and differentiate between Gram-positive and Gram-negative bloodstream infection in a duplex assay to detect RNA- amplicons. The sensitivity based on our current protocols in a simplex assay to detect specific ribosomal RNA sequences within total RNA extracted from S. aureus and E. coli cultures corresponds to tens of bacteria per ml. We furthermore show that the platform can detect RNA- amplicons from synthetic target DNA with 1 fM sensitivity in sample volumes that contain about 12 000 DNA molecules. These experiments demonstrate an alternative approach that can enable rapid and real-time microarray-based molecular diagnostics.

Carrier dynamics in silicon nanowires studied using optical-pump terahertz-probe spectroscopy

NASA Astrophysics Data System (ADS)

Beaudoin, Alexandre; Salem, Bassem; Baron, Thierry; Gentile, Pascal; Morris, Denis

2014-03-01

The advance of non-contact measurements involving pulsed terahertz radiation presents great interests for characterizing electrical properties of a large ensemble of nanowires. In this work, N-doped and undoped silicon nanowires (SiNWs) grown by chemical vapour deposition (CVD) on quartz substrate were characterized using optical-pump terahertz probe (OPTP) transmission experiments. Our results show that defects and ionized impurities introduced by N-doping the CVD-grown SiNWs tend to reduce the photoexcited carrier lifetime and degrade their conductivity properties. Capture mechanisms by the surface trap states play a key role on the photocarrier dynamics in theses small diameters' (~100 nm) SiNWs and the doping level is found to alter this dynamics. We propose convincing capture and recombination scenarios that explain our OPTP measurements. Fits of our photoconductivity data curves, from 0.5 to 2 THz, using a Drude-plasmon conductivity model allow determining photocarrier mobility values of 190 and 70 cm2/V .s, for the undoped and N-doped NWs samples, respectively.

Design of activated serine-containing catalytic triads with atomic level accuracy

PubMed Central

Rajagopalan, Sridharan; Wang, Chu; Yu, Kai; Kuzin, Alexandre P.; Richter, Florian; Lew, Scott; Miklos, Aleksandr E.; Matthews, Megan L.; Seetharaman, Jayaraman; Su, Min; Hunt, John. F.; Cravatt, Benjamin F.; Baker, David

2014-01-01

A challenge in the computational design of enzymes is that multiple properties must be simultaneously optimized -- substrate-binding, transition state stabilization, and product release -- and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads, and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate-reactivity. Following optimization by yeast-display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest the designs could provide the basis for a new class of organophosphate captures agents. PMID:24705591

Towards an animal model of callousness.

PubMed

Hernandez-Lallement, Julen; van Wingerden, Marijn; Kalenscher, Tobias

2016-12-28

Callous-unemotional traits - the insensitivity to other's welfare and well-being - are characterized by a lack of empathy. They are characteristic of psychopathy and can be found in other anti-social disorders, such as conduct disorder. Because of the increasing prevalence of anti-social disorders and the rising societal costs of violence and aggression, it is of great importance to elucidate the psychological and physiological mechanisms underlying callousness in the search for pharmacological treatments. One promising avenue is to create a relevant animal model to explore the neural bases of callousness. Here, we review recent advances in rodent models of pro-social choice that could be applied to probe the absence of pro-sociality as a proxy of callous behavior, and provide future directions for the exploration of the neural substrates of callousness. Copyright © 2016 Elsevier Ltd. All rights reserved.

Breathable NIPAAm Network with Controllable Hydration Supports Model Lipid Membrane

NASA Astrophysics Data System (ADS)

Jablin, Michael; Smith, Hillary; Zhernenkov, Mikhail; Vidyasagar, Ajay; Toomey, Ryan; Saiz, Jessica; Toperverg, Boris; Watkins, Erik; Kuhl, Tonya; Hurd, Alan; Majewski, Jaroslaw

2009-03-01

The interaction of a model lipid bilayer composed of DPPC with a surface-tethered poly(N-isopropylacrylamide) (NIPAAm) was explored with neutron reflectometry (NR). The Langmuir-Blodgett / Langmuir-Schaeffer method was used to deposit a lipid bilayer onto the polymer. NR measurements were used to probe the in- and out-of-plane structure of the system as a function of temperature. NR with fluorescence microscopy show that the polymer supports a lipid bilayer, and hydration of the support can be controlled. At low temp. the membrane develops out-of-plane undulations visible in off-specular scattering. Analysis of the off-specular reveals in-plane correlation of the bilayer fluctuations. The separation of the lipid bilayer from the solid support of a substrate constitutes a significant step towards a more realistic model of biological membranes.

Assessment of inhibitory effects on major human cytochrome P450 enzymes by spasmolytics used in the treatment of overactive bladder syndrome.

PubMed

Dahlinger, Dominik; Aslan, Sevinc; Pietsch, Markus; Frechen, Sebastian; Fuhr, Uwe

2017-07-01

The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride on the seven major human cytochrome P450 enzymes (CYP) by using a standardized and validated seven-in-one cytochrome P450 cocktail inhibition assay. An in vitro cocktail of seven highly selective probe substrates was incubated with human liver microsomes and varying concentrations of the seven test compounds. The major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Enzyme kinetics were estimated by determining IC 50 and K i values via nonlinear regression. Obtained K i values were used for predictions of potential clinical impact of the inhibition using a static mechanistic prediction model. In this study, 49 IC 50 experiments were conducted. In six cases, IC 50 values lower than the calculated threshold for drug-drug interactions (DDIs) in the gut wall were observed. In these cases, no increase in inhibition was determined after a 30 min preincubation. Considering a typical dosing regimen and applying the obtained K i values of 0.72 µM (darifenacin, 15 mg daily) and 7.2 µM [propiverine, 30 mg daily, immediate release (IR)] for the inhibition of CYP2D6 yielded a predicted 1.9-fold and 1.4-fold increase in the area under the curve (AUC) of debrisoquine (CYP2D6 substrate), respectively. Due to the inhibition of the particular intestinal CYP3A4, the obtained K i values of 14 µM of propiverine (30 mg daily, IR) resulted in a predicted doubling of the AUC for midazolam (CYP3A4 substrate). In vitro / in vivo extrapolation based on pharmacokinetic data and the conducted screening experiments yielded similar effects of darifenacin on CYP2D6 and propiverine on CYP3A4 as obtained in separately conducted in vivo DDI studies. As a novel finding, propiverine was identified to potentially inhibit CYP2D6 at clinically occurring concentrations.

Assessment of inhibitory effects on major human cytochrome P450 enzymes by spasmolytics used in the treatment of overactive bladder syndrome

PubMed Central

Dahlinger, Dominik; Aslan, Sevinc; Pietsch, Markus; Frechen, Sebastian; Fuhr, Uwe

2017-01-01

Background: The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride on the seven major human cytochrome P450 enzymes (CYP) by using a standardized and validated seven-in-one cytochrome P450 cocktail inhibition assay. Methods: An in vitro cocktail of seven highly selective probe substrates was incubated with human liver microsomes and varying concentrations of the seven test compounds. The major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Enzyme kinetics were estimated by determining IC50 and Ki values via nonlinear regression. Obtained Ki values were used for predictions of potential clinical impact of the inhibition using a static mechanistic prediction model. Results: In this study, 49 IC50 experiments were conducted. In six cases, IC50 values lower than the calculated threshold for drug–drug interactions (DDIs) in the gut wall were observed. In these cases, no increase in inhibition was determined after a 30 min preincubation. Considering a typical dosing regimen and applying the obtained Ki values of 0.72 µM (darifenacin, 15 mg daily) and 7.2 µM [propiverine, 30 mg daily, immediate release (IR)] for the inhibition of CYP2D6 yielded a predicted 1.9-fold and 1.4-fold increase in the area under the curve (AUC) of debrisoquine (CYP2D6 substrate), respectively. Due to the inhibition of the particular intestinal CYP3A4, the obtained Ki values of 14 µM of propiverine (30 mg daily, IR) resulted in a predicted doubling of the AUC for midazolam (CYP3A4 substrate). Conclusions: In vitro/in vivo extrapolation based on pharmacokinetic data and the conducted screening experiments yielded similar effects of darifenacin on CYP2D6 and propiverine on CYP3A4 as obtained in separately conducted in vivo DDI studies. As a novel finding, propiverine was identified to potentially inhibit CYP2D6 at clinically occurring concentrations. PMID:28747995

Identification of metabolically active bacteria in the gut of the generalist Spodoptera littoralis via DNA stable isotope probing using 13C-glucose.

PubMed

Shao, Yongqi; Arias-Cordero, Erika M; Boland, Wilhelm

2013-11-13

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction(1), boosting the immune response(2), pheromone production(3), as well as nutrition, including the synthesis of essential amino acids(4,) among others.     Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms. To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing (13)C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA(5). The incorporation of (13)C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled ((12)C) one. In the end, the (13)C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the (12)C-unlabeled similar one(6). Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species. Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, (13)C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

A simplified protocol employing elacridar in rodents: a screening model in drug discovery to assess P-gp mediated efflux at the blood brain barrier.

PubMed

Kallem, Rajareddy; Kulkarni, Chetan P; Patel, Dakshay; Thakur, Megha; Sinz, Michael; Singh, Sheelendra P; Mahammad, S Shahe; Mandlekar, Sandhya

2012-06-01

In the present study we have developed a simple, time, and cost effective in vivo rodent protocol to screen the susceptibility of a test compound for P-glycoprotein (P-gp) mediated efflux at the blood brain barrier (BBB) during early drug discovery. We used known P-gp substrates as test compounds (quinidine, digoxin, and talinolol) and elacridar (GF120918) as a chemical inhibitor to establish the model. The studies were carried out in both mice and rats. Elacridar was dosed intravenously at 5 mg/kg, 0.5 h prior to probe substrate administration. Plasma and brain samples were collected and analyzed using UPLC-MS/MS. In the presence of elacridar, the ratio of brain to plasma area under the curve (B/P) in mouse increased 2, 4, and 38-fold, respectively, for talinolol, digoxin, and quinidine; whereas in rat, a 70-fold increase was observed for quinidine. Atenolol, a non P-gp substrate, exhibited poor brain penetration in the presence or absence of elacridar in both species (B/P ratio ~ 0.1). Elacridar had no significant effect on the systemic clearance of digoxin or quinidine; however, a trend towards increasing volume of distribution and half life was observed. Our results support the utility of elacridar in evaluation of the influence of P-gp mediated efflux on drug distribution to the brain. Our protocol employing a single intravenous dose of elacridar and test compound provides a cost effective alternative to expensive P-gp knockout mice models during early drug discovery.

Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

PubMed Central

Martin, Teresa A.; Herman, Christine T.; Limpoco, Francis T.; Michael, Madeline C.; Potts, Gregory K.; Bailey, Ryan C.

2014-01-01

Methods for the generation of substrates presenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retention of function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool that will allow the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces. PMID:21793535

Morphologically manipulated Ag/ZnO nanostructures as surface enhanced Raman scattering probes for explosives detection

NASA Astrophysics Data System (ADS)

Shaik, Ummar Pasha; Hamad, Syed; Ahamad Mohiddon, Md.; Soma, Venugopal Rao; Ghanashyam Krishna, M.

2016-03-01

The detection of secondary explosive molecules (e.g., ANTA, FOX-7, and CL-20) using Ag decorated ZnO nanostructures as surface enhanced Raman scattering (SERS) probes is demonstrated. ZnO nanostructures were grown on borosilicate glass substrates by rapid thermal oxidation of metallic Zn films at 500 °C. The oxide nanostructures, including nanosheets and nanowires, emerged over the surface of the Zn film leaving behind the metal residue. We demonstrate that SERS measurements with concentrations as low as 10 μM, of the three explosive molecules ANTA, FOX-7, and CL-20 over ZnO/Ag nanostructures, resulted in enhancement factors of ˜107, ˜107, and ˜104, respectively. These measurements validate the high sensitivity of detection of explosive molecules using Ag decorated ZnO nanostructures as SERS substrates. The Zn metal residue and conditions of annealing play an important role in determining the detection sensitivity.

Reduced Sampling Size with Nanopipette for Tapping-Mode Scanning Probe Electrospray Ionization Mass Spectrometry Imaging

PubMed Central

Kohigashi, Tsuyoshi; Otsuka, Yoichi; Shimazu, Ryo; Matsumoto, Takuya; Iwata, Futoshi; Kawasaki, Hideya; Arakawa, Ryuichi

2016-01-01

Mass spectrometry imaging (MSI) with ambient sampling and ionization can rapidly and easily capture the distribution of chemical components in a solid sample. Because the spatial resolution of MSI is limited by the size of the sampling area, reducing sampling size is an important goal for high resolution MSI. Here, we report the first use of a nanopipette for sampling and ionization by tapping-mode scanning probe electrospray ionization (t-SPESI). The spot size of the sampling area of a dye molecular film on a glass substrate was decreased to 6 μm on average by using a nanopipette. On the other hand, ionization efficiency increased with decreasing solvent flow rate. Our results indicate the compatibility between a reduced sampling area and the ionization efficiency using a nanopipette. MSI of micropatterns of ink on a glass and a polymer substrate were also demonstrated. PMID:28101441

Scalable maskless patterning of nanostructures using high-speed scanning probe arrays

NASA Astrophysics Data System (ADS)

Chen, Chen; Akella, Meghana; Du, Zhidong; Pan, Liang

2017-08-01

Nanoscale patterning is the key process to manufacture important products such as semiconductor microprocessors and data storage devices. Many studies have shown that it has the potential to revolutionize the functions of a broad range of products for a wide variety of applications in energy, healthcare, civil, defense and security. However, tools for mass production of these devices usually cost tens of million dollars each and are only affordable to the established semiconductor industry. A new method, nominally known as "pattern-on-the- y", that involves scanning an array of optical or electrical probes at high speed to form nanostructures and offers a new low-cost approach for nanoscale additive patterning. In this paper, we report some progress on using this method to pattern self-assembled monolayers (SAMs) on silicon substrate. We also functionalize the substrate with gold nanoparticle based on the SAM to show the feasibility of preparing amphiphilic and multi-functional surfaces.

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

PubMed

Zuccaro, Laura; Tesauro, Cinzia; Kurkina, Tetiana; Fiorani, Paola; Yu, Hak Ki; Knudsen, Birgitta R; Kern, Klaus; Desideri, Alessandro; Balasubramanian, Kannan

2015-11-24

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

PubMed

Tylko, G; Karasiński, J; Wróblewski, R; Roomans, G M; Kilarski, W M

2000-01-01

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

NASA Astrophysics Data System (ADS)

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

2016-11-01

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Complementary and partially complementary DNA duplexes tethered to a functionalized substrate: a molecular dynamics approach to biosensing.

PubMed

Monti, Susanna; Cacelli, Ivo; Ferretti, Alessandro; Prampolini, Giacomo; Barone, Vincenzo

2011-07-21

Molecular dynamics simulations (90 ns) of different DNA complexes attached to a functionalized substrate in solution were performed in order to clarify the behavior of mismatched DNA sequences captured by a tethered DNA probe (biochip). Examination of the trajectories revealed that the substrate influence and a series of cooperative events, including recognition, reorientation and reorganization of the bases, could induce the formation of stable duplexes having non-canonical arrangements. Major adjustment of the structures was observed when the mutated base was located in the end region of the chain close to the surface. This journal is © the Owner Societies 2011

Functionalization of nanostructured gold substrates with chiral chromophores for SERS applications: The case of 5-Aza[5]helicene.

PubMed

Zanchi, Chiara; Lucotti, Andrea; Cancogni, Damiano; Fontana, Francesca; Trusso, Sebastiano; Ossi, Paolo M; Tommasini, Matteo

2018-05-31

Nanostructured gold thin films can be fabricated by controlled pulsed laser deposition to get efficient sensors, with uniform morphology and optimized plasmon resonance, to be employed as plasmonic substrates in surface enhanced Raman scattering spectroscopy. By attaching 5-aza[5]helicen-6-yl-6-hexanethiol to such gold nanostructures, used in a previous work for label-free drug sensing with biomedical purposes, we successfully prepared functionalized substrates with remarkable surface enhanced Raman scattering activity. The long-term motivation is to develop probes for drug detection at low concentrations, where sensitivity to specific chiral targets is required. © 2018 Wiley Periodicals, Inc.

Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase.

PubMed

Roldán, Raquel; Sanchez-Moreno, Israel; Scheidt, Thomas; Hélaine, Virgil; Lemaire, Marielle; Parella, Teodor; Clapés, Pere; Fessner, Wolf-Dieter; Guérard-Hélaine, Christine

2017-04-11

d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Flexible and mechanical strain resistant large area SERS active substrates

NASA Astrophysics Data System (ADS)

Singh, J. P.; Chu, Hsiaoyun; Abell, Justin; Tripp, Ralph A.; Zhao, Yiping

2012-05-01

We report a cost effective and facile way to synthesize flexible, uniform, and large area surface enhanced Raman scattering (SERS) substrates using an oblique angle deposition (OAD) technique. The flexible SERS substrates consist of 1 μm long, tilted silver nanocolumnar films deposited on flexible polydimethylsiloxane (PDMS) and polyethylene terephthalate (PET) sheets using OAD. The SERS enhancement activity of these flexible substrates was determined using 10-5 M trans-1,2-bis(4-pyridyl) ethylene (BPE) Raman probe molecules. The in situ SERS measurements on these flexible substrates under mechanical (tensile/bending) strain conditions were performed. Our results show that flexible SERS substrates can withstand a tensile strain (ε) value as high as 30% without losing SERS performance, whereas the similar bending strain decreases the SERS performance by about 13%. A cyclic tensile loading test on flexible PDMS SERS substrates at a pre-specified tensile strain (ε) value of 10% shows that the SERS intensity remains almost constant for more than 100 cycles. These disposable and flexible SERS substrates can be integrated with biological substances and offer a novel and practical method to facilitate biosensing applications.

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

PubMed

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Validation of a microdose probe drug cocktail for clinical drug interaction assessments for drug transporters and CYP3A.

PubMed

Prueksaritanont, T; Tatosian, D A; Chu, X; Railkar, R; Evers, R; Chavez-Eng, C; Lutz, R; Zeng, W; Yabut, J; Chan, G H; Cai, X; Latham, A H; Hehman, J; Stypinski, D; Brejda, J; Zhou, C; Thornton, B; Bateman, K P; Fraser, I; Stoch, S A

2017-04-01

A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Structural and phase transformation of A{sup III}B{sup V}(100) semiconductor surface in interaction with selenium

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

Bezryadin, N. N.; Kotov, G. I., E-mail: giktv@mail.ru; Kuzubov, S. V., E-mail: kuzub@land.ru

2015-03-15

Surfaces of GaAs(100), InAs(100), and GaP(100) substrates thermally treated in selenium vapor have been investigated by transmission electron microscopy and electron probe X-ray microanalysis. Some specific features and regularities of the formation of A{sub 3}{sup III}B{sub 4}{sup VI} (100)c(2 × 2) surface phases and thin layers of gallium or indium selenides A{sub 2}{sup III}B{sub 3}{sup VI} (100) on surfaces of different A{sup III}B{sup V}(100) semiconductors are discussed within the vacancy model of surface atomic structure.

Manipulation of nanoparticles of different shapes inside a scanning electron microscope

PubMed Central

Polyakov, Boris; Dorogin, Leonid M; Butikova, Jelena; Antsov, Mikk; Oras, Sven; Lõhmus, Rünno; Kink, Ilmar

2014-01-01

Summary In this work polyhedron-like gold and sphere-like silver nanoparticles (NPs) were manipulated on an oxidized Si substrate to study the dependence of the static friction and the contact area on the particle geometry. Measurements were performed inside a scanning electron microscope (SEM) that was equipped with a high-precision XYZ-nanomanipulator. To register the occurring forces a quartz tuning fork (QTF) with a glued sharp probe was used. Contact areas and static friction forces were calculated by using different models and compared with the experimentally measured force. The effect of NP morphology on the nanoscale friction is discussed. PMID:24605279

Selective femtosecond laser structuring of dielectric thin films with different band gaps: a time-resolved study of ablation mechanisms

NASA Astrophysics Data System (ADS)

Rapp, Stephan; Schmidt, Michael; Huber, Heinz P.

2016-12-01

Ultrashort pulse lasers have been increasingly gaining importance for the selective structuring of dielectric thin films in industrial applications. In a variety of works the ablation of thin SiO2 and SiNx films from Si substrates has been investigated with near infrared laser wavelengths with photon energies of about 1.2 eV where both dielectrics are transparent (E_{{gap,SiO2}}≈ 8 eV; E_{{gap,SiN}x}≈ 2.5 eV). In these works it was found that few 100 nm thick SiO2 films are selectively ablated with a "lift-off" initiated by confined laser ablation whereas the SiN_{{x}} films are ablated by a combination of confined and direct laser ablation. In the work at hand, ultrafast pump-probe imaging was applied to compare the laser ablation dynamics of the two thin film systems directly with the uncoated Si substrate—on the same setup and under identical parameters. On the SiO2 sample, results show the pulse absorption in the Si substrate, leading to the confined ablation of the SiO2 layer by the expansion of the substrate. On the SiN_{{x}} sample, direct absorption in the layer is observed leading to its removal by evaporation. The pump-probe measurements combined with reflectivity corrected threshold fluence investigations suggest that melting of the Si substrate is sufficient to initiate the lift-off of an overlaying transparent film—evaporation of the substrate seems not to be necessary.

Development of a detachable high speed miniature scanning probe microscope for large area substrates inspection

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

Sadeghian, Hamed, E-mail: hamed.sadeghianmarnani@tno.nl, E-mail: h.sadeghianmarnani@tudelft.nl; Department of Precision and Microsystems Engineering, Delft University of Technology, Mekelweg 2, 2628 CD Delft; Herfst, Rodolf

We have developed a high speed, miniature scanning probe microscope (MSPM) integrated with a Positioning Unit (PU) for accurately positioning the MSPM on a large substrate. This combination enables simultaneous, parallel operation of many units on a large sample for high throughput measurements. The size of the MSPM is 19 × 45 × 70 mm{sup 3}. It contains a one-dimensional flexure stage with counter-balanced actuation for vertical scanning with a bandwidth of 50 kHz and a z-travel range of more than 2 μm. This stage is mechanically decoupled from the rest of the MSPM by suspending it on specific dynamicallymore » determined points. The motion of the probe, which is mounted on top of the flexure stage is measured by a very compact optical beam deflection (OBD). Thermal noise spectrum measurements of short cantilevers show a bandwidth of 2 MHz and a noise of less than 15 fm/Hz{sup 1/2}. A fast approach and engagement of the probe to the substrate surface have been achieved by integrating a small stepper actuator and direct monitoring of the cantilever response to the approaching surface. The PU has the same width as the MSPM, 45 mm and can position the MSPM to a pre-chosen position within an area of 275×30 mm{sup 2} to within 100 nm accuracy within a few seconds. During scanning, the MSPM is detached from the PU which is essential to eliminate mechanical vibration and drift from the relatively low-resonance frequency and low-stiffness structure of the PU. Although the specific implementation of the MSPM we describe here has been developed as an atomic force microscope, the general architecture is applicable to any form of SPM. This high speed MSPM is now being used in a parallel SPM architecture for inspection and metrology of large samples such as semiconductor wafers and masks.« less

Electron spin echo envelope modulation studies of the Cu(II)-substituted derivative of isopenicillin N synthase: A structural and spectroscopic model

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

Feng Jiang; Peisach, J.; Lijune Ming

Electron spin echo envelope modulation spectroscopy (ESEEM) was used to study the active site structure of isopenicillin N synthase (IPNS) from Cephalosporium acremonium with Cu(II) as a spectroscopic probe. Fourier transform of the simulated electron spin-echo envelope for the Cu(II)-substituted enzyme, Cu(II)IPNS, revealed two nearly magnetically equivalent, equatorially coordinated His imidazoles. The superhyperfine coupling constant, A{sub iso}, for the remote {sup 14}N of each imidazole was 1.65 MHz. The binding of substrate to the enzyme altered the magnetic coupling so that A{sub iso} is 1.30 MHz for one nitrogen and 2.16 MHz for the other. From a comparison of themore » ESSEM of Cu(II)IPNS in D{sub 2}O and H{sub 2}O, it is suggested that water is a ligand of Cu(II) and this is displaced upon the addition of substrate.« less

Carbon Nanotubes Blended Hydroxyapatite Ethanol Sensor

NASA Astrophysics Data System (ADS)

Anjum, S. R.; Khairnar, R. S.

2016-12-01

Nano crystals of Hydroxyapatite (HAp) were synthesized by a wet chemical precipitation method. The nano composite materials were developed by doping various weight concentrations of carbon nanotubes in HAp, followed by characterization using scanning electron microscopy, and X-ray diffraction. Thick films of these materials were prepared by using screen printing technique. The ethanol sensing properties of these nano crystals and nano composite films were investigated by two probe electrical method. The gas sensing features such as operating temperature, response and recovery time, maximum gas detection limit, etc. were studied, since these parameters are of prime importance for sensor. The results revealed that at room temperature, the composite materials exhibited improved sensing performance towards 100 ppm ethanol with fast response times. It also showed shorter recovery time with higher vapor uptake capacity. The ethanol adsorption processes on doped and undoped substrates can be explained by surface chemical reactions as well as providing the possible adsorption models. The novelty of this work lies in developing reusable sensor substrates for room temperature sensing.

Interfaces between hexagonal and cubic oxides and their structure alternatives

DOE PAGES

Zhou, Hua; Wu, Lijun; Wang, Hui-Qiong; ...

2017-11-14

Multi-layer structure of functional materials often involves the integration of different crystalline phases. The film growth orientation thus frequently exhibits a transformation, owing to multiple possibilities caused by incompatible in-plane structural symmetry. Nevertheless, the detailed mechanism of the transformation has not yet been fully explored. Here we thoroughly probe the heteroepitaxially grown hexagonal zinc oxide (ZnO) films on cubic (001)-magnesium oxide (MgO) substrates using advanced scanning transition electron microscopy, X-ray diffraction and first principles calculations, revealing two distinct interface models of (001) ZnO/(001) MgO and (100) ZnO/(001) MgO. Here we have found that the structure alternatives are controlled thermodynamically bymore » the nucleation, while kinetically by the enhanced Zn adsorption and O diffusion upon the phase transformation. Finally, this work not only provides a guideline for the interface fabrication with distinct crystalline phases but also shows how polar and non-polar hexagonal ZnO films might be manipulated on the same cubic substrate.« less

Probing Dynamic Cell-Substrate Interactions using Photochemically Generated Surface-Immobilized Gradients: Application to Selectin-Mediated Leukocyte Rolling

PubMed Central

Herman, Christine T.; Potts, Gregory K.; Michael, Madeline C.; Tolan, Nicole V.

2014-01-01

Model substrates presenting biochemical cues immobilized in a controlled and well-defined manner are of great interest for their applications in biointerface studies that elucidate the molecular basis of cell receptor-ligand interactions. Herein, we describe a direct, photochemical method to generate one-component surface-immobilized biomolecular gradients that are applied to the study of selectin-mediated leukocyte rolling. The technique employs benzophenone-modified glass substrates, which upon controlled exposure to UV light (350 – 365 nm) in the presence of protein-containing solutions facilitate the generation of covalently immobilized protein gradients. Conditions were optimized to generate gradient substrates presenting P-selectin and PSGL-1 (P-selectin Glycoprotein Ligand-1) immobilized at site densities over a 5- to 10-fold range (from as low as ~200 molecules/μm2 to as high as 6000 molecules/μm2). The resulting substrates were quantitatively characterized via fluorescence analysis and radioimmunoassays before their use in the leukocyte rolling assays. HL-60 promyelocytes and Jurkat T lymphocytes were assessed for their ability to tether to and roll on substrates presenting immobilized P-selectin and PSGL-1 under conditions of physiologically relevant shear stress. The results of these flow assays reveal the combined effect of immobilized protein site density and applied wall shear stress on cell rolling behavior. Two-component substrates presenting P-selectin and ICAM-1 (intercellular adhesion molecule-1) were also generated to assess the interplay between these two proteins and their effect on cell rolling and adhesion. These proof-of-principle studies verify that the described gradient generation approach yields well-defined gradient substrates that present immobilized proteins over a large range of site densities that are applicable for investigation of cell-materials interactions, including multi-parameter leukocyte flow studies. Future applications of this enabling methodology may lead to new insights into the biophysical phenomena and molecular mechanism underlying complex biological processes such as leukocyte recruitment and the inflammatory response. PMID:21614364

Optical Probing of Low-Pressure Solution Grown GaN Crystal Properties

DTIC Science & Technology

2010-04-01

observed in Mg and Si doped epitaxial films deposited by MBE and MOCVD on freestanding GaN HVPE substrates [23–25]. Considering the purity of the precursors...bands with similar energy positions here reported, a dominant deeper acceptor impurity has been assigned to Zn , a well known deep acceptor in GaN . Room...00-00-2010 to 00-00-2010 4. TITLE AND SUBTITLE Optical probing of low-pressure solution grown GaN crystal properties 5a. CONTRACT NUMBER 5b

Probing of the interfacial Heisenberg and Dzyaloshinskii-Moriya exchange interaction by magnon spectroscopy.

PubMed

Zakeri, Khalil

2017-01-11

This Topical Review presents an overview of the recent experimental results on the quantitative determination of the magnetic exchange parameters in ultrathin magnetic films and multilayers grown on different substrates. The experimental approaches for probing both the symmetric Heisenberg and the antisymmetric Dzyaloshinskii-Moriya exchange interaction in ultrathin magnetic films and at interfaces are discussed in detail. It is explained how the experimental spectrum of magnetic excitations can be used to quantify the strength of these interactions.

Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

PubMed

Schmitt, Kyle C; Mamidyala, Sreeman; Biswas, Swati; Dutta, Aloke K; Reith, Maarten E A

2010-03-01

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

Characterization of surface roughness of laser deposited titanium alloy and copper using AFM

NASA Astrophysics Data System (ADS)

Erinosho, M. F.; Akinlabi, E. T.; Johnson, O. T.

2018-03-01

Laser Metal Deposition (LMD) is the process of using the laser beam of a nozzle to produce a melt pool on a metal surface usually the substrate and metal powder is been deposited into it thereby creating a fusion bond with the substrate to form a new material layer against the force gravity. A good metal laminate is formed when the wettability between the dropping metal powder and the substrate adheres. This paper reports the surface roughness of laser deposited titanium alloy and copper (Ti6Al4V + Cu) using the Atomic Force Microscopy (AFM). This AFM is employed in order to sense the surface and produce different manipulated images using the micro-fabricated mechanical tip under a probe cartridge of high resolution. The process parameters employed during the deposition routine determines the output of the deposit. A careful attention is given to the laser deposited Ti6Al4V + Cu samples under the AFM probe because of their single tracked layers with semi-circular pattern of deposition. This research work can be applicable in the surface modification of laser deposited samples for the marine industry.

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays

PubMed Central

Fixe, F.; Dufva, M.; Telleman, P.; Christensen, C. B. V.

2004-01-01

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray™ slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques. PMID:14718554

Activation of P-glycoprotein and CYP 3A by Coptidis Rhizoma in vivo: Using cyclosporine as a probe substrate in rats.

PubMed

Yu, Chung-Ping; Huang, Ching-Ya; Lin, Shiuan-Pey; Hou, Yu-Chi

2018-04-01

Coptidis Rhizoma (CR), the rhizome of Coptis chinensis FRANCH, is a popular Chinese herb. CR contains plenty of isoquinoline alkaloids such as berberine, coptisine and palmatine. Cyclosporine (CSP), an important immunosuppressant with narrow therapeutic window, is employed as a probe substrate of P-glycoprotein (P-gp) and CYP3A4 in order to investigate the in vivo modulation effect of CR on P-gp and CYP3A4. Three groups of rats were orally administered CSP without and with single dose or repeated dosing of CR in a parallel design. Blood samples were collected at specific time points and the blood CSP concentration was determined by a specific monoclonal fluorescence polarization immunoassay. The results showed that a single dose (1.0 g/kg) and the 7th dose (1.0 g/kg) of CR significantly decreased the C max of CSP by 56.9% and 70.4%, and reduced the AUC 0-540 by 56.4% and 68.7%, respectively. Cell study indicated that CR decoction, berberine, coptisine, palmatine all activated the efflux transport of P-gp. Ex-vivo study showed that the serum metabolites of CR activated CYP 3A4. In conclusion, through using CSP as an in vivo probe substrate, we have verified that oral intake of CR activated the functions of P-gp and CYP3A based on in vivo and in vitro studies. Copyright © 2017. Published by Elsevier B.V.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

PubMed

Dallas, Shannon; Salphati, Laurent; Gomez-Zepeda, David; Wanek, Thomas; Chen, Liangfu; Chu, Xiaoyan; Kunta, Jeevan; Mezler, Mario; Menet, Marie-Claude; Chasseigneaux, Stephanie; Declèves, Xavier; Langer, Oliver; Pierre, Esaie; DiLoreto, Karen; Hoft, Carolin; Laplanche, Loic; Pang, Jodie; Pereira, Tony; Andonian, Clara; Simic, Damir; Rode, Anja; Yabut, Jocelyn; Zhang, Xiaolin; Scheer, Nico

2016-05-01

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics.

PubMed

Mitchell, Gabriel J; Nelson, Daniel C; Weitz, Joshua S

2010-10-04

The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.

Selective Growth of Metallic and Semiconducting Single Walled Carbon Nanotubes on Textured Silicon.

PubMed

Jang, Mira; Lee, Jongtaek; Park, Teahee; Lee, Junyoung; Yang, Jonghee; Yi, Whikun

2016-03-01

We fabricated the etched Si substrate having the pyramidal pattern size from 0.5 to 4.2 μm by changing the texturing process parameters, i.e., KOH concentration, etching time, and temperature. Single walled carbon nanotubes (SWNTs) were then synthesized on the etched Si substrates with different pyramidal pattern by chemical vapor deposition. We investigated the optical and electronic properties of SWNT film grown on the etched Si substrates of different morphology by using scanning electron microscopy, Raman spectroscopy and conducting probe atomic force microscopy. We confirmed that the morphology of substrate strongly affected the selective growth of the SWNT film. Semiconducting SWNTs were formed on larger pyramidal sized Si wafer with higher ratio compared with SWNTs on smaller pyramidal sized Si.

Tuning Fluorescence Direction with Plasmonic Metal–Dielectric– Metal Substrates

PubMed Central

Choudhury, Sharmistha Dutta; Badugu, Ramachandram; Nowaczyk, Kazimierz; Ray, Krishanu; Lakowicz, Joseph R.

2013-01-01

Controlling the emission properties of fluorophores is essential for improving the performance of fluorescence-based techniques in modern biochemical research, medical diagnosis, and sensing. Fluorescence emission is isotropic in nature, which makes it difficult to capture more than a small fraction of the total emission. Metal– dielectric–metal (MDM) substrates, discussed in this Letter, convert isotropic fluorescence into beaming emission normal to the substrate. This improves fluorescence collection efficiency and also opens up new avenues for a wide range of fluorescence-based applications. We suggest that MDM substrates can be readily adapted for multiple uses, such as in microarray formats, for directional fluorescence studies of multiple probes or for molecule-specific sensing with a high degree of spatial control over the fluorescence emission. SECTION: Physical Processes in Nanomaterials and Nanostructures PMID:24013521

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

Bosserman, Mary A.; Downey, Theresa; Noinaj, Nicholas

Baeyer–Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C–C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed usmore » to probe the substrate binding pocket of MtmOIV and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.« less

Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.

PubMed

Andersen, Rokhsana J; Brask, Jesper

2016-06-01

Three racemic fluorogenic triglycerides are synthesized and evaluated as lipase assay substrates. The presented synthesis route goes through a key triglyceride intermediate which can be chemoselectively functionalized with a wide range of different probes. Hence the substrate can be tailor-made for a specific assay, or focus can be on low cost in larger scale for applications in high-throughput screening (HTS) assays. In the specific examples, TG-ED, TG-FD and TG-F2 are assembled with the Edans-Dabcyl or the fluorescein-Dabcyl FRET pair, or relying on fluorescein self-quenching, respectively. Proof-of-concept assays allowed determination of 1st order kinetic parameters (kcat/KM) of 460s(-1)M(-1), 59s(-1)M(-1) and 346s(-1)M(-1), respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204s(-1)M(-1). Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Performance Analysis and Modeling of Thermally Sprayed Resistive Heaters

NASA Astrophysics Data System (ADS)

Lamarre, Jean-Michel; Marcoux, Pierre; Perrault, Michel; Abbott, Richard C.; Legoux, Jean-Gabriel

2013-08-01

Many processes and systems require hot surfaces. These are usually heated using electrical elements located in their vicinity. However, this solution is subject to intrinsic limitations associated with heating element geometry and physical location. Thermally spraying electrical elements directly on surfaces can overcome these limitations by tailoring the geometry of the heating element to the application. Moreover, the element heat transfer is maximized by minimizing the distance between the heater and the surface to be heated. This article is aimed at modeling and characterizing resistive heaters sprayed on metallic substrates. Heaters were fabricated by using a plasma-sprayed alumina dielectric insulator and a wire flame-sprayed iron-based alloy resistive element. Samples were energized and kept at a constant temperature of 425 °C for up to 4 months. SEM cross-sectional observations revealed the formation of cracks at very specific locations in the alumina layer after thermal use. Finite-element modeling shows that these cracks originate from high local thermal stresses and can be predicted according to the considered geometry. The simulation model was refined using experimental parameters obtained by several techniques such as emissivity and time-dependent temperature profile (infra-red camera), resistivity (four-probe technique), thermal diffusivity (laser flash method), and mechanical properties (micro and nanoindentation). The influence of the alumina thickness and the substrate material on crack formation was evaluated.

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

Nakayama, T.; Kubo, O.; Shingaya, Y.

the research of advanced materials based on nanoscience and nanotechnology, it is often desirable to measure nanoscale local electrical conductivity at a designated position of a given sample. For this purpose, multiple-probe scanning probe microscopes (MP-SPMs), in which two, three or four scanning tunneling microscope (STM) or atomic force microscope (AFM) probes are operated independently, have been developed. Each probe in an MP-SPM is used not only for observing high-resolution STM or AFM images but also for forming an electrical contact enabling nanoscale local electrical conductivity measurement. The world's first double-probe STM (DP-STM) developed by the authors, which was subsequentlymore » modified to a triple-probe STM (TP-STM), has been used to measure the conductivities of one-dimensional metal nanowires and carbon nanotubes and also two-dimensional molecular films. A quadruple-probe STM (QP-STM) has also been developed and used to measure the conductivity of two-dimensional molecular films without the ambiguity of contact resistance between the probe and sample. Moreover, a quadruple-probe AFM (QP-AFM) with four conductive tuning-fork-type self-detection force sensing probes has been developed to measure the conductivity of a nanostructure on an insulating substrate. A general-purpose computer software to control four probes at the same time has also been developed and used in the operation of the QP-AFM. These developments and applications of MP-SPMs are reviewed in this paper.« less

Resistance irrelevant CYP417A2v2 was found degrading insecticide in Laodelphax striatellus.

PubMed

Miah, Mohammad Asaduzzaman; Elzaki, Mohammed Esmail Abdalla; Han, Zhaojun

2017-07-01

Cytochrome P450 monooxygenases (CYPs) usually overexpressed in resistant strain were found involved in oxidative detoxification of insecticides. In this study, an investigation was conducted to confirm if resistance irrelevant CYPs which were not overexpressed in resistant strain before, were capable of degrading insecticides. Three resistance irrelevant CYPs viz. CYP417A2v2, CYP425A1v2, and CYP4DJ1 from CYP4 family of Laodelphax striatellus were randomly selected for experiments. CYP417A2v2 and CYP425A1v2 were found expressed successfully in Sf9 cell line while CYP4DJ1 was not expressed successfully and out of two expressed CYPs, only CYP417A2v2 showed its efficient catalytic activity. For catalytic activity, three traditional model probe substrates and five insecticides were assayed. For the probe substrates screened, p -nitroanisole and ethoxycoumarin were preferentially metabolized by CYP417A2v2 (specific activity 3.76 ± 1.22 and 1.63 ± 0.37 nmol min -1  mg protein -1 , respectively) and they may be potential diagnostic probes for this enzyme. Among insecticides, only imidacloprid was efficiently degraded by CYP417A2v2. Incubation of imidacloprid with CYP417A2v2 of L. striatellus and subsequent HPLC, LC-MS, and MS/MS analysis revealed the formation of imidacloprid metabolites, that is, 4' or 5'hydroxy-imidacloprid by hydroxylation. This result implies the exemption of CYPs character that it is not always, all the CYPs degrading insecticides being selected and overexpressed in resistant strains and the degrading CYPs without mutations to upregulate could be candidates during insecticide resistance evolution. This characterization of individual insect CYPs in insecticide degradation can provide insight for better understand of insecticide resistance development.

The preparation method of terahertz monolithic integrated device

NASA Astrophysics Data System (ADS)

Zhang, Cong; Su, Bo; He, Jingsuo; Zhang, Hongfei; Wu, Yaxiong; Zhang, Shengbo; Zhang, Cunlin

2018-01-01

The terahertz monolithic integrated device is to integrate the pumping area of the terahertz generation, the detection area of the terahertz receiving and the metal waveguide of terahertz transmission on the same substrate. The terahertz generation and detection device use a photoconductive antenna structure the metal waveguide use a microstrip line structure. The evanescent terahertz-bandwidth electric field extending above the terahertz transmission line interacts with, and is modified by, overlaid dielectric samples, thus enabling the characteristic vibrational absorption resonances in the sample to be probed. In this device structure, since the semiconductor substrate of the photoconductive antenna is located between the strip conductor and the dielectric layer of the microstrip line, and the semiconductor substrate cannot grow on the dielectric layer directly. So how to prepare the semiconductor substrate of the photoconductive antenna and how to bond the semiconductor substrate to the dielectric layer of the microstrip line is a key step in the terahertz monolithic integrated device. In order to solve this critical problem, the epitaxial wafer structure of the two semiconductor substrates is given and transferred to the desired substrate by two methods, respectively.

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker.

PubMed

He, Dan; Xie, Xiao; Yang, Fan; Zhang, Heng; Su, Haomiao; Ge, Yun; Song, Haiping; Chen, Peng R

2017-11-13

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring.

PubMed Central

Bej, A K; McCarty, S C; Atlas, R M

1991-01-01

Multiplex polymerase chain reaction (PCR) and gene probe detection of target lacZ and uidA genes were used to detect total coliform bacteria and Escherichia coli, respectively, for determining water quality. In tests of environmental water samples, the lacZ PCR method gave results statistically equivalent to those of the plate count and defined substrate methods accepted by the U.S. Environmental Protection Agency for water quality monitoring and the uidA PCR method was more sensitive than 4-methylumbelliferyl-beta-D-glucuronide-based defined substrate tests for specific detection of E. coli. Images PMID:1768116

Long residence times revealed by Aurora A kinase-targeting fluorescent probes derived from inhibitors MLN8237 and VX-689.

PubMed

Lavogina, Darja; Enkvist, Erki; Viht, Kaido; Uri, Asko

2014-02-10

We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well-known inhibitors MLN8237 and VX-689 (MK-5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (kon 10(5)-10(7) M(-1) s(-1), koff 10(-3)-10(-4) s(-1); residence time 500-3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on-rate and increase in the off-rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX-689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Force modulation and electrochemical gating of conductance in a cytochrome

NASA Astrophysics Data System (ADS)

Davis, Jason J.; Peters, Ben; Xi, Wang

2008-09-01

Scanning probe methods have been used to measure the effect of electrochemical potential and applied force on the tunnelling conductance of the redox metalloprotein yeast iso-1-cytochrome c (YCC) at a molecular level. The interaction of a proximal probe with any sample under test will, at this scale, be inherently perturbative. This is demonstrated with conductive probe atomic force microscopy (CP-AFM) current-voltage spectroscopy in which YCC, chemically adsorbed onto pristine Au(111) via its surface cysteine residue, is observed to become increasingly compressed as applied load is increased, with concomitant decrease in junction resistance. Electrical contact at minimal perturbation, where probe-molecule coupling is comparable to that in scanning tunnelling microscopy, brings with it the observation of negative differential resistance, assigned to redox-assisted probe-substrate tunnelling. The role of the redox centre in conductance is also resolved in electrochemical scanning tunnelling microscopy assays where molecular conductance is electrochemically gateable through more than an order of magnitude.

In situ identification of the synthrophic protein fermentative Coprothermobacter spp. involved in the thermophilic anaerobic digestion process.

PubMed

Gagliano, Maria Cristina; Braguglia, Camilla Maria; Rossetti, Simona

2014-09-01

Thermophilic bacteria have recently attracted great attention because of their potential application in improving different biochemical processes such as anaerobic digestion of various substrates, wastewater treatment or hydrogen production. In this study we report on the design of a specific 16S rRNA-targeted oligonucleotide probe for detecting members of Coprothermobacter genus characterized by a strong protease activity to degrade proteins and peptides. The newly designed CTH485 probe and helper probes hCTH429 and hCTH439 were optimized for use in fluorescence in situ hybridization (FISH) on thermophilic anaerobic sludge samples. In situ probing revealed that thermo-adaptive mechanisms shaping the 16S rRNA gene may affect the identification of thermophilic microorganisms. The novel developed FISH probe extends the possibility to study the widespread thermophilic syntrophic interaction of Coprothermobacter spp. with hydrogenotrophic methanogenic archaea, whose establishment is a great benefit for the whole anaerobic system. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Influence of surface topology and electrostatic potential on water/electrode systems

NASA Astrophysics Data System (ADS)

Siepmann, J. Ilja; Sprik, Michiel

1995-01-01

We have used the classical molecular dynamics technique to simulate the ordering of a water film adsorbed on an atomic model of a tip of a scanning tunneling microscope approaching a planar metal surface. For this purpose, we have developed a classical model for the water-substrate interactions that solely depends on the coordinates of the particles and does not require the definition of geometrically smooth boundary surfaces or image planes. The model includes both an electrostatic induction for the metal atoms (determined by means of an extended Lagrangian technique) and a site-specific treatment of the water-metal chemisorption. As a validation of the model we have investigated the structure of water monolayers on metal substrates of various topology [the (111), (110), and (100) crystallographic faces] and composition (Pt, Ag, Cu, and Ni), and compared the results to experiments. The modeling of the electrostatic induction is compatible with a finite external potential imposed on the metal. This feature is used to investigate the structural rearrangements of the water bilayer between the pair of scanning tunneling microscope electrodes in response to an applied external voltage difference. We find significant asymmetry in the dependence on the sign of the applied voltage. Another result of the calculation is an estimate of the perturbation to the work function caused by the wetting film. For the conditions typical for operation of a scanning tunneling microscope probe, the change in the work function is found to be comparable to the applied voltage (a few hundred millivolts).

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

GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

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

Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

1994-09-01

GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by amore » sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.« less

Assessing Telomere Length Using Surface Enhanced Raman Scattering

NASA Astrophysics Data System (ADS)

Zong, Shenfei; Wang, Zhuyuan; Chen, Hui; Cui, Yiping

2014-11-01

Telomere length can provide valuable insight into telomeres and telomerase related diseases, including cancer. Here, we present a brand-new optical telomere length measurement protocol using surface enhanced Raman scattering (SERS). In this protocol, two single strand DNA are used as SERS probes. They are labeled with two different Raman molecules and can specifically hybridize with telomeres and centromere, respectively. First, genome DNA is extracted from cells. Then the telomere and centromere SERS probes are added into the genome DNA. After hybridization with genome DNA, excess SERS probes are removed by magnetic capturing nanoparticles. Finally, the genome DNA with SERS probes attached is dropped onto a SERS substrate and subjected to SERS measurement. Longer telomeres result in more attached telomere probes, thus a stronger SERS signal. Consequently, SERS signal can be used as an indicator of telomere length. Centromere is used as the inner control. By calibrating the SERS intensity of telomere probe with that of the centromere probe, SERS based telomere measurement is realized. This protocol does not require polymerase chain reaction (PCR) or electrophoresis procedures, which greatly simplifies the detection process. We anticipate that this easy-operation and cost-effective protocol is a fine alternative for the assessment of telomere length.

Subcellular localization of rat CYP2E1 impacts metabolic efficiency toward common substrates.

PubMed

Hartman, Jessica H; Martin, H Cass; Caro, Andres A; Pearce, Amy R; Miller, Grover P

2015-12-02

Cytochrome P450 2E1 (CYP2E1) detoxifies or bioactivates many low molecular-weight compounds. Most knowledge about CYP2E1 activity relies on studies of the enzyme localized to endoplasmic reticulum (erCYP2E1); however, CYP2E1 undergoes transport to mitochondria (mtCYP2E1) and becomes metabolically active. We report the first comparison of in vitro steady-state kinetic profiles for erCYP2E1 and mtCYP2E1 oxidation of probe substrate 4-nitrophenol and pollutants styrene and aniline using subcellular fractions from rat liver. For all substrates, metabolic efficiency changed with substrate concentration for erCYP2E1 reflected in non-hyperbolic kinetic profiles but not for mtCYP2E1. Hyperbolic kinetic profiles for the mitochondrial enzyme were consistent with Michaelis-Menten mechanism in which metabolic efficiency was constant. By contrast, erCYP2E1 metabolism of 4-nitrophenol led to a loss of enzyme efficiency at high substrate concentrations when substrate inhibited the reaction. Similarly, aniline metabolism by erCYP2E1 demonstrated negative cooperativity as metabolic efficiency decreased with increasing substrate concentration. The opposite was observed for erCYP2E1 oxidation of styrene; the sigmoidal kinetic profile indicated increased efficiency at higher substrate concentrations. These mechanisms and CYP2E1 levels in mitochondria and endoplasmic reticulum were used to estimate the impact of CYP2E1 subcellular localization on metabolic flux of pollutants. Those models showed that erCYP2E1 mainly carries out aniline metabolism at all aniline concentrations. Conversely, mtCYP2E1 dominates styrene oxidation at low styrene concentrations and erCYP2E1 at higher concentrations. Taken together, subcellular localization of CYP2E1 results in distinctly different enzyme activities that could impact overall metabolic clearance and/or activation of substrates and thus impact the interpretation and prediction of toxicological outcomes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Plasma monitoring of the RLVIP-process with a Langmuir probe

NASA Astrophysics Data System (ADS)

Huber, D.; Hallbauer, A.; Pulker, H. K.

2005-09-01

The aim of this investigation was to study the characteristics of a reactive-low-voltage-high-current-ion-plating plasma and to correlate the observed plasma data with the properties of films deposited under such conditions. A Langmuir probe system (Smart Probe - Scientific Systems) was inserted into a Balzers BAP 800 ion plating plant above the e-gun evaporation source close to the insulated substrate holder. In this position during RLVIP deposition, plasma potential, floating potential, self-bias voltage, electron temperature, ion current density, and particle number density were measured and calculated, respectively. All measurements were performed in dependence of arc current (20-80A) and oxygen partial pressure (1 - 36 x 10-4mbar). With rising arc current the number of charged particles, the self-bias voltage between plasma and substrates as well as the energy of the condensing and bombarding species were increased. These data explain the increase of density, refractive index and mechanical stress of RLVIP-metal-oxide-layers, like Ta2O5 and Nb2O5, deposited with higher arc currents. An increase of gas pressure decreased the energy of the particles and therefore reduced slightly film density and refractive index. However, it improved chemistry and eliminated unwanted residual optical absorption and also decreased compressive mechanical film stress.

Conductive scanning probe microscopy of the semicontinuous gold film and its SERS enhancement toward two-step photo-induced charge transfer and effect of the supportive layer

NASA Astrophysics Data System (ADS)

Sinthiptharakoon, K.; Sapcharoenkun, C.; Nuntawong, N.; Duong, B.; Wutikhun, T.; Treetong, A.; Meemuk, B.; Kasamechonchung, P.; Klamchuen, A.

2018-05-01

The semicontinuous gold film, enabling various electronic applications including development of surface-enhanced Raman scattering (SERS) substrate, is investigated using conductive atomic force microscopy (CAFM) and Kelvin probe force microscopy (KPFM) to reveal and investigate local electronic characteristics potentially associated with SERS generation of the film material. Although the gold film fully covers the underlying silicon surface, CAFM results reveal that local conductivity of the film is not continuous with insulating nanoislands appearing throughout the surface due to incomplete film percolation. Our analysis also suggests the two-step photo-induced charge transfer (CT) play the dominant role in the enhancement of SERS intensity with strong contribution from free electrons of the silicon support. Silicon-to-gold charge transport is illustrated by KPFM results showing that Fermi level of the gold film is slightly inhomogeneous and far below the silicon conduction band. We propose that inhomogeneity of the film workfunction affecting chemical charge transfer between gold and Raman probe molecule is associated with the SERS intensity varying across the surface. These findings provide deeper understanding of charge transfer mechanism for SERS which can help in design and development of the semicontinuous gold film-based SERS substrate and other electronic applications.

Urokinase injection-triggered clearance enhancement of a 4-arm PEG-conjugated 64Cu-bombesin analog tetramer: A novel approach for the improvement of PET imaging contrast.

PubMed

Matsumura, Kazushi; Zouda, Maki; Wada, Yasuhiro; Yamashita, Fumiyoshi; Hashida, Mitsuru; Watanabe, Yasuyoshi; Mukai, Hidefumi

2018-05-07

Radiolabeled antibodies, polyethylene glycol-conjugated (PEGylated) peptides, liposomes, and other materials were investigated as positron-emission tomography (PET) probes. These substances accumulate in tumors but often remain too long in circulation. We investigated the combination of intravenous urokinase injection and its substrate linker as a triggered radioisotope clearance enhancement system to improve imaging contrast. To this end, we synthesized a four-arm PEGylated 64 Cu-bombesin analog tetramer with a urokinase substrate linker. In mouse blood, it was almost perfectly cleaved and degraded into smaller radioactive fragments in vitro with urokinase (≥20,000 IU/mL). In mouse blood circulation, ∼50-65% of the probe was rapidly degraded after the urokinase injection and the radioactive fragments were eliminated mainly from the kidney. In contrast, tumor radioactivity levels did not change, and therefore, the tumors were clearly visualized. The tumor/blood ratio, an indicator of imaging contrast, increased 2.5 times, while elimination of the radioisotope from the blood was enhanced. This approach has the potential to improve imaging contrast using various PET probes. It could also shorten the time required to obtain sufficient contrast and decrease patient radiation exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

Study of adhesion and friction properties on a nanoparticle gradient surface: transition from JKR to DMT contact mechanics.

PubMed

Ramakrishna, Shivaprakash N; Nalam, Prathima C; Clasohm, Lucy Y; Spencer, Nicholas D

2013-01-08

We have previously investigated the dependence of adhesion on nanometer-scale surface roughness by employing a roughness gradient. In this study, we correlate the obtained adhesion forces on nanometer-scale rough surfaces to their frictional properties. A roughness gradient with varying silica particle (diameter ≈ 12 nm) density was prepared, and adhesion and frictional forces were measured across the gradient surface in perfluorodecalin by means of atomic force microscopy with a polyethylene colloidal probe. Similarly to the pull-off measurements, the frictional forces initially showed a reduction with decreasing particle density and later an abrupt increase as the colloidal sphere began to touch the flat substrate beneath, at very low particle densities. The friction-load relation is found to depend on the real contact area (A(real)) between the colloid probe and the underlying particles. At high particle density, the colloidal sphere undergoes large deformations over several nanoparticles, and the contact adhesion (JKR type) dominates the frictional response. However, at low particle density (before the colloidal probe is in contact with the underlying surface), the colloidal sphere is suspended by a few particles only, resulting in local deformations of the colloid sphere, with the frictional response to the applied load being dominated by long-range, noncontact (DMT-type) interactions with the substrate beneath.

Probing long-range structural order in SnPc/Ag(111) by umklapp process assisted low-energy angle-resolved photoelectron spectroscopy

NASA Astrophysics Data System (ADS)

Jauernik, Stephan; Hein, Petra; Gurgel, Max; Falke, Julian; Bauer, Michael

2018-03-01

Laser-based angle-resolved photoelectron spectroscopy is performed on tin-phthalocyanine (SnPc) adsorbed on silver Ag(111). Upon adsorption of SnPc, strongly dispersing bands are observed which are identified as secondary Mahan cones formed by surface umklapp processes acting on photoelectrons from the silver substrate as they transit through the ordered adsorbate layer. We show that the photoemission data carry quantitative structural information on the adsorbate layer similar to what can be obtained from a conventional low-energy electron diffraction (LEED) study. More specifically, we compare photoemission data and LEED data probing an incommensurate-to-commensurate structural phase transition of the adsorbate layer. Based on our results we propose that Mahan-cone spectroscopy operated in a pump-probe configuration can be used in the future to probe structural dynamics at surfaces with a temporal resolution in the sub-100-fs regime.

Revealing Nanostructures through Plasmon Polarimetry.

PubMed

Kleemann, Marie-Elena; Mertens, Jan; Zheng, Xuezhi; Cormier, Sean; Turek, Vladimir; Benz, Felix; Chikkaraddy, Rohit; Deacon, William; Lombardi, Anna; Moshchalkov, Victor V; Vandenbosch, Guy A E; Baumberg, Jeremy J

2017-01-24

Polarized optical dark-field spectroscopy is shown to be a versatile noninvasive probe of plasmonic structures that trap light to the nanoscale. Clear spectral polarization splittings are found to be directly related to the asymmetric morphology of nanocavities formed between faceted gold nanoparticles and an underlying gold substrate. Both experiment and simulation show the influence of geometry on the coupled system, with spectral shifts Δλ = 3 nm from single atoms. Analytical models allow us to identify the split resonances as transverse cavity modes, tightly confined to the nanogap. The direct correlation of resonance splitting with atomistic morphology allows mapping of subnanometre structures, which is crucial for progress in extreme nano-optics involving chemistry, nanophotonics, and quantum devices.

Finite Element Analysis of Surface Residual Stress in Functionally Gradient Cemented Carbide Tool

NASA Astrophysics Data System (ADS)

Su, Chuangnan; Liu, Deshun; Tang, Siwen; Li, Pengnan; Qiu, Xinyi

2018-03-01

A component distribution model is proposed for three-component functionally gradient cemented carbide (FGCC) based on electron probe microanalysis results obtained for gradient layer thickness, microstructure, and elemental distribution. The residual surface stress of FGCC-T5 tools occurring during the fabrication process is analyzed using an ANSYS-implemented finite element method (FEM) and X-ray diffraction. A comparison of the experimental and calculated values verifies the feasibility of using FEM to analyze the residual surface stress in FGCC-T5 tools. The effects of the distribution index, geometrical shape, substrate thickness, gradient layer thickness, and position of the cobalt-rich layer on residual surface stress are studied in detail.

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.

PubMed

Zhong, Wei; Kuntz, Douglas A; Ember, Brian; Singh, Harminder; Moremen, Kelley W; Rose, David R; Boons, Geert-Jan

2008-07-16

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.

LTCC-based differential photo acoustic cell for ppm gas sensing

NASA Astrophysics Data System (ADS)

Karioja, P.; Keränen, K.; Kautio, K.; Ollila, J.; Heikkinen, M.; Kauppinen, I.; Kuusela, T.; Matveev, B.; McNie, M. E.; Jenkins, R. M.; Palve, J.

2010-04-01

Silicon MEMS cantilever-based photoacoustic technology allows for the sensing of ultra low gas concentrations with very wide dynamic range. The sensitivity enhancement is achieved with a cantilever microphone system in which the cantilever displacement is probed with an optical interferometer providing a pico-meter resolution. In the gas sensor, the silicon cantilever microphone is placed in a two-chamber differential gas cell. By monitoring differential pressure changes between the two chambers, the differential cell operates as a differential infra-red detector for optical absorption signals through a measurement and reference path. The differential pressure signal is proportional to gas concentration in the optical measurement path. We have designed, implemented and tested a differential photo acoustic gas cell based on Low Temperature Co-fired Ceramic (LTCC) multilayer substrate technology. Standard LTCC technology enables implementation of 2.5D structures including holes, cavities and channels into the electronic substrate. The implemented differential photoacoustic gas cell structure includes two 10 mm long cylindrical cells, diameter of 2.4 mm. Reflectance measurements of the cell showed that reflectivity of the substrate material can be improved by a factor 15 - 90 in the 3 - 8 μm spectral region using gold or silver paste coatings. A transparent window is required in the differential gas cell structure in order to probe the displacement of the silicon cantilever. The transparent sapphire window was sealed to the LTCC substrate using two methods: screen printed Au80/Sn20 solder paste and pre-attached glass solder paste (Diemat DM2700P/H848). Both methods were shown to provide hermetic sealing of sapphire windows to LTCC substrate. The measured He-leak rate for the 10 sealed test samples implemented using glass paste were less than 2.0 ×10-9 atm×cm3/s, which meets the requirement for the leak rate according to MIL-STD 883. The achieved hermetic level suggests that the proof-of-principle packaging demonstrator paves the way for implementing a novel differential photoacoustic gas cell for a future miniature gas sensor module. The future module consisting of a sample gas cell and immersion lens IR-LEDs together with interferometric probing of the cantilever microphone is expected to be capable of measuring ultra low concentrations of a wide range of gases with their fundamental absorption bands at 3 - 7 μm wavelength, such as CO, CO2 and CH4.

Sensitive and fast detection of fructose in complex media via symmetry breaking and signal amplification using surface-enhanced Raman spectroscopy.

PubMed

Sun, Fang; Bai, Tao; Zhang, Lei; Ella-Menye, Jean-Rene; Liu, Sijun; Nowinski, Ann K; Jiang, Shaoyi; Yu, Qiuming

2014-03-04

A new strategy is proposed to sensitively and rapidly detect analytes with weak Raman signals in complex media using surface-enhanced Raman spectroscopy (SERS) via detecting the SERS signal changes of the immobilized probe molecules on SERS-active substrates upon binding of the analytes. In this work, 4-mercaptophenylboronic acid (4-MPBA) was selected as the probe molecule which was immobilized on the gold surface of a quasi-three-dimensional plasmonic nanostructure array (Q3D-PNA) SERS substrate to detect fructose. The molecule of 4-MPBA possesses three key functions: molecule recognition and reversible binding of the analyte via the boronic acid group, amplification of SERS signals by the phenyl group and thus shielding of the background noise of complex media, and immobilization on the surface of SERS-active substrates via the thiol group. Most importantly, the symmetry breaking of the 4-MPBA molecule upon fructose binding leads to the change of area ratio between totally symmetric 8a ring mode and nontotally symmetric 8b ring mode, which enables the detection. The detection curves were obtained in phosphate-buffered saline (PBS) and in undiluted artificial urine at clinically relevant concentrations, and the limit of detection of 0.05 mM was achieved.

Effect of surface roughness and subsurface damage on grazing-incidence x-ray scattering and specular reflectance.

PubMed

Lodha, G S; Yamashita, K; Kunieda, H; Tawara, Y; Yu, J; Namba, Y; Bennett, J M

1998-08-01

Grazing-incidence specular reflectance and near-specular scattering were measured at Al-K(alpha) (1.486-keV, 8.34-?) radiation on uncoated dielectric substrates whose surface topography had been measured with a scanning probe microscope and a mechanical profiler. Grazing-incidence specular reflectance was also measured on selected substrates at the Cu-K(alpha) (8.047-keV, 1.54-?) wavelength. Substrates included superpolished and conventionally polished fused silica; SiO(2) wafers; superpolished and precision-ground Zerodur; conventionally polished, float-polished, and precision-ground BK-7 glass; and superpolished and precision-ground silicon carbide. Roughnesses derived from x-ray specular reflectance and scattering measurements were in good agreement with topographic roughness values measured with a scanning probe microscope (atomic force microscope) and a mechanical profiler that included similar ranges of surface spatial wavelengths. The specular reflectance was also found to be sensitive to the density of polished surface layers and subsurface damage down to the penetration depth of the x rays. Density gradients and subsurface damage were found in the superpolished fused-silica and precision-ground Zerodur samples. These results suggest that one can nondestructively evaluate subsurface damage in transparent materials using grazing-incidence x-ray specular reflectance in the 1.5-8-keV range.

Link between microbial composition and carbon substrate-uptake preferences in a PHA-storing community

PubMed Central

Albuquerque, Maria G E; Carvalho, Gilda; Kragelund, Caroline; Silva, Ana F; Barreto Crespo, Maria T; Reis, Maria A M; Nielsen, Per H

2013-01-01

The microbial community of a fermented molasses-fed sequencing batch reactor (SBR) operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was identified and quantified through a 16 S rRNA gene clone library and fluorescence in situ hybridization (FISH). The microbial enrichment was found to be composed of PHA-storing populations (84% of the microbial community), comprising members of the genera Azoarcus, Thauera and Paracoccus. The dominant PHA-storing populations ensured the high functional stability of the system (characterized by high PHA-storage efficiency, up to 60% PHA content). The fermented molasses contained primarily acetate, propionate, butyrate and valerate. The substrate preferences were determined by microautoradiography-FISH and differences in the substrate-uptake capabilities for the various probe-defined populations were found. The results showed that in the presence of multiple substrates, microbial populations specialized in different substrates were selected, thereby co-existing in the SBR by adapting to different niches. Azoarcus and Thauera, primarily consumed acetate and butyrate, respectively. Paracoccus consumed a broader range of substrates and had a higher cell-specific substrate uptake. The relative species composition and their substrate specialization were reflected in the substrate removal rates of different volatile fatty acids in the SBR reactor. PMID:22810062

Controlled Zn-mediated grafting of thin layers of bipodal diazonium salt on gold and carbon substrates.

PubMed

Torréns, Mabel; Ortiz, Mayreli; Turner, Anthony P F; Beni, Valerio; O'Sullivan, Ciara K

2015-01-07

A controlled, rapid, and potentiostat-free method has been developed for grafting the diazonium salt (3,5-bis(4-diazophenoxy)benzoic acid tetrafluoroborate (DCOOH)) on gold and carbon substrates, based on a Zn-mediated chemical dediazonation. The highly stable thin layer organic platforms obtained were characterized by cyclic voltammetry, AFM, impedance, XP, and Raman spectroscopies. A dediazonation mechanism based on radical formation is proposed. Finally, DCOOH was proved as a linker to an aminated electroactive probe. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thin film absorption characterization by focus error thermal lensing

NASA Astrophysics Data System (ADS)

Domené, Esteban A.; Schiltz, Drew; Patel, Dinesh; Day, Travis; Jankowska, E.; Martínez, Oscar E.; Rocca, Jorge J.; Menoni, Carmen S.

2017-12-01

A simple, highly sensitive technique for measuring absorbed power in thin film dielectrics based on thermal lensing is demonstrated. Absorption of an amplitude modulated or pulsed incident pump beam by a thin film acts as a heat source that induces thermal lensing in the substrate. A second continuous wave collimated probe beam defocuses after passing through the sample. Determination of absorption is achieved by quantifying the change of the probe beam profile at the focal plane using a four-quadrant detector and cylindrical lenses to generate a focus error signal. This signal is inherently insensitive to deflection, which removes noise contribution from point beam stability. A linear dependence of the focus error signal on the absorbed power is shown for a dynamic range of over 105. This technique was used to measure absorption loss in dielectric thin films deposited on fused silica substrates. In pulsed configuration, a single shot sensitivity of about 20 ppm is demonstrated, providing a unique technique for the characterization of moving targets as found in thin film growth instrumentation.

Effect of dopant density on contact potential difference across n-type GaAs homojunctions using Kelvin Probe Force Microscopy

NASA Astrophysics Data System (ADS)

Boumenou, C. Kameni; Urgessa, Z. N.; Djiokap, S. R. Tankio; Botha, J. R.; Nel, J.

2018-04-01

In this study, cross-sectional surface potential imaging of n+/semi-insulating GaAs junctions is investigated by using amplitude mode kelvin probe force microscopy. The measurements have shown two different potential profiles, related to the difference in surface potential between the semi-insulating (SI) substrate and the epilayers. It is shown that the contact potential difference (CPD) between the tip and the sample is higher on the semi-insulating substrate side than on the n-type epilayer side. This change in CPD across the interface has been explained by means of energy band diagrams indicating the relative Fermi level positions. In addition, it has also been found that the CPD values across the interface are much smaller than the calculated values (on average about 25% of the theoretical values) and increase with the electron density. Therefore, the results presented in study are only in qualitative agreement with the theory.

Designing topological defects in 2D materials using scanning probe microscopy and a self-healing mechanism: a density functional-based molecular dynamics study

NASA Astrophysics Data System (ADS)

Popov, Igor; Đurišić, Ivana; Belić, Milivoj R.

2017-12-01

Engineering of materials at the atomic level is one of the most important aims of nanotechnology. The unprecedented ability of scanning probe microscopy to address individual atoms opened up the possibilities for nanomanipulation and nanolitography of surfaces and later on of two-dimensional materials. While the state-of-the-art scanning probe lithographic methods include, primarily, adsorption, desorption and repositioning of adatoms and molecules on substrates or tailoring nanoribbons by etching of trenches, the precise modification of the intrinsic atomic structure of materials is yet to be advanced. Here we introduce a new concept, scanning probe microscopy with a rotating tip, for engineering of the atomic structure of membranes based on two-dimensional materials. In order to indicate the viability of the concept, we present our theoretical research, which includes atomistic modeling, molecular dynamics simulations, Fourier analysis and electronic transport calculations. While stretching can be employed for fabrication of atomic chains only, our comprehensive molecular dynamics simulations indicate that nanomanipulation by scanning probe microscopy with a rotating tip is capable of assembling a wide range of topological defects in two-dimensional materials in a rather controllable and reproducible manner. We analyze two possibilities. In the first case the probe tip is retracted from the membrane while in the second case the tip is released beneath the membrane allowing graphene to freely relax and self-heal the pore made by the tip. The former approach with the tip rotation can be achieved experimentally by rotation of the sample, which is equivalent to rotation of the tip, whereas irradiation of the membrane by nanoclusters can be utilized for the latter approach. The latter one has the potential to yield a yet richer diversity of topological defects on account of a lesser determinacy. If successfully realized experimentally the concept proposed here could be an important step toward controllable nanostructuring of two-dimensional materials.

Designing topological defects in 2D materials using scanning probe microscopy and a self-healing mechanism: a density functional-based molecular dynamics study.

PubMed

Popov, Igor; Đurišić, Ivana; Belić, Milivoj R

2017-12-08

Engineering of materials at the atomic level is one of the most important aims of nanotechnology. The unprecedented ability of scanning probe microscopy to address individual atoms opened up the possibilities for nanomanipulation and nanolitography of surfaces and later on of two-dimensional materials. While the state-of-the-art scanning probe lithographic methods include, primarily, adsorption, desorption and repositioning of adatoms and molecules on substrates or tailoring nanoribbons by etching of trenches, the precise modification of the intrinsic atomic structure of materials is yet to be advanced. Here we introduce a new concept, scanning probe microscopy with a rotating tip, for engineering of the atomic structure of membranes based on two-dimensional materials. In order to indicate the viability of the concept, we present our theoretical research, which includes atomistic modeling, molecular dynamics simulations, Fourier analysis and electronic transport calculations. While stretching can be employed for fabrication of atomic chains only, our comprehensive molecular dynamics simulations indicate that nanomanipulation by scanning probe microscopy with a rotating tip is capable of assembling a wide range of topological defects in two-dimensional materials in a rather controllable and reproducible manner. We analyze two possibilities. In the first case the probe tip is retracted from the membrane while in the second case the tip is released beneath the membrane allowing graphene to freely relax and self-heal the pore made by the tip. The former approach with the tip rotation can be achieved experimentally by rotation of the sample, which is equivalent to rotation of the tip, whereas irradiation of the membrane by nanoclusters can be utilized for the latter approach. The latter one has the potential to yield a yet richer diversity of topological defects on account of a lesser determinacy. If successfully realized experimentally the concept proposed here could be an important step toward controllable nanostructuring of two-dimensional materials.

Identification of genes and gene pathways associated with major depressive disorder by integrative brain analysis of rat and human prefrontal cortex transcriptomes

PubMed Central

Malki, K; Pain, O; Tosto, M G; Du Rietz, E; Carboni, L; Schalkwyk, L C

2015-01-01

Despite moderate heritability estimates, progress in uncovering the molecular substrate underpinning major depressive disorder (MDD) has been slow. In this study, we used prefrontal cortex (PFC) gene expression from a genetic rat model of MDD to inform probe set prioritization in PFC in a human post-mortem study to uncover genes and gene pathways associated with MDD. Gene expression differences between Flinders sensitive (FSL) and Flinders resistant (FRL) rat lines were statistically evaluated using the RankProd, non-parametric algorithm. Top ranking probe sets in the rat study were subsequently used to prioritize orthologous selection in a human PFC in a case–control post-mortem study on MDD from the Stanley Brain Consortium. Candidate genes in the human post-mortem study were then tested against a matched control sample using the RankProd method. A total of 1767 probe sets were differentially expressed in the PFC between FSL and FRL rat lines at (q⩽0.001). A total of 898 orthologous probe sets was found on Affymetrix's HG-U95A chip used in the human study. Correcting for the number of multiple, non-independent tests, 20 probe sets were found to be significantly dysregulated between human cases and controls at q⩽0.05. These probe sets tagged the expression profile of 18 human genes (11 upregulated and seven downregulated). Using an integrative rat–human study, a number of convergent genes that may have a role in pathogenesis of MDD were uncovered. Eighty percent of these genes were functionally associated with a key stress response signalling cascade, involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), AP-1 (activator protein 1) and ERK/MAPK, which has been systematically associated with MDD, neuroplasticity and neurogenesis. PMID:25734512

Power Flow Angles for Slanted Finger Surface Acoustic Wave Filters on Langasite Substrate

NASA Astrophysics Data System (ADS)

Goto, Mikihiro; Yatsuda, Hiromi; Chiba, Takao

2007-07-01

Power flow angles (PFAs) on a langasite (LGS) substrate with Euler angles of (0{\\degree}, 138.5{\\degree}, \\psi), \\psi=25.7 to 27.7° are investigated for slanted finger interdigital transducer (SFIT) surface acoustic wave (SAW) filters by an electrical and optical methods. In the electrical method, several tilted SFIT SAW filters with different tilt angles for (0{\\degree}, 138.5{\\degree}, \\psi) LGS substrates were designed, and the frequency responses of the filters were measured. In the optical method, the PFAs were directly measured by optical probing for a parallel interdigital transducer (IDT) with wide propagation area on the substrate. As a result, a good correlation between electrical and optical measurements of the PFAs is obtained, but the calculated PFAs are slightly different from the measured PFAs. A good frequency response of a tilted 380 MHz SFIT SAW filter with an appropriate tilt angle corresponding to the PFA on the substrate is obtained even though the aperture is small.

Engineered jadomycin analogues with altered sugar moieties revealing JadS as a substrate flexible O-glycosyltransferase.

PubMed

Li, Liyuan; Pan, Guohui; Zhu, Xifen; Fan, Keqiang; Gao, Wubin; Ai, Guomin; Ren, Jinwei; Shi, Mingxin; Olano, Carlos; Salas, José A; Yang, Keqian

2017-07-01

Glycosyltransferases (GTs)-mediated glycodiversification studies have drawn significant attention recently, with the goal of generating bioactive compounds with improved pharmacological properties by diversifying the appended sugars. The key to achieving glycodiversification is to identify natural and/or engineered flexible GTs capable of acting upon a broad range of substrates. Here, we report the use of a combinatorial biosynthetic approach to probe the substrate flexibility of JadS, the GT in jadomycin biosynthesis, towards different non-native NDP-sugar substrates, enabling us to identify six jadomycin B analogues with different sugar moieties. Further structural engineering by precursor-directed biosynthesis allowed us to obtain 11 new jadomycin analogues. Our results for the first time show that JadS is a flexible O-GT that can utilize both L- and D- sugars as donor substrates, and tolerate structural changes at the C2, C4 and C6 positions of the sugar moiety. JadS may be further exploited to generate novel glycosylated jadomycin molecules in future glycodiversification studies.

Probing metabolic processes of intact soil microbial communities using position-specific 13C-labeled glucose

NASA Astrophysics Data System (ADS)

Fairbanks, D. E.; Hungate, B. A.; KOCH, G. W.; Schwartz, E.; Dijkstra, P.

2012-12-01

Soils represent one of the largest carbon pools in the terrestrial biosphere and fluxes into or out of this pool may feedback to current climate change. Understanding the mechanisms behind microbial processes regulating C cycling, microbial turnover, and soil organic matter stabilization is hindered by our lack of understanding of the details of microbial physiology in soils. Position-specific 13C labeled metabolic tracers are proposed as a new way to probe microbial community energy production, biosynthesis, C use efficiency (the proportion of substrate incorporated into microbial biomass), and enables the determination of C fluxes through the various C metabolic pathways. We determined the 13CO2 production from microbial communities within a one hour time frame by adding six isotopomers (1-13C, 2-13C, 3-13C, 4-13C, 5-13C, 6-13C) of glucose in parallel incubations using a young volcanic soil (Pinyon-juniper wood, near Sunset Crater, Flagstaff, Arizona). We compared the measured rates of position-specific 13CO2 production with modeled results based on glucose (1-13C and U-13C) and pyruvate (1-13C and 2,3-13C) incubations. These labeling and modeling techniques may improve our ability to analyze the biochemistry and ecophysiology of intact soil microbial communities.

On the use of SPM to probe the interplay between polymer surface chemistry and polymer surface mechanics

NASA Astrophysics Data System (ADS)

Brogly, Maurice; Noel, Olivier; Awada, Houssein; Castelein, Gilles

2007-03-01

Adhesive properties of a polymer surface results from the complex contribution of surface chemistry and activation of sliding and dissipating mechanisms within the polymer surface layer. The purpose of this study is to dissociate the different contributions (chemical and mechanical) included in an AFM force-distance curve in order to establish relationships between the surface viscoelastic properties of the polymer, the surface chemistry of functionalized polymer surfaces and the adhesive forces, as determined by C-AFM experiments. Indeed we are interested in the measurements of local attractive or adhesive forces in AFM contact mode, of controlled chemical and mechanical model substrates. In order to investigate the interplay between mechanical or viscoelastic mechanisms and surface chemistry during the tip - polymer contact, we achieved force measurements on model PDMS polymer networks, whose surfaces are chemically controlled with the same functional groups as before (silicon substrates). On the basis of AFM nano-indentation experiments, surface Young moduli have been determined. The results show that the viscoelastic contribution is dominating in the adhesion force measurement. We propose an original model, which express the local adhesion force to the energy dissipated within the contact and the surface properties of the material (thermodynamic work of adhesion). Moreover we show that the dissipation function is related to Mc, the mass between crosslinks of the network.

Electric-field distribution near rectangular microstrip radiators for hyperthermia heating: theory versus experiment in water.

PubMed

Underwood, H R; Peterson, A F; Magin, R L

1992-02-01

A rectangular microstrip antenna radiator is investigated for its near-zone radiation characteristics in water. Calculations of a cavity model theory are compared with the electric-field measurements of a miniature nonperturbing diode-dipole E-field probe whose 3 mm tip was positioned by an automatic three-axis scanning system. These comparisons have implications for the use of microstrip antennas in a multielement microwave hyperthermia applicator. Half-wavelength rectangular microstrip patches were designed to radiate in water at 915 MHz. Both low (epsilon r = 10) and high (epsilon r = 85) dielectric constant substrates were tested. Normal and tangential components of the near-zone radiated electric field were discriminated by appropriate orientation of the E-field probe. Low normal to transverse electric-field ratios at 3.0 cm depth indicate that the radiators may be useful for hyperthermia heating with an intervening water bolus. Electric-field pattern addition from a three-element linear array of these elements in water indicates that phase and amplitude adjustment can achieve some limited control over the distribution of radiated power.

Probing Mechanoregulation of Neuronal Differentiation by Plasma Lithography Patterned Elastomeric Substrates

NASA Astrophysics Data System (ADS)

Nam, Ki-Hwan; Jamilpour, Nima; Mfoumou, Etienne; Wang, Fei-Yue; Zhang, Donna D.; Wong, Pak Kin

2014-11-01

Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions.

Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

PubMed

Fellner, Matthias; Aloi, Sekotilani; Tchesnokov, Egor P; Wilbanks, Sigurd M; Jameson, Guy N L

2016-03-08

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

PubMed

Stawikowski, Maciej J; Stawikowska, Roma; Fields, Gregg B

2015-05-19

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

Combining Solvent Isotope Effects with Substrate Isotope Effects in Mechanistic Studies of Alcohol and Amine Oxidation by Enzymes*

PubMed Central

Fitzpatrick, Paul F.

2014-01-01

Oxidation of alcohols and amines is catalyzed by multiple families of flavin-and pyridine nucleotide-dependent enzymes. Measurement of solvent isotope effects provides a unique mechanistic probe of the timing of the cleavage of the OH and NH bonds, necessary information for a complete description of the catalytic mechanism. The inherent ambiguities in interpretation of solvent isotope effects can be significantly decreased if isotope effects arising from isotopically labeled substrates are measured in combination with solvent isotope effects. The application of combined solvent and substrate (mainly deuterium) isotope effects to multiple enzymes is described here to illustrate the range of mechanistic insights that such an approach can provide. PMID:25448013

Transmission X-ray scattering as a probe for complex liquid-surface structures

DOE PAGES

Fukuto, Masafumi; Yang, Lin; Nykypanchuk, Dmytro; ...

2016-01-28

The need for functional materials calls for increasing complexity in self-assembly systems. As a result, the ability to probe both local structure and heterogeneities, such as phase-coexistence and domain morphologies, has become increasingly important to controlling self-assembly processes, including those at liquid surfaces. The traditional X-ray scattering methods for liquid surfaces, such as specular reflectivity and grazing-incidence diffraction, are not well suited to spatially resolving lateral heterogeneities due to large illuminated footprint. A possible alternative approach is to use scanning transmission X-ray scattering to simultaneously probe local intermolecular structures and heterogeneous domain morphologies on liquid surfaces. To test the feasibilitymore » of this approach, transmission small- and wide-angle X-ray scattering (TSAXS/TWAXS) studies of Langmuir films formed on water meniscus against a vertically immersed hydrophilic Si substrate were recently carried out. First-order diffraction rings were observed in TSAXS patterns from a monolayer of hexagonally packed gold nanoparticles and in TWAXS patterns from a monolayer of fluorinated fatty acids, both as a Langmuir monolayer on water meniscus and as a Langmuir–Blodgett monolayer on the substrate. The patterns taken at multiple spots have been analyzed to extract the shape of the meniscus surface and the ordered-monolayer coverage as a function of spot position. These results, together with continual improvement in the brightness and spot size of X-ray beams available at synchrotron facilities, support the possibility of using scanning-probe TSAXS/TWAXS to characterize heterogeneous structures at liquid surfaces.« less

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

PubMed

Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

2014-09-26

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface-enhanced Raman scattering from graphene covered gold nanocap arrays

NASA Astrophysics Data System (ADS)

Long, Kailin; Luo, Xiaoguang; Nan, Haiyan; Du, Deyang; Zhao, Weiwei; Ni, Zhenhua; Qiu, Teng

2013-11-01

This work reports an efficient method to fabricate large-area flexible substrates for surface enhanced Raman scattering (SERS) application. Our technique is based on a single-step direct imprint process via porous anodic alumina stamps. Periodic hexagonal arrangements of porous anodic alumina stamps are transferred to the polyethylene terephthalate substrates by mechanically printing process. Printed nanocaps will turn into "hot spots" for electromagnetic enhancement with a deposited gold film by high vacuum evaporation. The gaps between the nanocaps are controllable with a tight correspondence to the thickness of the deposited gold, which dramatically influence the enhancement factor. After covered with a single-layer graphene sheet, the gold nanocap substrate can be further optimized with an extra enhancement of Raman signals, and it is available for the trace detection of probe molecules. This convenient, simple, and low-cost method of making flexible SERS-active substrates potentially opens a way towards biochemical analysis and disease detection.

Rectification of Ion Current in Nanopipettes by External Substrates

PubMed Central

Shi, Wenqing; Baker, Lane A.

2014-01-01

We describe ion distribution and the current-voltage (i-V) response of nanopipettes at different probe-to-substrate distances (Dps) as simulated by finite-element methods. Results suggest electrostatic interactions between a charged substrate and the nanopipette dominate electrophoretic ion transport through the nanopipette when Dps is within one order of magnitude of the Debye length (~10 nm for a 1 mM solution as employed in the simulation). Ion current rectification (ICR) and permselectivity associated with a neutral or charged nanopipette can be reversibly enhanced or reduced dependent on Dps, charge polarity and charge density (σ) of the substrate. Regulation of nanopipette current is a consequence of the enrichment or depletion of ions within the nanopipette interior which influences conductivity of the nanopipette. When the external substrate is less negatively charged than the nanopipette, the substrate first reduces, and then enhances the ICR as Dps decreases. Surprisingly, both experimental and simulated data show that a neutral substrate was also able to reduce and reverse the ICR of a slightly negatively charged nanopipette. Simulated results ascribe such effects to the elimination of ion depletion within the nanopipette at positive potentials. PMID:24200344

Rectification of ion current in nanopipettes by external substrates.

PubMed

Sa, Niya; Lan, Wen-Jie; Shi, Wenqing; Baker, Lane A

2013-12-23

We describe ion distribution and the current-voltage (i-V) response of nanopipettes at different probe-to-substrate distances (Dps) as simulated by finite-element methods. Results suggest electrostatic interactions between a charged substrate and the nanopipette dominate electrophoretic ion transport through the nanopipette when Dps is within 1 order of magnitude of the Debye length (∼10 nm for a 1 mM solution as employed in the simulation). Ion current rectification (ICR) and permselectivity associated with a neutral or charged nanopipette can be reversibly enhanced or reduced dependent on Dps, charge polarity, and charge density (σ) of the substrate. Regulation of nanopipette current is a consequence of the enrichment or depletion of ions within the nanopipette interior, which influences conductivity of the nanopipette. When the external substrate is less negatively charged than the nanopipette, the substrate first reduces, and then enhances the ICR as Dps decreases. Surprisingly, both experimental and simulated data show that a neutral substrate was also able to reduce and reverse the ICR of a slightly negatively charged nanopipette. Simulated results ascribe such effects to the elimination of ion depletion within the nanopipette at positive potentials.

P(VDF/TrFE) morphologies and crystalline lamellae orientations dependence on substrates characterized by scanning probe microscopy

NASA Astrophysics Data System (ADS)

Lakbita, Imane; El-Hami, Khalil

2018-02-01

Ultra-thin films of the polyvinylidene fluoride and trifluoroethylene (P(VDF/TrFE)) copolymer were elaborated on various different substrates by the spin coating method. The purpose of this paper is to study the P(VDF/TrFE) morphologies and crystalline lamellae orientation dependence on substrates. We chose the potassium chloride (KCl), Sodium Chloride (NaCl) and Potassium Bromide (KBr) with the [110] direction and the highly ordered pyrolytic graphite (HOPG) substrates because they present different crystallographic structures. The atomic force microscopy is used for imaging P(VDF/TrFE) morphologies with nanometer resolution and determining the surface roughness. The analysis of the AFM topography images revealed that the P(VDF/TrFE) film has, almost, the same texture on KCl, NaCl or on KBr substrates and their crystalline lamellae had grown in two preferred orientations. Unlike the HOPG substrate, their crystalline lamellae were entangled, randomly oriented and positioned adjacent to each other. The growth texture of the P(VDF/TrFE) copolymer showed experimentally a strong dependence on substrate types. Since the P(VDF/TrFE) is ferroelectric, piezoelectric and pyroelectric, this finding may lead to potential applications.

APPLICATION OF FLUORESCENCE SPECTROSCOPIC TECHNIQUES AND PROBES TO THE DETECTION OF BIOPOLYMER DEGRADATION IN NATURAL ENVIRONMENTS. (R825159)

EPA Science Inventory

The activities and substrate specificities of extracellular enzymes in natural systems are not well understood, despite their critical role in microbial remineralization of organic carbon. These enzymes initiate organic carbon degradation by selectively hydrolyzing high molecular...

Nanostructure and Dynamics of Polymers and Thin Polymer Films: Studies by Positron Annihilation Spectroscopy

NASA Astrophysics Data System (ADS)

Yee, Albert F.

1997-03-01

The relaxational, mechanical and transport properties of glassy polymers are strongly influenced by the nanostructural and dynamical characteristics of each material. In very thin polymer films such characteristics may be affected by the presence of a free surface or a substrate. Positron Annihilation Lifetime Spectroscopy (PALS) is a useful and in some ways unique tool for probing these important characteristics. Conventional PALS on several bulk polymers over an extended temperature range are used to illustrate how these characteristics are obtained(HA Hristov, B Bolan, AF Yee, L Xie, and DW Gidley, accepted by Macromolecules.). A new technique, which we shall call "beam-PALS", and the results of its application on nm-thick polystyrene films supported on one side by a Si substrate are described. In beam-PALS the lifetime, τ _3, and formation fraction, I_3, of triplet positronium decaying in the void volume near the polymer surface are measured versus the positron implantation energy, E. The strong E dependence of I3 supports a spur-electron capture model of Ps formation with deduced spur sizes ranging from 200 to 660 ÅThin film measurements indicate that the mean probe depth can be much smaller, given mainly by the average positron implantation distance, Z(E)(L Xie, GB DeMaggio, WE Frieze, J DeVries, DW Gidley, HA Hristov and AF Yee, PRL 74, 4947 (1995).). The thermal expansion behaviors of thin, Si-supported polystyrene films near the glass transition temperature, Tg were also measured. A reduction in void volume expansion is correlated with a reduction in the apparent Tg as film thickness decreases. Our results can be fitted using a 3-layer model incorporating a 50 Åconstrained layer at the Si interface and a 20 Åsurface region with reduced T_g(GB DeMaggio, WE Frieze, DW Gidley, M Zhu, HA Hristov, and AF Yee, accepted by PRL.).

Functional Na+ Channels in Cell Adhesion probed by Transistor Recording

PubMed Central

Schmidtner, Markus; Fromherz, Peter

2006-01-01

Cell membranes in a tissue are in close contact to each other, embedded in the extracellular matrix. Standard electrophysiological methods are not able to characterize ion channels under these conditions. Here we consider the area of cell adhesion on a solid substrate as a model system. We used HEK 293 cells cultured on fibronectin and studied the activation of NaV1.4 sodium channels in the adherent membrane with field-effect transistors in a silicon substrate. Under voltage clamp, we compared the transistor response with the whole-cell current. We observed that the extracellular voltage in the cell-chip contact was proportional to the total membrane current. The relation was calibrated by alternating-current stimulation. We found that Na+ channels are present in the area of cell adhesion on fibronectin with a functionality and a density that is indistinguishable from the free membrane. The experiment provides a basis for studying selective accumulation and depletion of ion channels in cell adhesion and also for a development of cell-based biosensoric devices and neuroelectronic systems. PMID:16227504

Probing the effect of charge transfer enhancement in off resonance mode SERS via conjugation of the probe dye between silver nanoparticles and metal substrates.

PubMed

Selvakannan, Pr; Ramanathan, Rajesh; Plowman, Blake J; Sabri, Ylias M; Daima, Hemant K; O'Mullane, Anthony P; Bansal, Vipul; Bhargava, Suresh K

2013-08-21

The charge transfer-mediated surface enhanced Raman scattering (SERS) of crystal violet (CV) molecules that were chemically conjugated between partially polarized silver nanoparticles and optically smooth gold and silver substrates has been studied under off-resonant conditions. Tyrosine molecules were used as a reducing agent to convert silver ions into silver nanoparticles where oxidised tyrosine caps the silver nanoparticle surface with its semiquinone group. This binding through the quinone group facilitates charge transfer and results in partially oxidised silver. This establishes a chemical link between the silver nanoparticles and the CV molecules, where the positively charged central carbon of CV molecules can bind to the terminal carboxylate anion of the oxidised tyrosine molecules. After drop casting Ag nanoparticles bound with CV molecules it was found that the free terminal amine groups tend to bind with the underlying substrates. Significantly, only those CV molecules that were chemically conjugated between the partially polarised silver nanoparticles and the underlying gold or silver substrates were found to show SERS under off-resonant conditions. The importance of partial charge transfer at the nanoparticle/capping agent interface and the resultant conjugation of CV molecules to off resonant SERS effects was confirmed by using gold nanoparticles prepared in a similar manner. In this case the capping agent binds to the nanoparticle through the amine group which does not facilitate charge transfer from the gold nanoparticle and under these conditions SERS enhancement in the sandwich configuration was not observed.

Simultaneous Nanoscale Surface Charge and Topographical Mapping.

PubMed

Perry, David; Al Botros, Rehab; Momotenko, Dmitry; Kinnear, Sophie L; Unwin, Patrick R

2015-07-28

Nanopipettes are playing an increasingly prominent role in nanoscience, for sizing, sequencing, delivery, detection, and mapping interfacial properties. Herein, the question of how to best resolve topography and surface charge effects when using a nanopipette as a probe for mapping in scanning ion conductance microscopy (SICM) is addressed. It is shown that, when a bias modulated (BM) SICM scheme is used, it is possible to map the topography faithfully, while also allowing surface charge to be estimated. This is achieved by applying zero net bias between the electrode in the SICM tip and the one in bulk solution for topographical mapping, with just a small harmonic perturbation of the potential to create an AC current for tip positioning. Then, a net bias is applied, whereupon the ion conductance current becomes sensitive to surface charge. Practically this is optimally implemented in a hopping-cyclic voltammetry mode where the probe is approached at zero net bias at a series of pixels across the surface to reach a defined separation, and then a triangular potential waveform is applied and the current response is recorded. Underpinned with theoretical analysis, including finite element modeling of the DC and AC components of the ionic current flowing through the nanopipette tip, the powerful capabilities of this approach are demonstrated with the probing of interfacial acid-base equilibria and high resolution imaging of surface charge heterogeneities, simultaneously with topography, on modified substrates.

Spectrophotometric and ultrasensitive DNA bioassay by circular-strand displacement polymerization reaction.

PubMed

Yu, Luxin; Wu, Wei; Chen, Junhua; Xiao, Zhuo; Ge, Chenchen; Lie, Puchang; Fang, Zhiyuan; Chen, Lingbo; Zhang, Ya; Zeng, Lingwen

2013-12-07

We demonstrated a new spectrophotometric DNA detection approach based on a circular strand-displacement polymerization reaction for the quantitative detection of sequence specific DNA. In this assay, the hybridization of an immobilized hairpin probe on the microtiter plate, to target DNA, results in a conformational change and leads to a stem separation. A short primer thus anneals with the open stem and triggers a polymerization reaction, allowing a cyclic reaction comprising the release of target DNA and hybridization of the target with the remaining immobilized hairpin probe. Through this cyclical process, a large number of duplex DNA complexes are produced. Finally, the biotin modified duplex DNA products can be detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophotometer. As a proof of concept, a short DNA sequence (20-nt) related to the South East Asia (SEA) type deletion of α-thalassemia was chosen as the model target. This proposed assay has a very high sensitivity and selectivity with a dynamic response ranging from 0.1 fM to 10 nM and the detection limit was 8 aM. It can be performed within 2 hours, and it can differentiate target SEA DNA from wild-type DNA. By substituting the hairpin probes used in the present work, this assay can be used to detect other subtypes of genetic disorders.

Analysis of non-contact and contact probe-to-sample thermal exchange for quantitative measurements of thin film and nanostructure thermal conductivity by the scanning hot probe method

NASA Astrophysics Data System (ADS)

Wilson, Adam A.

The ability to measure thermal properties of thin films and nanostructured materials is an important aspect of many fields of academic study. A strategy especially well-suited for nanoscale investigations of these properties is the scanning hot probe technique, which is unique in its ability to non-destructively interrogate the thermal properties with high resolution, both laterally as well as through the thickness of the material. Strategies to quantitatively determine sample thermal conductivity depend on probe calibration. State of the art calibration strategies assume that the area of thermal exchange between probe and sample does not vary with sample thermal conductivity. However, little investigation has gone into determining whether or not that assumption is valid. This dissertation provides a rigorous study into the probe-to-sample heat transfer through the air gap at diffusive distances for a variety of values of sample thermal conductivity. It is demonstrated that the thermal exchange radius and gap/contact thermal resistance varies with sample thermal conductivity as well as tip-to-sample clearance in non-contact mode. In contact mode, it is demonstrated that higher thermal conductivity samples lead to a reduction in thermal exchange radius for Wollaston probe tips. Conversely, in non-contact mode and in contact mode for sharper probe tips where air contributes the most to probe-to-sample heat transfer, the opposite trend occurs. This may be attributed to the relatively strong solid-to-solid conduction occurring between probe and sample for the Wollaston probes. A three-dimensional finite element (3DFE) model was developed to investigate how the calibrated thermal exchange parameters vary with sample thermal conductivity when calibrating the probe via the intersection method in non-contact mode at diffusive distances. The 3DFE model was then used to explore the limits of sensitivity of the experiment for a range of simulated experimental conditions. It is determined that, when operating the scanning hot probe technique in air at standard temperature and pressure using Wollaston probes, the technique is capable of measuring, within 20% uncertainty, samples with values of thermal conductivity up to 10 Wm-1K-1 in contact mode and up to 2 Wm-1K-1 in non-contact mode. By increasing the thermal conductivity of the probe's surroundings (i.e. changing air to a more conductive gas), sensitivity in non-contact mode to sample thermal conductivity is improved, which suggests potential for future investigations using non-contact scanning hot probe to measure thermal conductivity of higher thermal conductivity samples. The ability of the technique to differentiate thin films from the substrate is investigated, and the sensitivity of the technique to thin films and samples with anisotropic properties is explored. The models (both analytical and finite element) developed and reported in this dissertation lead to the ability to measure samples which, by the standard procedure before this work, were unable to be accurately measured. While other techniques failed to be able to successfully interrogate the film thermal conductivity of a full set of double-wall carbon nanotubes infused into polymers, the methods developed in this work allowed non-contact scanning hot probe measurements to be successfully performed to obtain the film thermal conductivity for each sample. Finite element simulations accounting for the anisotropy of these thin film on sample materials show similar trends with independently measured in-plane thermal conductivity for the only two (of five) samples in the set which were successfully able to be measured by the independent technique. Investigations in contact mode with high resolution Pd probes, whose probe-to-sample clearance is difficult to control in a repeatable fashion, show that surface roughness affects the thermal contact resistance. This can lead to values of reported sample thermal conductivity which are misleading, when using the standard calibrated thermal exchange parameters on samples with significantly different surface roughness than the calibration samples. This affect was taken into account to report sample thermal conductivity of Bi2Te3 nanoflakes.

An integrated eddy current detection and imaging system on a silicon chip

NASA Technical Reports Server (NTRS)

Henderson, H. Thurman; Kartalia, K. P.; Dury, Joseph D.

1991-01-01

Eddy current probes have been used for many years for numerous sensing applications including crack detection in metals. However, these applications have traditionally used the eddy current effect in the form of a physically wound single or different probe pairs which of necessity must be made quite large compared to microelectronics dimensions. Also, the traditional wound probe can only take a point reading, although that point might include tens of individual cracks or crack arrays; thus, conventional eddy current probes are beset by two major problems: (1) no detailed information can be obtained about the crack or crack array; and (2) for applications such as quality assurance, a vast amount of time must be taken to scan a complete surface. Laboratory efforts have been made to fabricate linear arrays of single turn probes in a thick film format on a ceramic substrate as well as in a flexible cable format; however, such efforts inherently suffer from relatively large size requirements as well as sensitivity issues. Preliminary efforts to fully extend eddy current probing from a point or single dimensional level to a two dimensional micro-eddy current format on a silicon chip, which might overcome all of the above problems, are presented.

Design and verification of a pangenome microarray oligonucleotide probe set for Dehalococcoides spp.

PubMed

Hug, Laura A; Salehi, Maryam; Nuin, Paulo; Tillier, Elisabeth R; Edwards, Elizabeth A

2011-08-01

Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an oligonucleotide microarray probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This "pangenome" probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome probe set was compared to probe sets designed independently for each of the five Dehalococcoides strains. The pangenome probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific probe sets on nontarget strains with <99% average nucleotide identity. An in silico analysis of the expected probe hybridization against the recently released Dehalococcoides strain GT genome and additional KB-1 metagenome sequence data indicated that the pangenome probe set performs more robustly than the combined strain-specific probe sets in the detection of genes not included in the original design. The pangenome probe set represents a highly specific, universal tool for the detection and characterization of Dehalococcoides from contaminated sites. It has the potential to become a common platform for Dehalococcoides-focused research, allowing meaningful comparisons between microarray experiments regardless of the strain examined.

Unravelling RNA-substrate interactions in a ribozyme-catalysed reaction using fluorescent turn-on probes.

PubMed

Gaffarogullari, Ece Cazibe; Greulich, Peter; Kobitski, Andrei Yu; Nierth, Alexander; Nienhaus, G Ulrich; Jäschke, Andres

2015-04-07

The Diels-Alder reaction is one of the most important C-C bond-forming reactions in organic chemistry, and much effort has been devoted to controlling its enantio- and diastereoselectivity. The Diels-Alderase ribozyme (DAse) catalyses the reaction between anthracene dienes and maleimide dienophiles with multiple-turnover, stereoselectivity, and up to 1100-fold rate acceleration. Here, a new generation of anthracene-BODIPY-based fluorescent probes was developed to monitor catalysis by the DAse. The brightness of these probes increases up to 93-fold upon reaction with N-pentylmaleimide (NPM), making these useful tools for investigating the stereochemistry of the ribozyme-catalysed reaction. With these probes, we observed that the DAse catalyses the reaction with >91% de and >99% ee. The stereochemistry of the major product was determined unambiguously by rotating-frame nuclear Overhauser NMR spectroscopy (ROESY-NMR) and is in agreement with crystallographic structure information. The pronounced fluorescence change of the probes furthermore allowed a complete kinetic analysis, which revealed an ordered bi uni type reaction mechanism, with the dienophile binding first. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new method for mapping the three-dimensional atomic distribution within nanoparticles by atom probe tomography (APT).

PubMed

Kim, Se-Ho; Kang, Phil Woong; Park, O Ok; Seol, Jae-Bok; Ahn, Jae-Pyoung; Lee, Ji Yeong; Choi, Pyuck-Pa

2018-07-01

We present a new method of preparing needle-shaped specimens for atom probe tomography from freestanding Pd and C-supported Pt nanoparticles. The method consists of two steps, namely electrophoresis of nanoparticles on a flat Cu substrate followed by electrodeposition of a Ni film acting as an embedding matrix for the nanoparticles. Atom probe specimen preparation can be subsequently carried out by means of focused-ion-beam milling. Using this approach, we have been able to perform correlative atom probe tomography and transmission electron microscopy analyses on both nanoparticle systems. Reliable mass spectra and three-dimensional atom maps could be obtained for Pd nanoparticle specimens. In contrast, atom probe samples prepared from C-supported Pt nanoparticles showed uneven field evaporation and hence artifacts in the reconstructed atom maps. Our developed method is a viable means of mapping the three-dimensional atomic distribution within nanoparticles and is expected to contribute to an improved understanding of the structure-composition-property relationships of various nanoparticle systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Wideband Microstrip Antenna-Feeding Array

NASA Technical Reports Server (NTRS)

Huang, John

1990-01-01

Special impedance-matching probes help reduce feed complexity. Lightweight array of microstrip antenna elements designed to transmit and illuminate reflector antenna with circularly polarized radiation at 1,545 to 1,550 MHz and to receive circularly polarized radiation at 1,646 to 1,660 MHz. Microstrip array is cluster of 7 subarrays containing total of 28 microstrip patches. Produces cicularly polarized beam with suitable edge taper to illuminate reflector antenna. Teardrop-shaped feed probe provides gradual change of field from coaxial transmission line into microstrip substrate. Intended to be part of larger overlapping-cluster array generating multiple contiguous beams.

Electronic Disorder in Organic Semiconducting Films Observed with Kelvin Probe Force Microscopy

NASA Astrophysics Data System (ADS)

Hoffman, Benjamin Carl

This work is a study into electronic disorder within organic semiconducting (OSC) films from a scan-probe perspective. Organic electronics are an exciting technology poised for use in next generation devices with unique applications such as transparent displays and ultrathin flexible solar cells. Understanding and mapping electronic disorder in OSC has a high degree of relevance towards recognizing the properties of charge trapping that hinders transport and diminishes device performance. Evidence of surface potential inhomogeneity is identified by using Kelvin probe force microscopy (KPFM) to measure the contact potential difference (CPD) between probe and sample. OSC films are grown via organic molecular beam deposition (OMBD) to create well-ordered crystals with precise control of nominal thickness. Further research methods involve the study of diffraction peaks from grazing-incidence wide-angle x-ray scattering (GIWAXS) for crystallographic analysis as well as use of a probe station for transfer characteristics of fabricated thin film transistors. Initial research into this subject involved thin films of the novel organic molecule 2,8- diflouro-5,11-bis(triethylsilylethynyl)-anthradithiophene (diF-TES-ADT) that were grown on silicon substrates with a native oxide layer and analyzed with GIWAXS and KPFM. The crystallography of the films is that of a uniform (001) orientation. Variations in surface potential in diF-TES-ADT crystallites are observed to be unique from variations in the substrate. Nevertheless, surface potential variations in thick films are influenced by chemical passivation of the substrate and so the source of CPD variations are assigned to be intrinsic defects. Chemical treatment and processing methods control the growth kinetics which are linked to charge traps locally distorting the surface potential in OSC films. To continue the research into identifying charge trapping in ultra-thin films, 1.5 monolayer thick films of alpha-sexithiophene (6T) were grown on silicon substrates with a thick oxide layer to compare the surface potential of the first monolayer with that of the bilayer. The temperature of the substrate during film growth was varied and found to influence the interlayer surface potential contrast. Higher temperature samples have a bilayer with CPD above that of the monolayer, while lower temperature samples have a bilayer with CPD below that of the monolayer. GIWAXS data collected shows that the lower temperature sample has no observed out-of-plane order which we identify as the source of interlayer trapping formed between the first and second monolayers of the 6T films. The final project into the specifics of disorder involves using ultrathin films of 6T in an operational transistor with grounded source-drain electrodes to study the influence of gate bias on surface potential. Using the mean and standard deviation of CPD for a series of gate biases, the trap density of states (DOS) is calculated directly while allowing for the quantification of spatial variations. CPD histograms from the series of images illustrate the screening of deep traps as the transistor is turned on. While in the 'off' regime the images are electrostatic, after transitioning to the 'on' state the images show instability after threshold voltage. This dynamic 'on' state offers a unique view of shallow trap states being filled and then thermally released.

Distributed force probe bending model of critical dimension atomic force microscopy bias

NASA Astrophysics Data System (ADS)

Ukraintsev, Vladimir A.; Orji, Ndubuisi G.; Vorburger, Theodore V.; Dixson, Ronald G.; Fu, Joseph; Silver, Rick M.

2013-04-01

Critical dimension atomic force microscopy (CD-AFM) is a widely used reference metrology technique. To characterize modern semiconductor devices, small and flexible probes, often 15 to 20 nm in diameter, are used. Recent studies have reported uncontrolled and significant probe-to-probe bias variation during linewidth and sidewall angle measurements. To understand the source of these variations, tip-sample interactions between high aspect ratio features and small flexible probes, and their influence on measurement bias, should be carefully studied. Using theoretical and experimental procedures, one-dimensional (1-D) and two-dimensional (2-D) models of cylindrical probe bending relevant to carbon nanotube (CNT) AFM probes were developed and tested. An earlier 1-D bending model was refined, and a new 2-D distributed force (DF) model was developed. Contributions from several factors were considered, including: probe misalignment, CNT tip apex diameter variation, probe bending before snapping, and distributed van der Waals-London force. A method for extracting Hamaker probe-surface interaction energy from experimental probe-bending data was developed. Comparison of the new 2-D model with 1-D single point force (SPF) model revealed a difference of about 28% in probe bending. A simple linear relation between biases predicted by the 1-D SPF and 2-D DF models was found. The results suggest that probe bending can be on the order of several nanometers and can partially explain the observed CD-AFM probe-to-probe variation. New 2-D and three-dimensional CD-AFM data analysis software is needed to take full advantage of the new bias correction modeling capabilities.

Mg2+-dependent conformational changes and product release during DNA-catalyzed RNA ligation monitored by Bimane fluorescence

PubMed Central

Turriani, Elisa; Höbartner, Claudia; Jovin, Thomas M.

2015-01-01

Among the deoxyribozymes catalyzing the ligation of two RNA substrates, 7S11 generates a branched RNA containing a 2′,5′-linkage. We have attached the small fluorogenic probe Bimane to the triphosphate terminated RNA substrate and utilized emission intensity and anisotropy to follow structural rearrangements leading to a catalytically active complex upon addition of Mg2+. Bimane coupled to synthetic oligonucleotides is quenched by nearby guanines via photoinduced electron transfer. The degree of quenching is sensitive to changes in the base pairing of the residues involved and in their distances to the probe. These phenomena permit the characterization of various sequential processes in the assembly and function of 7S11: binding of Mg2+ to the triphosphate moiety, release of quenching of the probe by the 5′-terminal G residues of R-RNA as they engage in secondary base-pair interactions, local rearrangement into a distinct active conformation, and continuous release of the Bimane-labeled pyrophosphate during the course of reaction at 37°C. It was possible to assign equilibrium and rate constants and structural interpretations to the sequence of conformational transitions and catalysis, information useful for optimizing the design of next generation deoxyribozymes. The fluorescent signatures, thermodynamic equilibria and catalytic function of numerous mutated (base/substituted) molecules were examined. PMID:25505142

"Off-On"switching electrochemiluminescence biosensor for mercury(II) detection based on molecular recognition technology.

PubMed

Cheng, Lin; Wei, BingGuo; He, Ling Ling; Mao, Ling; Zhang, Jie; Ceng, JinXiang; Kong, DeRong; Chen, ChaDan; Cui, HanFeng; Hong, Nian; Fan, Hao

2017-02-01

A novel "off-On" electrogenerated chemiluminescence (ECL) biosensor has been developed for the detection of mercury(II) based on molecular recognition technology. The ECL mercury(II) biosensor comprises two main parts: an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Ruthenium(II) tris-(bipyridine)(Ru(bpy) 3 2+ )/Cyclodextrins-Au nanoparticles(CD-AuNps)/Nafion on the surface of glass carbon electrode (GCE), and the ECL intensity switch is the single hairpin DNA probe designed according to the "molecular recognition" strategy which was functionalized with ferrocene tag at one end and attached to Cyclodextrins (CD) on modified GCE through supramolecular noncovalent interaction. We demonstrated that, in the absence of Hg(II) ion, the probe keeps single hairpin structure and resulted in a quenching of ECL of Ru(bpy) 3 2+ . Whereas, in the presence of Hg(II) ion, the probe prefers to form the T-Hg(II)-T complex and lead to an obvious recovery of ECL of Ru(bpy) 3 2+ , which provided a sensing platform for the detection of Hg(II) ion. Using this sensing platform, a simple, rapid and selective "off-On" ECL biosensor for the detection of mercury(II) with a detection limit of 0.1 nM has been developed. Copyright © 2016. Published by Elsevier Inc.

Tunable SERS signals of Rhodamine B molecules on Fe3O4@Au nanocomposite substrates controlled by magnetic field

NASA Astrophysics Data System (ADS)

Song, Hao; Fang, Rui Yang; Li, Ling

2018-02-01

In this work, we experimentally synthesized the Fe3O4@Au nanocomposites and used them as surface-enhanced Raman scattering (SERS) substrates. The Fe3O4@Au nanocomposites retained the metallic plasmon resonant effect and possessed the magnetic field controllable characteristics. The Raman spectra of Rhodamine B (RhB) probe molecules were studied under different external magnetic field. The magnitude of external magnetic field varied from 0 Gs to 700 Gs (1 Gs = 10-4 T) with intervals of 100 Gs. When the magnetic field magnitude increased, the Raman intensity of RhB probe molecules at 1356 cm-1 increased linearly. The slope of the linear fitting curves for the Raman intensity and area were 0.118/Gs and 3.700/Gs. The Raman enhancement could raise up to 7 times for RhB probe molecules when the magnetic field magnitude increased to 700 Gs. After removing the external magnetic field, the Raman peaks returned to its original intensity in several minutes. Under the external magnetic field, the Fe3O4@Au nanocomposites were concentrated, leading to the increase number of SERS "hot spots" and the surface Au density. The results show that the magnetic field controlled Fe3O4@Au nanocomposites can realize the enhanced and controllable SERS effect, which can be used in the reversible optical sensing and bio-medical applications.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

PubMed

Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

2016-04-12

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

[18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

PubMed Central

Kim, Woosuk; Le, Thuc M.; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T.; Abt, Evan R.; Capri, Joseph R.; Austin, Wayne R.; Van Valkenburgh, Juno S.; Steele, Dalton; Gipson, Raymond M.; Slavik, Roger; Cabebe, Anthony E.; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S.; Lee, Jason T.; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F.; Witte, Owen N.; Donahue, Timothy R.; Phelps, Michael E.; Herschman, Harvey R.; Herrmann, Ken; Czernin, Johannes; Radu, Caius G.

2016-01-01

Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds—[18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) and 2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-guanine ([18F]F-AraG)—for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [18F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [18F]F-AraG is a better substrate for dGK than for dCK. [18F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [18F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [18F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [18F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [18F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [18F]CFA PET as a new cancer biomarker for treatment stratification and monitoring. PMID:27035974

Functionalized apertures for the detection of chemical and biological materials

DOEpatents

Letant, Sonia E.; van Buuren, Anthony W.; Terminello, Louis J.; Thelen, Michael P.; Hope-Weeks, Louisa J.; Hart, Bradley R.

2010-12-14

Disclosed are nanometer to micron scale functionalized apertures constructed on a substrate made of glass, carbon, semiconductors or polymeric materials that allow for the real time detection of biological materials or chemical moieties. Many apertures can exist on one substrate allowing for the simultaneous detection of numerous chemical and biological molecules. One embodiment features a macrocyclic ring attached to cross-linkers, wherein the macrocyclic ring has a biological or chemical probe extending through the aperture. Another embodiment achieves functionalization by attaching chemical or biological anchors directly to the walls of the apertures via cross-linkers.

Preparation of Cu-doped nickel oxide thin films and their properties

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

Gowthami, V.; Meenakshi, M.; Anandhan, N.

2014-04-24

Copper doped Nickel oxide film was preferred on glass substrate by simple nebulizer technique keeping the substrate temperature at 350°C and characterized by X-ray diffraction (XRD), Photoluminescence (PL) and Four probe resistivity measurements. XRD studies indicated cubic structure and the crystallites are preferentially oriented along the [111] direction. Interesting results have been obtained from the study of PL spectra. A peak corresponding to 376nm in the emission spectra for 0%, 5% and 10% copper doped samples. The samples show sharp and strong UV emission corresponding to the near band edge emission under excitation of 275nm.

Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

DOEpatents

Erlanger, B.F.; Chen, B.X.

1997-07-22

The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest. 8 figs.

Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

DOEpatents

Erlanger, Bernard F.; Chen, Bi-Xing

1997-01-01

The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest.

On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

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

Brokaw, Jason B.; Chu, Jhih-wei

2010-11-17

We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier worksmore » of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro177 on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro177 hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.« less

Ultrafast relaxation dynamics in BiFeO 3/YBa 2Cu 3O 7 bilayers

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

Springer, D.; Nair, Saritha K.; He, Mi

The temperature dependence of the relaxation dynamics in the bilayer thin film heterostructure composed of multiferroic BiFeO 3 (BFO) and superconducting YBa 2Cu 3O 7 (YBCO) grown on (001) SrTiO 3 substrate is studied by time-resolved pump-probe technique, and compared with that of pure YBCO thin film grown under the same growth conditions. The superconductivity of YBCO is found to be retained in the heterostructure. We observe a speeding up of the YBCO recombination dynamics in the superconducting state of the heterostructure, and attribute it to the presence of weak ferromagnetism at the BFO/YBCOinterface as observed inmagnetization data. An extensionmore » of the Rothwarf-Taylor model is used to fit the ultrafast dynamics of BFO/YBCO, that models an increased quasiparticle occupation of the ferromagnetic interfacial layer in the superconducting state of YBCO.« less

Ultrafast relaxation dynamics in BiFeO 3/YBa 2Cu 3O 7 bilayers

DOE PAGES

Springer, D.; Nair, Saritha K.; He, Mi; ...

2016-02-12

The temperature dependence of the relaxation dynamics in the bilayer thin film heterostructure composed of multiferroic BiFeO 3 (BFO) and superconducting YBa 2Cu 3O 7 (YBCO) grown on (001) SrTiO 3 substrate is studied by time-resolved pump-probe technique, and compared with that of pure YBCO thin film grown under the same growth conditions. The superconductivity of YBCO is found to be retained in the heterostructure. We observe a speeding up of the YBCO recombination dynamics in the superconducting state of the heterostructure, and attribute it to the presence of weak ferromagnetism at the BFO/YBCOinterface as observed inmagnetization data. An extensionmore » of the Rothwarf-Taylor model is used to fit the ultrafast dynamics of BFO/YBCO, that models an increased quasiparticle occupation of the ferromagnetic interfacial layer in the superconducting state of YBCO.« less

An analytical model for floating probes in AC plasma and its application to double probes for high density, high power RF discharges

NASA Astrophysics Data System (ADS)

Caneses, Juan Francisco; Blackwell, Boyd; Plasma Research Laboratory Team

2013-10-01

In this work we provide an analytical model that allows one to quantitatively assess the RF compensation performance and suitability of the double probe technique for use in RF generated plasma. The model is based in the theory of the self-bias effect as described in Braithwaite's work, which we extend to include the time resolved behavior of floating probes. We provide experimental verification for this model and show that the theory of transient RF self-bias probes and harmonic current detection probes are limiting cases of this extended model. Furthermore, the model shows that the RF compensation is solely dependent on the sheath impedance, the probe's stray capacitance to ground and RF frequency. In addition, we use these results to implement a double probe system for use in high density helicon plasma where heat loads could potentially damage the intricate components in an RF compensating circuit. Finally we use this model to (1) recommend ways to extend the operational regime of double probes where the plasma conditions would render them unsuitable and to (2) comment on the use of this model to aid design of RF compensated Langmuir probes.

Simultaneous detection of multiple biomarkers by means of SERS on polymer nanopillar gold arrays

NASA Astrophysics Data System (ADS)

Morasso, Carlo; Picciolini, Silvia; Mehn, Dora; Pellacani, Paola; Frangolho, Ana; Marchesini, Gerardo; Vanna, Renzo; Gualerzi, Alice; Bedoni, Marzia; Marabelli, Franco; Gramatica, Furio

2016-03-01

The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.

On CD-AFM bias related to probe bending

NASA Astrophysics Data System (ADS)

Ukraintsev, V. A.; Orji, N. G.; Vorburger, T. V.; Dixson, R. G.; Fu, J.; Silver, R. M.

2012-03-01

Critical Dimension AFM (CD-AFM) is a widely used reference metrology. To characterize modern semiconductor devices, very small and flexible probes, often 15 nm to 20 nm in diameter, are now frequently used. Several recent publications have reported on uncontrolled and significant probe-to-probe bias variation during linewidth and sidewall angle measurements [1,2]. Results obtained in this work suggest that probe bending can be on the order of several nanometers and thus potentially can explain much of the observed CD-AFM probe-to-probe bias variation. We have developed and experimentally tested one-dimensional (1D) and two-dimensional (2D) models to describe the bending of cylindrical probes. An earlier 1D bending model reported by Watanabe et al. [3] was refined. Contributions from several new phenomena were considered, including: probe misalignment, diameter variation near the carbon nanotube tip (CNT) apex, probe bending before snapping, distributed van der Waals-London force, etc. The methodology for extraction of the Hamaker probe-surface interaction energy from experimental probe bending data was developed. To overcome limitations of the 1D model, a new 2D distributed force (DF) model was developed. Comparison of the new model with the 1D single point force (SPF) model revealed about 27 % difference in probe bending bias between the two. A simple linear relation between biases predicted by the 1D SPF and 2D DF models was found. This finding simplifies use of the advanced 2D DF model of probe bending in various CD-AFM applications. New 2D and three-dimensional (3D) CDAFM data analysis software is needed to take full advantage of the new bias correction modeling capabilities.

Modeling Central Carbon Metabolic Processes in Soil Microbial Communities: Comparing Measured With Modeled

NASA Astrophysics Data System (ADS)

Dijkstra, P.; Fairbanks, D.; Miller, E.; Salpas, E.; Hagerty, S.

2013-12-01

Understanding the mechanisms regulating C cycling is hindered by our inability to directly observe and measure the biochemical processes of glycolysis, pentose phosphate pathway, and TCA cycle in intact and complex microbial communities. Position-specific 13C labeled metabolic tracer probing is proposed as a new way to study microbial community energy production, biosynthesis, C use efficiency (the proportion of substrate incorporated into microbial biomass), and enables the quantification of C fluxes through the central C metabolic network processes (Dijkstra et al 2011a,b). We determined the 13CO2 production from U-13C, 1-13C, 2-13C, 3-13C, 4-13C, 5-13C, and 6-13C labeled glucose and 1-13C and 2,3-13C pyruvate in parallel incubations in three soils along an elevation gradient. Qualitative and quantitative interpretation of the results indicate a high pentose phosphate pathway activity in soils. Agreement between modeled and measured CO2 production rates for the six C-atoms of 13C-labeled glucose indicate that the metabolic model used is appropriate for soil community processes, but that improvements can be made. These labeling and modeling techniques may improve our ability to analyze the biochemistry and (eco)physiology of intact microbial communities. Dijkstra, P., Blankinship, J.C., Selmants, P.C., Hart, S.C., Koch, G.W., Schwartz, E., Hungate, B.A., 2011a. Probing C flux patterns of soil microbial metabolic networks using parallel position-specific tracer labeling. Soil Biology & Biochemistry 43, 126-132. Dijkstra, P., Dalder, J.J., Selmants, P.C., Hart, S.C., Koch, G.W., Schwartz, E., Hungate, B.A., 2011b. Modeling soil metabolic processes using isotopologue pairs of position-specific 13C-labeled glucose and pyruvate. Soil Biology & Biochemistry 43, 1848-1857.

Development of scanning graphene Hall probes for magnetic microscopy

NASA Astrophysics Data System (ADS)

Schaefer, Brian T.; Wang, Lei; McEuen, Paul L.; Nowack, Katja C.

We discuss our progress on developing scanning Hall probes fabricated from hexagonal boron nitride (hBN)-encapsulated graphene, with the goal to image magnetic fields with submicron resolution. In contrast to scanning superconducting quantum interference device (SQUID) microscopy, this technique is compatible with a large applied magnetic field and not limited to cryogenic temperatures. The field sensitivity of a Hall probe depends inversely on carrier density, while the primary source of noise in the measurement is Johnson noise originating from the device resistance. hBN-encapsulated graphene demonstrates high carrier mobility at low carrier densities, therefore making it an ideal material for sensitive Hall probes. Furthermore, engineering the dielectric environment of graphene by encapsulating in hBN reduces low-frequency charge noise and disorder from the substrate. We outline our plans for adapting these devices for scanning, including characterization of the point spread function with a scanned current loop and fabrication of a deep-etched structure that enables positioning the sensitive area within 100 nanometers of the sample surface.

Development of dual-emission ratiometric probe-based on fluorescent silica nanoparticle and CdTe quantum dots for determination of glucose in beverages and human body fluids.

PubMed

Zhai, Hong; Feng, Ting; Dong, Lingyu; Wang, Liyun; Wang, Xiangfeng; Liu, Hailing; Liu, Yuan; Chen, Luan; Xie, MengXia

2016-08-01

A novel dual emission ratiometric fluorescence probe for determination of glucose has been developed. The reference dye fluorescence isothiocyanate (FITC) has been encapsulated in the silica nanoparticles and then the red emission CdTe QDs were grafted on the surface of the silica particles to obtain the fluorescence probe. With glucose and dopamine as substrates, the glucose level was proportional to the fluorescence ratio change of above probe caused by dopamine oxidation, which was produced via bienzyme catalysis (glucose oxidase and horseradish peroxidase). The established approach was sensitive and selective, and has been applied to determine the glucose in beverage, urine and serum samples. The average recoveries of the glucose at various spiking levels ranged from 95.5% to 108.9% with relative standard deviations from 1.5% to 4.3%. The results provided a clue to develop sensors for rapid determination of the target analytes from complex matrices. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ultrasonic generator and detector using an optical mask having a grating for launching a plurality of spatially distributed, time varying strain pulses in a sample

DOEpatents

Maris, Humphrey J.

2003-01-01

A method and a system are disclosed for determining at least one characteristic of a sample that contains a substrate and at least one film disposed on or over a surface of the substrate. The method includes a first step of placing a mask over a free surface of the at least one film, where the mask has a top surface and a bottom surface that is placed adjacent to the free surface of the film. The bottom surface of the mask has formed therein or thereon a plurality of features for forming at least one grating. A next step directs optical pump pulses through the mask to the free surface of the film, where individual ones of the pump pulses are followed by at least one optical probe pulse. The pump pulses are spatially distributed by the grating for launching a plurality of spatially distributed, time varying strain pulses within the film, which cause a detectable change in optical constants of the film. A next step detects a reflected or a transmitted portion of the probe pulses, which are also spatially distributed by the grating. A next step measures a change in at least one characteristic of at least one of reflected or transmitted probe pulses due to the change in optical constants, and a further step determines the at least one characteristic of the sample from the measured change in the at least one characteristic of the probe pulses. An optical mask is also disclosed herein, and forms a part of these teachings.

Ultrasonic generator and detector using an optical mask having a grating for launching a plurality of spatially distributed, time varying strain pulses in a sample

DOEpatents

Maris, Humphrey J.

2002-01-01

A method and a system are disclosed for determining at least one characteristic of a sample that contains a substrate and at least one film disposed on or over a surface of the substrate. The method includes a first step of placing a mask over a free surface of the at least one film, where the mask has a top surface and a bottom surface that is placed adjacent to the free surface of the film. The bottom surface of the mask has formed therein or thereon a plurality of features for forming at least one grating. A next step directs optical pump pulses through the mask to the free surface of the film, where individual ones of the pump pulses are followed by at least one optical probe pulse. The pump pulses are spatially distributed by the grating for launching a plurality of spatially distributed, time varying strain pulses within the film, which cause a detectable change in optical constants of the film. A next step detects a reflected or a transmitted portion of the probe pulses, which are also spatially distributed by the grating. A next step measures a change in at least one characteristic of at least one of reflected or transmitted probe pulses due to the change in optical constants, and a further step determines the at least one characteristic of the sample from the measured change in the at least one characteristic of the probe pulses. An optical mask is also disclosed herein, and forms a part of these teachings.

Pitavastatin is a more sensitive and selective organic anion-transporting polypeptide 1B clinical probe than rosuvastatin

PubMed Central

Prueksaritanont, Thomayant; Chu, Xiaoyan; Evers, Raymond; Klopfer, Stephanie O; Caro, Luzelena; Kothare, Prajakti A; Dempsey, Cynthia; Rasmussen, Scott; Houle, Robert; Chan, Grace; Cai, Xiaoxin; Valesky, Robert; Fraser, Iain P; Stoch, S Aubrey

2014-01-01

Aims Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion-transporting polypeptide (OATP) 1B, but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral (PO) and intravenous (IV) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters. Methods The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV. The in vitro transporter studies were performed using hepatocytes and recombinant expression systems. Results Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration–time curve were 5.7- and 7.6-fold for pitavastatin and 4.4- and 3.3-fold for rosuvastatin, after PO and IV rifampicin, respectively. In vitro studies showed that rifampicin was an inhibitor of OATP1B1 and OATP1B3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3. Conclusions The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose. PMID:24617605

Urinary p-cresol diagnosis using nanocomposite of ZnO/MoS2 and molecular imprinted polymer on optical fiber based lossy mode resonance sensor.

PubMed

Usha, Sruthi P; Gupta, Banshi D

2018-03-15

A lossy mode resonance (LMR) based sensor for urinary p-cresol testing on optical fiber substrate is developed. The sensor probe fabrication includes dip coating of nanocomposite layer of zinc oxide and molybdenum sulphide (ZnO/MoS 2 ) over unclad core of optical fiber as the transducer layer followed by the layer of molecular imprinted polymer (MIP) as the recognition medium. The addition of molybdenum sulphide in the transducer layer increases the absorption of light in the medium which enhances the LMR properties of zinc oxide thereby increasing the conductivity and hence the sensitivity of the sensor. The sensor probe is characterized for p-cresol concentration range from 0µM (reference sample) to 1000µM in artificially prepared urine. Optimizations of various probe fabrication parameters are carried to bring out the sensor's optimal performance with a sensitivity of 11.86nm/µM and 28nM as the limit of detection (LOD). A two-order improvement in LOD is obtained as compared to the recently reported p-cresol sensor. The proposed sensor possesses a response time of 15s which is 8 times better than that reported in the literature utilizing electrochemical method. Its response time is also better than the p-cresol sensor currently available in the market for the medical field. Thus, with a fast response, significant stability and repeatability, the proposed sensor holds practical implementation possibilities in the medical field. Further, the realization of sensor probe over optical fiber substrate adds remote sensing and online monitoring feasibilities. Copyright © 2017 Elsevier B.V. All rights reserved.

The effect of substrate temperature on the microstructural, electrical and optical properties of Sn-doped indium oxide thin films

NASA Astrophysics Data System (ADS)

Raoufi, Davood; Taherniya, Atefeh

2015-06-01

In this work, Sn doping In2O3 (ITO) thin films with a thickness of 200 nm were deposited on glass substrates by electron beam evaporation (EBE) method at different substrate temperatures. The crystal structure of these films was studied by X-ray diffraction technique. The sheet resistance was measured by a four-point probe. Van der Pauw method was used to measure carrier density and mobility of ITO films. The optical transmittance spectra were recorded in the wavelength region of 300-800 nm. Scanning electron microscope (SEM) has been used for the surface morphology analysis. The prepared ITO films exhibited body-centered cubic (BCC) structure with preferred orientation of growth along the (2 2 2) crystalline plane. The grain size of the films increases by rising the substrate temperature. Transparency of the films, over the visible light region, is increased with increasing the substrate temperature. It is found that the electrical properties of ITO films are significantly affected by substrate temperature. The electrical resistivity decreases with increasing substrate temperature, whereas the carrier density and mobility are enhanced with an increase in substrate temperature. The evaluated values of energy band gap Eg for ITO films were increase from 3.84 eV to 3.91 eV with increasing the substrate temperatures from 200 °C to 500 °C. The SEM micrographs of the films revealed a homogeneous growth without perceptible cracks with particles which are well covered on the substrate.

Quantitative isotope incorporation reveals substrate partitioning in a coastal microbial community.

PubMed

Mayali, Xavier; Weber, Peter K

2018-05-01

To quantitatively link microbial identity with biogeochemical function, we carried out 14 simultaneous stable isotope probing experiments with organic and inorganic C and N substrates to measure the isotope incorporation by over one hundred co-occurring eukaryotic and prokaryotic populations in a coastal community. We found that nitrate was the most commonly incorporated substrate, and that light-driven carbon fixation was carried out by some bacterial taxa from the Flavobacteriales and OM60 (NOR5) clade, in addition to photoautotrophic phytoplankton. We found that organisms that incorporated starch, maltose, glucose, lactose and bicarbonate were phylogenetically clustered, suggesting that specific bacterial lineages specialized in the incorporation of these substrates. The data further revealed that coastal microorganisms spanned a range of resource utilization strategies from generalists to specialists and demonstrated a high level of substrate partitioning, with two thirds of taxa exhibiting unique substrate incorporation patterns and the remaining third shared by no more than three OTUs each. Specialists exhibited more extreme incorporation levels (high or low), whereas generalists displayed more intermediate activity levels. These results shed valuable insights into the bottom-up ecological strategies enabling the persistence of high microbial diversity in aquatic ecosystems.

Highly Sensitive, Uniform, and Reproducible Surface-Enhanced Raman Spectroscopy Substrate with Nanometer-Scale Quasi-periodic Nanostructures.

PubMed

Jin, Yuanhao; Wang, Yingcheng; Chen, Mo; Xiao, Xiaoyang; Zhang, Tianfu; Wang, Jiaping; Jiang, Kaili; Fan, Shoushan; Li, Qunqing

2017-09-20

We introduce a simple and cost-effective approach for fabrication of effective surface-enhanced Raman spectroscopy (SERS) substrates. It is shown that the as-fabricated substrates show excellent SERS effects in various probe molecules with high sensitivity, that is, picomolar level detection, and also good reliability. With a SERS enhancement factor beyond 10 8 and excellent reproducibility (deviation less than 5%) of signal intensity, the fabrication of the SERS substrate is realized on a four-inch wafer and proven to be effective in pesticide residue detection. The SERS substrate is realized first through the fabrication of quasi-periodic nanostructured silicon with dimension features in tens of nanometers using superaligned carbon nanotubes networks as an etching mask, after which a large amount of hot spots with nanometer gaps are formed through deposition of a gold film. With rigorous nanostructure design, the enhanced performance of electromagnetic field distribution for nanostructures is optimized. With the advantage of cost-effective large-area preparation, it is believed that the as-fabricated SERS substrate could be used in a wide variety of actual applications where detection of trace amounts is necessary.

Efficient surface enhanced Raman scattering on confeito-like gold nanoparticle-adsorbed self-assembled monolayers.

PubMed

Chang, Chia-Chi; Imae, Toyoko; Chen, Liang-Yih; Ujihara, Masaki

2015-12-28

Confeito-like gold nanoparticles (AuNPs; average diameter = 80 nm) exhibiting a plasmon absorption band at 590 nm were adsorbed through immersion-adsorption on two self-assembled monolayers (SAMs) of 3-aminopropyltriethoxysilane (APTES-SAM) and polystyrene spheres coated with amine-terminated poly(amido amine) dendrimers (DEN/PS-SAM). The surface enhanced Raman scattering (SERS) effect on the SAM substrates was examined using the molecules of a probe dye, rhodamine 6G (R6G). The Raman scattering was strongly intensified on both substrates, but the enhancement factor (>10,000) of the AuNP/DEN/PS-SAM hierarchy substrate was 5-10 times higher than that of the AuNP/APTES-SAM substrate. This strong enhancement is attributed to the large surface area of the substrate and the presence of hot spots. Furthermore, analyzing the R6G concentration dependence of SERS suggested that the enhancement mechanism effectively excited the R6G molecules in the first layer on the hot spots and invoked the strong SERS effect. These results indicate that the SERS activity of confeito-like AuNPs on SAM substrates has high potential in molecular electronic devices and ultrasensitive analyses.

Modulation of Rat Hepatic CYP1A and 2C Activity by Honokiol and Magnolol: Differential Effects on Phenacetin and Diclofenac Pharmacokinetics In Vivo.

PubMed

Kim, Sang-Bum; Kim, Kyu-Sang; Ryu, Heon-Min; Hong, Seong-Ho; Kim, Bo-Kyoung; Kim, Dae-Duk; Park, Jin Woo; Yoon, In-Soo

2018-06-17

Honokiol (2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol) and magnolol (4-Allyl-2-(5-allyl-2-hydroxy-phenyl)phenol) are the major active polyphenol constituents of Magnolia officinalis (Magnoliaceae) bark, which has been widely used in traditional Chinese medicine (Houpu Tang) for the treatment of various diseases, including anxiety, stress, gastrointestinal disorders, infection, and asthma. The aim of this study was to investigate the direct effects of honokiol and magnolol on hepatic CYP1A and 2C-mediated metabolism in vitro using rat liver microsomes and in vivo using the Sprague-Dawley rat model. Honokiol and magnolol inhibited in vitro CYP1A activity (probe substrate: phenacetin) more potently than CYP2C activity (probe substrate: diclofenac): The mean IC 50 values of honokiol for the metabolism of phenacetin and diclofenac were 8.59 μM and 44.7 μM, while those of magnolol were 19.0 μM and 47.3 μM, respectively. Notably, the systemic exposure (AUC and C max ) of phenacetin, but not of diclofenac, was markedly enhanced by the concurrent administration of intravenous honokiol or magnolol. The differential effects of the two phytochemicals on phenacetin and diclofenac in vivo pharmacokinetics could at least be partly attributed to their lower IC 50 values for the inhibition of phenacetin metabolism than for diclofenac metabolism. In addition, the systemic exposure, CL, and V ss of honokiol and magnolol tended to be similar between the rat groups receiving phenacetin and diclofenac. These findings improve our understanding of CYP-mediated drug interactions with M. officinalis and its active constituents.

Acoustic Transducers as Passive Cooperative Targets for Wireless Sensing of the Sub-Surface World: Challenges of Probing with Ground Penetrating RADAR

PubMed Central

Martin, Gilles; Goavec-Mérou, Gwenhael; Rabus, David; Alzuaga, Sébastien; Arapan, Lilia; Sagnard, Marianne; Carry, Émile

2018-01-01

Passive wireless transducers are used as sensors, probed by a RADAR system. A simple way to separate the returning signal from the clutter is to delay the response, so that the clutter decays before the echoes are received. This can be achieved by introducing a fixed delay in the sensor design. Acoustic wave transducers are ideally suited as cooperative targets for passive, wireless sensing. The incoming electromagnetic pulse is converted into an acoustic wave, propagated on the sensor substrate surface, and reflected as an electromagnetic echo. According to a known law, the acoustic wave propagation velocity depends on the physical quantity under investigation, which is then measured as an echo delay. Both conversions between electromagnetic and acoustic waves are based on the piezoelectric property of the substrate of which the sensor is made. Investigating underground sensing, we address the problems of using GPR (Ground-Penetrating RADAR) for probing cooperative targets. The GPR is a good candidate for this application because it provides an electromagnetic source and receiver, as well as echo recording tools. Instead of designing dedicated electronics, we choose a commercially available, reliable and rugged instrument. The measurement range depends on parameters like antenna radiation pattern, radio spectrum matching between GPR and the target, antenna-sensor impedance matching and the transfer function of the target. We demonstrate measurements at depths ranging from centimeters to circa 1 m in a sandbox. In our application, clutter rejection requires delays between the emitted pulse and echoes to be longer than in the regular use of the GPR for geophysical measurements. This delay, and the accuracy needed for sensing, challenge the GPR internal time base. In the GPR units we used, the drift turns out to be incompatible with the targeted application. The available documentation of other models and brands suggests that this is a rather general limitation. We solved the problem by replacing the analog ramp generator defining the time base with a fully digital solution, whose time accuracy and stability relies on a quartz oscillator. The resulting stability is acceptable for sub-surface cooperative sensor measurement. PMID:29337914

Acoustic Transducers as Passive Cooperative Targets for Wireless Sensing of the Sub-Surface World: Challenges of Probing with Ground Penetrating RADAR.

PubMed

Friedt, Jean-Michel; Martin, Gilles; Goavec-Mérou, Gwenhael; Rabus, David; Alzuaga, Sébastien; Arapan, Lilia; Sagnard, Marianne; Carry, Émile

2018-01-16

Passive wireless transducers are used as sensors, probed by a RADAR system. A simple way to separate the returning signal from the clutter is to delay the response, so that the clutter decays before the echoes are received. This can be achieved by introducing a fixed delay in the sensor design. Acoustic wave transducers are ideally suited as cooperative targets for passive, wireless sensing. The incoming electromagnetic pulse is converted into an acoustic wave, propagated on the sensor substrate surface, and reflected as an electromagnetic echo. According to a known law, the acoustic wave propagation velocity depends on the physical quantity under investigation, which is then measured as an echo delay. Both conversions between electromagnetic and acoustic waves are based on the piezoelectric property of the substrate of which the sensor is made. Investigating underground sensing, we address the problems of using GPR (Ground-Penetrating RADAR) for probing cooperative targets. The GPR is a good candidate for this application because it provides an electromagnetic source and receiver, as well as echo recording tools. Instead of designing dedicated electronics, we choose a commercially available, reliable and rugged instrument. The measurement range depends on parameters like antenna radiation pattern, radio spectrum matching between GPR and the target, antenna-sensor impedance matching and the transfer function of the target. We demonstrate measurements at depths ranging from centimeters to circa 1 m in a sandbox. In our application, clutter rejection requires delays between the emitted pulse and echoes to be longer than in the regular use of the GPR for geophysical measurements. This delay, and the accuracy needed for sensing, challenge the GPR internal time base. In the GPR units we used, the drift turns out to be incompatible with the targeted application. The available documentation of other models and brands suggests that this is a rather general limitation. We solved the problem by replacing the analog ramp generator defining the time base with a fully digital solution, whose time accuracy and stability relies on a quartz oscillator. The resulting stability is acceptable for sub-surface cooperative sensor measurement.

Chronic neural probe for simultaneous recording of single-unit, multi-unit, and local field potential activity from multiple brain sites

NASA Astrophysics Data System (ADS)

Pothof, F.; Bonini, L.; Lanzilotto, M.; Livi, A.; Fogassi, L.; Orban, G. A.; Paul, O.; Ruther, P.

2016-08-01

Objective. Drug resistant focal epilepsy can be treated by resecting the epileptic focus requiring a precise focus localisation using stereoelectroencephalography (SEEG) probes. As commercial SEEG probes offer only a limited spatial resolution, probes of higher channel count and design freedom enabling the incorporation of macro and microelectrodes would help increasing spatial resolution and thus open new perspectives for investigating mechanisms underlying focal epilepsy and its treatment. This work describes a new fabrication process for SEEG probes with materials and dimensions similar to clinical probes enabling recording single neuron activity at high spatial resolution. Approach. Polyimide is used as a biocompatible flexible substrate into which platinum electrodes and leads are integrated with a minimal feature size of 5 μm. The polyimide foils are rolled into the cylindrical probe shape at a diameter of 0.8 mm. The resulting probe features match those of clinically approved devices. Tests in saline solution confirmed the probe stability and functionality. Probes were implanted into the brain of one monkey (Macaca mulatta), trained to perform different motor tasks. Suitable configurations including up to 128 electrode sites allow the recording of task-related neuronal signals. Main results. Probes with 32 and 64 electrode sites were implanted in the posterior parietal cortex. Local field potentials and multi-unit activity were recorded as early as one hour after implantation. Stable single-unit activity was achieved for up to 26 days after implantation of a 64-channel probe. All recorded signals showed modulation during task execution. Significance. With the novel probes it is possible to record stable biologically relevant data over a time span exceeding the usual time needed for epileptic focus localisation in human patients. This is the first time that single units are recorded along cylindrical polyimide probes chronically implanted 22 mm deep into the brain of a monkey, which suggests the potential usefulness of this probe for human applications.

Chronic neural probe for simultaneous recording of single-unit, multi-unit, and local field potential activity from multiple brain sites.

PubMed

Pothof, F; Bonini, L; Lanzilotto, M; Livi, A; Fogassi, L; Orban, G A; Paul, O; Ruther, P

2016-08-01

Drug resistant focal epilepsy can be treated by resecting the epileptic focus requiring a precise focus localisation using stereoelectroencephalography (SEEG) probes. As commercial SEEG probes offer only a limited spatial resolution, probes of higher channel count and design freedom enabling the incorporation of macro and microelectrodes would help increasing spatial resolution and thus open new perspectives for investigating mechanisms underlying focal epilepsy and its treatment. This work describes a new fabrication process for SEEG probes with materials and dimensions similar to clinical probes enabling recording single neuron activity at high spatial resolution. Polyimide is used as a biocompatible flexible substrate into which platinum electrodes and leads are integrated with a minimal feature size of 5 μm. The polyimide foils are rolled into the cylindrical probe shape at a diameter of 0.8 mm. The resulting probe features match those of clinically approved devices. Tests in saline solution confirmed the probe stability and functionality. Probes were implanted into the brain of one monkey (Macaca mulatta), trained to perform different motor tasks. Suitable configurations including up to 128 electrode sites allow the recording of task-related neuronal signals. Probes with 32 and 64 electrode sites were implanted in the posterior parietal cortex. Local field potentials and multi-unit activity were recorded as early as one hour after implantation. Stable single-unit activity was achieved for up to 26 days after implantation of a 64-channel probe. All recorded signals showed modulation during task execution. With the novel probes it is possible to record stable biologically relevant data over a time span exceeding the usual time needed for epileptic focus localisation in human patients. This is the first time that single units are recorded along cylindrical polyimide probes chronically implanted 22 mm deep into the brain of a monkey, which suggests the potential usefulness of this probe for human applications.

Analyzing the biosensor signal in flows: studies with glucose optrodes.

PubMed

Kivirand, K; Floren, A; Kagan, M; Avarmaa, T; Rinken, T; Jaaniso, R

2015-01-01

Responses of enzymatic bio-optrodes in flow regime were studied and an original model was proposed with the aim of establishing a reliable method for a quick determination of biosensor signal parameters, applicable for biosensor calibration. A dual-optrode glucose biosensor, comprising of a glucose bio-optrode and a reference oxygen optrode, both placed into identical flow channels, was developed and used as a model system. The signal parameters of this biosensor at different substrate concentrations were not dependent on the speed of the probe flow and could be determined from the initial part of the biosensor transient phase signal, providing a valuable tool for rapid analysis. In addition, the model helped to design the biosensor system with reduced impact of enzyme inactivation to the system stability (20% decrease of the enzyme activity lead to only a 1% decrease of the slope of the calibration curve) and hence significantly prolong the effective lifetime of bio-optrodes. Copyright © 2014 Elsevier B.V. All rights reserved.

Template-based preparation of free-standing semiconducting polymeric nanorod arrays on conductive substrates.

PubMed

Haberkorn, Niko; Weber, Stefan A L; Berger, Rüdiger; Theato, Patrick

2010-06-01

We describe the synthesis and characterization of a cross-linkable siloxane-derivatized tetraphenylbenzidine (DTMS-TPD), which was used for the fabrication of semiconducting highly ordered nanorod arrays on conductive indium tin oxide or Pt-coated substrates. The stepwise process allow fabricating of macroscopic areas of well-ordered free-standing nanorod arrays, which feature a high resistance against organic solvents, semiconducting properties and a good adhesion to the substrate. Thin films of the TPD derivate with good hole-conducting properties could be prepared by cross-linking and covalently attaching to hydroxylated substrates utilizing an initiator-free thermal curing at 160 degrees C. The nanorod arrays composed of cross-linked DTMS-TPD were fabricated by an anodic aluminum oxide (AAO) template approach. Furthermore, the nanorod arrays were investigated by a recently introduced method allowing to probe local conductivity on fragile structures. It revealed that more than 98% of the nanorods exhibit electrical conductance and consequently feature a good electrical contact to the substrate. The prepared nanorod arrays have the potential to find application in the fabrication of multilayered device architectures for building well-ordered bulk-heterojunction solar cells.

Piezoelectric substrate effect on electron-acoustic phonon scattering in bilayer graphene

NASA Astrophysics Data System (ADS)

Ansari, Mohd Meenhaz; Ashraf, SSZ

2018-05-01

We have studied the effect of piezoelectric scattering as a function of electron temperature and distance between the sample and the substrate on electron-acoustic phonon scattering rate in Bilayer Graphene sitting on a piezoelectric substrate. We obtain approximate analytical result by neglecting the chiral nature of carriers and then proceed to obtain unapproximated numerical results for the scattering rate incorporating chirality of charge carriers. We find that on the incorporation of full numerical computation the magnitude as well as the power exponent both is affected with the power exponent changed from T3 to T3.31 in the low temperature range and to T6.98 dependence in the temperature range (>5K). We also find that the distance between the sample and substrate begins to strongly affect the scattering rate at temperatures above 10K. These calculation not only suggest the influencing effect of piezoelectric substrate on the transport properties of Dirac Fermions at very low temperatures but also open a channel to study low dimension structures by probing piezoelectric acoustical phonons.

An Einzel lens apparatus for deposition of levitated graphene on a substrate in UHV

NASA Astrophysics Data System (ADS)

Coppock, Joyce; Nagornykh, Pavel; McAdams, Ian; Kane, Bruce

The goal of our research is to levitate a charged micron-scale graphene flake in an electrical AC quadrupole trap in ultra-high vacuum (UHV) in order to study its properties and dynamics while decoupled from any substrate. As a complement to the optical measurements that can be performed on the levitated flake, we are developing a method of depositing the same flake on a substrate, which can be removed from the system for further study using such probes as atomic force microscopy (AFM) and scanning tunneling microscopy (STM). As the flake is released from the trap and propelled toward the substrate, its trajectory will be controlled by an Einzel (electrostatic) lens to achieve accurate positioning on the substrate. This talk will discuss the design of the lens as well as particle tracing simulations to determine the proper lens voltage to focus the particle's trajectory. In the future, deposited graphene may be used to passivate H-terminated silicon. The method is expected to be generalizable to achieve deposition of 2D materials on surfaces in a clean UHV environment.

Deposition of Nanostructured CdS Thin Films by Thermal Evaporation Method: Effect of Substrate Temperature

PubMed Central

Memarian, Nafiseh; Rozati, Seyeed Mohammad; Concina, Isabella

2017-01-01

Nanocrystalline CdS thin films were grown on glass substrates by a thermal evaporation method in a vacuum of about 2 × 10−5 Torr at substrate temperatures ranging between 25 °C and 250 °C. The physical properties of the layers were analyzed by transmittance spectra, XRD, SEM, and four-point probe measurements, and exhibited strong dependence on substrate temperature. The XRD patterns of the films indicated the presence of single-phase hexagonal CdS with (002) orientation. The structural parameters of CdS thin films (namely crystallite size, number of grains per unit area, dislocation density and the strain of the deposited films) were also calculated. The resistivity of the as-deposited films were found to vary in the range 3.11–2.2 × 104 Ω·cm, depending on the substrate temperature. The low resistivity with reasonable transmittance suggest that this is a reliable way to fine-tune the functional properties of CdS films according to the specific application. PMID:28773133

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase*

PubMed Central

Graham, Brian W.; Tao, Yeqing; Dodge, Katie L.; Thaxton, Carly T.; Olaso, Danae; Young, Nicolas L.; Marshall, Alan G.

2016-01-01

The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect single-stranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5–30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding. PMID:27044751

Substrate viscosity enhances correlation in epithelial sheet movement.

PubMed

Murrell, Michael; Kamm, Roger; Matsudaira, Paul

2011-07-20

The movement of the epithelium plays vital roles in the development and renewal of complex tissues, from the separation of tissues in the early embryo, to turnover in the homeostasis of the gastrointestinal mucosa. Yet, despite its importance, a clear interpretation of the mechanism for collective motion in epithelial sheets remains elusive. This interpretation is prohibited by the lack of understanding of the relationship between motion and cell-cell contact, and their mediation by the mechanical properties of the underlying substrate. To better mimic physiological substrates that have inherent viscosity, we probe this relationship using polydimethylsiloxane, a substrate whose mechanical properties can be tuned from predominantly elastic to viscous by altering its cross-linking content. We therefore characterize the comparative spatiotemporal correlations in cell velocity during the movement of an epithelial monolayer as a function of the viscoelasticity of the substrate. Our results show that high correlation in cell velocity is achieved when the substrate G''(ω) is ~0.4 × G'(ω). This correlation is driven by a balance between cell-cell contact and the adhesion and contraction of the extracellular matrix. For G'(ω) > G'(ω), this balance shifts, and contraction of the tissue drives the substrate to flow, further elevating the correlation in movement. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Neurocircuitry models of posttraumatic stress disorder and extinction: human neuroimaging research--past, present, and future.

PubMed

Rauch, Scott L; Shin, Lisa M; Phelps, Elizabeth A

2006-08-15

The prevailing neurocircuitry models of anxiety disorders have been amygdalocentric in form. The bases for such models have progressed from theoretical considerations, extrapolated from research in animals, to in vivo human imaging data. For example, one current model of posttraumatic stress disorder (PTSD) has been highly influenced by knowledge from rodent fear conditioning research. Given the phenomenological parallels between fear conditioning and the pathogenesis of PTSD, we have proposed that PTSD is characterized by exaggerated amygdala responses (subserving exaggerated acquisition of fear associations and expression of fear responses) and deficient frontal cortical function (mediating deficits in extinction and the capacity to suppress attention/response to trauma-related stimuli), as well as deficient hippocampal function (mediating deficits in appreciation of safe contexts and explicit learning/memory). Neuroimaging studies have yielded convergent findings in support of this model. However, to date, neuroimaging investigations of PTSD have not principally employed conditioning and extinction paradigms per se. The recent development of such imaging probes now sets the stage for directly testing hypotheses regarding the neural substrates of fear conditioning and extinction abnormalities in PTSD.

Differential photo-acoustic gas cell based on LTCC for ppm gas sensing

NASA Astrophysics Data System (ADS)

Keränen, K.; Kautio, K.; Ollila, J.; Heikkinen, M.; Kauppinen, I.; Kuusela, T.; Matveev, B.; McNie, M. E.; Jenkins, R. M.; Karioja, P.

2010-02-01

Silicon MEMS cantilever-based photoacoustic technology allows for the sensing of ultra low gas concentrations with very wide dynamic range. The sensitivity enhancement is achieved with a cantilever microphone system in which the cantilever displacement is probed with an optical interferometer providing a pico-meter resolution. In the gas sensor, the silicon cantilever microphone is placed in a two-chamber differential gas cell. By monitoring differential pressure changes between the two chambers, the differential cell operates as a differential infra-red detector for optical absorption signals through a measurement and reference path. The differential pressure signal is proportional to gas concentration in the optical measurement path. We have designed, implemented and tested a differential photo-acoustic gas cell based on Low Temperature Co-fired Ceramic (LTCC) multilayer substrate technology. Standard LTCC technology enables implementation of 2.5D structures including holes, cavities and channels into the electronic substrate. The implemented differential photoacoustic gas cell structure includes two 10 mm long cylindrical cells, diameter of 2.4 mm. Reflectance measurements of the cell showed that reflectivity of the substrate material can be improved by a factor 15 - 90 in the 3 - 8 μm spectral region using gold or silver paste coatings. A transparent window is required in the differential gas cell structure in order to probe the displacement of the silicon cantilever. The transparent sapphire window was sealed to the LTCC substrate using two methods: screen printed Au80/Sn20 solder paste and pre-attached glass solder paste (Diemat DM2700P/H848). Both methods were shown to provide hermetic sealing of sapphire windows to LTCC substrate. The measured He-leak rate for the 10 sealed test samples implemented using glass paste were under 2.0 ×10-9 atm×cm3/s, which meets the requirement for the leak rate according to MIL-STD 883. The achieved hermeticity level suggests that the proof-of-principle packaging demonstrator paves the way for implementing a novel differential photoacoustic gas cell for a future miniature gas sensor module. The future module consisting of a sample gas cell and immersion lens IR LEDs together with interferometric probing of the cantilever microphone is expected to be capable of measuring ultra low concentrations of a wide range of gases with their fundamental absorption bands at 3 - 7 μm wavelength, such as CO, CO2 and CH4.

Measurements of the asymmetric dynamic sheath around a pulse biased sphere immersed in flowing metal plasma

NASA Astrophysics Data System (ADS)

Wu, Hongchen; Anders, André

2008-08-01

A long-probe technique was utilized to record the expansion and retreat of the dynamic sheath around a spherical substrate immersed in pulsed cathode arc metal plasma. Positively biased, long cylindrical probes were placed on the side and downstream of a negatively pulsed biased stainless steel sphere of 1 in. (25.4 mm) diameter. The amplitude and width of the negative high voltage pulses (HVPs) were 2 kV, 5 kV, 10 kV, and 2 µs, 4 µs, 10 µs, respectively. The variation of the probe (electron) current during the HVP is a direct measure for the sheath expansion and retreat. Maximum sheath sizes were determined for the different parameters of the HVP. The expected rarefaction zone behind the biased sphere (wake) due to the fast plasma flow was clearly established and quantified.

Prefrontal Control of Familiarity and Recollection in Working Memory

ERIC Educational Resources Information Center

Feredoes, Eva; Postle, Bradley R.

2010-01-01

Left inferior frontal gyrus (IFG) is a critical neural substrate for the resolution of proactive interference (PI) in working memory. We hypothesized that left IFG achieves this by controlling the influence of familiarity- versus recollection-based information about memory probes. Consistent with this idea, we observed evidence for an "early" (200…

Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

PubMed

Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

2015-01-25

Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

Dynamic and label-free high-throughput detection of biomolecular interactions based on phase-shift interferometry

NASA Astrophysics Data System (ADS)

Li, Qiang; Huang, Guoliang; Gan, Wupeng; Chen, Shengyi

2009-08-01

Biomolecular interactions can be detected by many established technologies such as fluorescence imaging, surface plasmon resonance (SPR)[1-4], interferometry and radioactive labeling of the analyte. In this study, we have designed and constructed a label-free, real-time sensing platform and its operating imaging instrument that detects interactions using optical phase differences from the accumulation of biological material on solid substrates. This system allows us to monitor biomolecular interactions in real time and quantify concentration changes during micro-mixing processes by measuring the changes of the optical path length (OPD). This simple interferometric technology monitors the optical phase difference resulting from accumulated biomolecular mass. A label-free protein chip that forms a 4×4 probe array was designed and fabricated using a commercial microarray robot spotter on solid substrates. Two positive control probe lines of BSA (Bovine Serum Albumin) and two experimental human IgG and goat IgG was used. The binding of multiple protein targets was performed and continuously detected by using this label-free and real-time sensing platform.

Base-modified GDP-mannose derivatives and their substrate activity towards a yeast mannosyltransferase.

PubMed

Collier, Alice; Wagner, Gerd K

2017-11-27

We have previously developed a new class of inhibitors and chemical probes for glycosyltransferases through base-modification of the sugar-nucleotide donor. The key feature of these donor analogues is the presence of an additional substituent at the nucleobase. To date, the application of this general concept has been limited to UDP-sugars and UDP-sugar-dependent glycosyltransferases. Herein, we report for the first time the application of our approach to a GDP-mannose-dependent mannosyltransferase. We have prepared four GDP-mannose derivatives with an additional substituent at either position 6 or 8 of the nucleobase. These donor analogues were recognised as donor substrates by the mannosyltransferase Kre2p from yeast, albeit with significantly lower turnover rates than the natural donor GDP-mannose. The presence of the additional substituent also redirected enzyme activity from glycosyl transfer to donor hydrolysis. Taken together, our results suggest that modification of the donor nucleobase is, in principle, a viable strategy for probe and inhibitor development against GDP-mannose-dependent GTs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural Studies of the Initial Stages of Fluoride Epitaxy on Silicon and GERMANIUM(111)

NASA Astrophysics Data System (ADS)

Denlinger, Jonathan David

The epitaxial growth of ionic insulators on semiconductor substrates is of interest due to fundamental issues of interface bonding and structure as well as to potential technological applications. The initial stages of Group IIa fluoride insulator growth on (111) Si and Ge substrates by molecular beam epitaxy are studied with the in situ combination of X-ray Photoelectron Spectroscopy (XPS) and Diffraction (XPD). While XPS probes the electronic structure, XPD reveals atomic structure. In addition, low energy electron diffraction (LEED) is used to probe surface order and a separate study using X-ray standing wave (XSW) fluorescence reveals interface cation bonding sites. Following the formation of a chemically-reacted interface layer in CaF_2 epitaxy on Si(111), the morphology of the subsequent bulk layers is found to be dependent on substrate temperature and incident flux rate. At temperatures >=600 ^circC a transition from three -dimensional island formation at low flux to laminar growth at higher flux is observed with bulk- and interface-resolved XPD. At lower substrate temperatures, laminar growth is observed at all fluxes, but with different bulk nucleation behavior due to changes in the stoichiometry of the interface layer. This new observation of kinetic effects on the initial nucleation in CaF_2 epitaxy has important ramifications for the formation of thicker heterostructures for scientific or device applications. XPS and XPD are also used to identify for the first time, surface core-level species of Ca and F, and a secondary interface-shifted F Auger component arising from a second-layer site directly above interface-layer Ca atoms. The effects of lattice mismatch (from -3% to 8%) are investigated with various growths of Ca_{rm x}Sr _{rm 1-x}F_2 on Si and Ge (111) substrates. Triangulation of (111) and (220) XSW indicates a predominance of 3-fold hollow Sr bonding sites coexisting with 4-fold top sites for monolayers of SrF_2 on Si. XSW and LEED reveal a lateral discommensuration of the overlayer for lattice mismatches of >5% relative to the substrate. XPD also reveals a transition from single - to mixed-domains of overlayer crystallographic orientation for mismatches >=3.5%.

Laser Altimeter for Flight Simulator

NASA Technical Reports Server (NTRS)

Webster, L. D.

1986-01-01

Height of flight-simulator probe above model of terrain measured by automatic laser triangulation system. Airplane simulated by probe that moves over model of terrain. Altitude of airplane scaled from height of probe above model. Height measured by triangulation of laser beam aimed at intersection of model surface with plumb line of probe.

Microstructure evolution of the Ir-inserted Ni silicides with additional annealing

NASA Astrophysics Data System (ADS)

Yoon, Kijeong; Song, Ohsung

2009-02-01

Thermally-evaporated 10 nm-Ni/1 nm-Ir/(poly)Si structures were fabricated in order to investigate the thermal stability of Ir-inserted nickel silicide after additional annealing. The silicide samples underwent rapid thermal annealing at 300 ° C to 1200 ° C for 40 s, followed by 30 min annealing at the given RTA temperatures. Silicides suitable for the salicide process were formed on the top of the single crystal and polycrystalline silicon substrates, mimicking actives and gates. The sheet resistance was measured using a four-point probe. High resolution x-ray diffraction and Auger depth profiling were used for phase and chemical composition analysis, respectively. Transmission electron microscope and scanning probe microscope were used to determine the cross-section structure and surface roughness. The silicide, which formed on single crystal silicon substrate with surface agglomeration after additional annealing, could defer the transformation of Ni(Ir)Si to Ni(Ir)Si2 and was stable at temperatures up to 1200 °C. Moreover, the silicide thickness doubled. There were no outstanding changes in the silicide thickness on polycrystalline silicon. However, after additional annealing, the silicon-silicide mixing became serious and showed high resistance at temperatures >700 °C. Auger depth profiling confirmed the increased thickness of the silicide layers after additional annealing without a change in composition. For a single crystal silicon substrate, the sheet resistance increased slightly due to the significant increases in surface roughness caused by surface agglomeration after additional annealing. Otherwise, there were almost no changes in surface roughness on the polycrystalline silicon substrate. The Ir-inserted nickel monosilicide was able to maintain a low resistance in a wide temperature range and is considered suitable for the nano-thick silicide process.

ZnO films on /001/-cut (110)-propagating GaAs substrates for surface acoustic wave device applications

NASA Technical Reports Server (NTRS)

Hickernell, Frederick S.; Higgins, Robert J.; Jen, Cheng-Kuei; Kim, Yoonkee; Hunt, William D.

1995-01-01

A potential application for piezoelectric films substrates is the monolithic integration of surface acoustic wave (SAW) devices with GaAs electronics. Knowledge of the SAW properties of the layered structure is critical for the optimum and accurate design of such devices. The acoustic properties of ZnO films sputtered on /001/-cut group of (110) zone axes-propagating GaAs substrates are investigated in this article, including SAW velocity, effective piezoelectric coupling constant, propagation loss, diffraction, velocity surface, and reflectivity of shorted and open metallic gratings. The measurements of these essential SAW properties for the frequency range between 180 and 360 MHz have been performed using a knife-edge laser probe for film thicknesses over the range of 1.6-4 micron and with films of different grain sizes. The high quality of dc triode sputtered films was observed as evidenced by high K(sup 2) and low attenuation. The measurements of the velocity surface, which directly affects the SAW diffraction, on the bare and metalized ZnO on SiO2 or Si3N4 on /001/-cut GaAs samples are reported using two different techniques: (1) knife-edge laser probe, (2) line-focus-beam scanning acoustic microscope. It was found that near the group of (110) zone axes propagation direction, the focusing SAW property of the bare GaAs changes into a nonfocusing one for the layered structure, but a reversed phenomenon exists near the (100) direction. Furthermore, to some extent the diffraction of the substrate can be controlled with the film thickness. The reflectivity of shorted and open gratings are also analyzed and measured. Zero reflectivity is observed for a shorted grating. There is good agreement between the measured data and theoretical values.

ZnO Films on {001}-Cut <110>-Propagating GaAs Substrates for Surface Acoustic Wave Device Applications

NASA Technical Reports Server (NTRS)

Kim, Yoonkee; Hunt, William D.; Hickernell, Frederick S.; Higgins, Robert J.; Jen, Cheng-Kuei

1995-01-01

A potential application for piezoelectric films on GaAs substrates is the monolithic integration of surface acoustic wave (SAW) devices with GaAs electronics. Knowledge of the SAW properties of the layered structure is critical for the optimum and accurate design of such devices. The acoustic properties of ZnO films sputtered on {001}-cut <110> -propagating GaAs substrates are investigated in this article, including SAW Velocity effective piezoelectric coupling constant, propagation loss. diffraction, velocity surface, and reflectivity of shorted and open metallic gratings. The measurements of these essential SAW properties for the frequency range between 180 and 360 MHz have been performed using a knife-edge laser probe for film thicknesses over the range of 1.6-4 micron and with films or different grain sizes. The high quality of dc triode sputtered films was observed as evidenced by high K(exp 2) and low attenuation. The measurements of the velocity surface, which directly affects the SAW diffraction, on the bare and metalized ZnO on SiO2, or Si3N4 on {001}-cut GaAs samples are reported using two different techniques: 1) knife-edge laser probe, 2) line-focus-beam scanning acoustic microscope. It was found that near the <110> propagation direction, the focusing SAW property of the bare GaAs changes into a nonfocusing one for the layered structure, but a reversed phenomenon exists near the <100> direction. Furthermore, to some extent the diffraction of the substrate can be controlled with the film thickness. The reflectivity of shorted and open gratings are also analyzed and measured. Zero reflectivity is observed for a shorted grating. There is good agreement between the measured data and theoretical values.

Integrated microfluidic probe station.

PubMed

Perrault, C M; Qasaimeh, M A; Brastaviceanu, T; Anderson, K; Kabakibo, Y; Juncker, D

2010-11-01

The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution--thus hydrodynamically confining the microjet--and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

Carbon-Based Solid-State Calcium Ion-Selective Microelectrode and Scanning Electrochemical Microscopy: A Quantitative Study of pH-Dependent Release of Calcium Ions from Bioactive Glass.

PubMed

Ummadi, Jyothir Ganesh; Downs, Corey J; Joshi, Vrushali S; Ferracane, Jack L; Koley, Dipankar

2016-03-15

Solid-state ion-selective electrodes are used as scanning electrochemical microscope (SECM) probes because of their inherent fast response time and ease of miniaturization. In this study, we report the development of a solid-state, low-poly(vinyl chloride), carbon-based calcium ion-selective microelectrode (Ca(2+)-ISME), 25 μm in diameter, capable of performing an amperometric approach curve and serving as a potentiometric sensor. The Ca(2+)-ISME has a broad linear response range of 5 μM to 200 mM with a near Nernstian slope of 28 mV/log[a(Ca(2+))]. The calculated detection limit for Ca(2+)-ISME is 1 μM. The selectivity coefficients of this Ca(2+)-ISME are log K(Ca(2+),A) = -5.88, -5.54, and -6.31 for Mg(2+), Na(+), and K(+), respectively. We used this new type of Ca(2+)-ISME as an SECM probe to quantitatively map the chemical microenvironment produced by a model substrate, bioactive glass (BAG). In acidic conditions (pH 4.5), BAG was found to increase the calcium ion concentration from 0.7 mM ([Ca(2+)] in artificial saliva) to 1.4 mM at 20 μm above the surface. In addition, a solid-state dual SECM pH probe was used to correlate the release of calcium ions with the change in local pH. Three-dimensional pH and calcium ion distribution mapping were also obtained by using these solid-state probes. The quantitative mapping of pH and Ca(2+) above the BAG elucidates the effectiveness of BAG in neutralizing and releasing calcium ions in acidic conditions.

Dual-colored graphene quantum dots-labeled nanoprobes/graphene oxide: functional carbon materials for respective and simultaneous detection of DNA and thrombin

NASA Astrophysics Data System (ADS)

Qian, Zhao Sheng; Shan, Xiao Yue; Chai, Lu Jing; Chen, Jian Rong; Feng, Hui

2014-10-01

Convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Herein, we report the respective and simultaneous detection of DNA and bovine α-thrombin (thrombin) entirely based on biocompatible carbon materials through a specially designed fluorescence on-off-on process. Colorful fluorescence, high emission efficiency, good photostability and excellent compatibility enables graphene quantum dots (GQDs) as the best choice for fluorophores in bioprobes, and thus two-colored GQDs as labeling fluorophores were chemically bonded with specific oligonucleotide sequence and aptamer to prepare two probes targeting the DNA and thrombin, respectively. Each probe can be assembled on the graphene oxide (GO) platform spontaneously by π-π stacking and electrostatic attraction; as a result, fast electron transfer in the assembly efficiently quenches the fluorescence of probe. The presence of DNA or thrombin can trigger the self-recognition between capturing a nucleotide sequence and its target DNA or between thrombin and its aptamer due to their specific hybridization and duplex DNA structures or the formation of apatamer-substrate complex, which is taken advantage of in order to achieve a separate quantitative analysis of DNA and thrombin. A dual-functional biosensor for simultaneous detection of DNA and thrombin was also constructed by self-assembly of two probes with distinct colors and GO platform, and was further evaluated with the presence of various concentrations of DNA and thrombin. Both biosensors serving as a general detection model for multiple species exhibit outstanding analytical performance, and are expected to be applied in vivo because of the excellent biocompatibility of their used materials.

Silver Nanoparticle-Decorated Shape-Memory Polystyrene Sheets as Highly Sensitive Surface-Enhanced Raman Scattering Substrates with a Thermally Inducible Hot Spot Effect.

PubMed

Mengesha, Zebasil Tassew; Yang, Jyisy

2016-11-15

In this study, an active surface-enhanced Raman scattering (SERS) substrate with a thermally inducible hot spot effect for sensitive measurement of Raman-active molecules was successfully fabricated from silver nanoparticle (AgNP)-decorated shape-memory polystyrene (SMP) sheets. To prepare the SERS substrate, SMP sheets were first pretreated with n-octylamine for effective decoration with AgNPs. By varying the formulation and condition of the reduction reaction, AgNP-decorated SMP (Ag@SMP) substrates were successfully prepared with optimized particle gaps to produce inducible hot spot effects on thermal shrink. High-quality SERS spectra were easily obtained with enhancement factors higher than 10 8 by probing with aromatic thiols. Several Ag@SMP substrates produced under different reaction conditions were explored for the creation of inducible hot spot effects. The results indicated that AgNP spacing is crucial for strong hot spot effects. The suitability of Ag@SMP substrates for quantification was also evaluated according to the detection of adenine. Results confirmed that prepared Ag@SMP substrates were highly suitable for quantitative analysis because they yielded an estimated limit of detection as low as 120 pg/cm 2 , a linear range of up to 7 ng/cm 2 , and a regression coefficient (R 2 ) of 0.9959. Ag@SMP substrates were highly reproducible; the average relative standard deviation for all measurements was less than 10%.

Preparation of nanowire specimens for laser-assisted atom probe tomography

NASA Astrophysics Data System (ADS)

Blumtritt, H.; Isheim, D.; Senz, S.; Seidman, D. N.; Moutanabbir, O.

2014-10-01

The availability of reliable and well-engineered commercial instruments and data analysis software has led to development in recent years of robust and ergonomic atom-probe tomographs. Indeed, atom-probe tomography (APT) is now being applied to a broader range of materials classes that involve highly important scientific and technological problems in materials science and engineering. Dual-beam focused-ion beam microscopy and its application to the fabrication of APT microtip specimens have dramatically improved the ability to probe a variety of systems. However, the sample preparation is still challenging especially for emerging nanomaterials such as epitaxial nanowires which typically grow vertically on a substrate through metal-catalyzed vapor phase epitaxy. The size, morphology, density, and sensitivity to radiation damage are the most influential parameters in the preparation of nanowire specimens for APT. In this paper, we describe a step-by-step process methodology to allow a precisely controlled, damage-free transfer of individual, short silicon nanowires onto atom probe microposts. Starting with a dense array of tiny nanowires and using focused ion beam, we employed a sequence of protective layers and markers to identify the nanowire to be transferred and probed while protecting it against Ga ions during lift-off processing and tip sharpening. Based on this approach, high-quality three-dimensional atom-by-atom maps of single aluminum-catalyzed silicon nanowires are obtained using a highly focused ultraviolet laser-assisted local electrode atom probe tomograph.

Computational substrates of norms and their violations during social exchange.

PubMed

Xiang, Ting; Lohrenz, Terry; Montague, P Read

2013-01-16

Social norms in humans constrain individual behaviors to establish shared expectations within a social group. Previous work has probed social norm violations and the feelings that such violations engender; however, a computational rendering of the underlying neural and emotional responses has been lacking. We probed norm violations using a two-party, repeated fairness game (ultimatum game) where proposers offer a split of a monetary resource to a responder who either accepts or rejects the offer. Using a norm-training paradigm where subject groups are preadapted to either high or low offers, we demonstrate that unpredictable shifts in expected offers creates a difference in rejection rates exhibited by the two responder groups for otherwise identical offers. We constructed an ideal observer model that identified neural correlates of norm prediction errors in the ventral striatum and anterior insula, regions that also showed strong responses to variance-prediction errors generated by the same model. Subjective feelings about offers correlated with these norm prediction errors, and the two signals displayed overlapping, but not identical, neural correlates in striatum, insula, and medial orbitofrontal cortex. These results provide evidence for the hypothesis that responses in anterior insula can encode information about social norm violations that correlate with changes in overt behavior (changes in rejection rates). Together, these results demonstrate that the brain regions involved in reward prediction and risk prediction are also recruited in signaling social norm violations.

Computational Substrates of Norms and Their Violations during Social Exchange

PubMed Central

Xiang, Ting; Lohrenz, Terry; Montague, P. Read

2013-01-01

Social norms in humans constrain individual behaviors to establish shared expectations within a social group. Previous work has probed social norm violations and the feelings that such violations engender; however, a computational rendering of the underlying neural and emotional responses has been lacking. We probed norm violations using a two-party, repeated fairness game (ultimatum game) where proposers offer a split of a monetary resource to a responder who either accepts or rejects the offer. Using a norm-training paradigm where subject groups are preadapted to either high or low offers, we demonstrate that unpredictable shifts in expected offers creates a difference in rejection rates exhibited by the two responder groups for otherwise identical offers. We constructed an ideal observer model that identified neural correlates of norm prediction errors in the ventral striatum and anterior insula, regions that also showed strong responses to variance-prediction errors generated by the same model. Subjective feelings about offers correlated with these norm prediction errors, and the two signals displayed overlapping, but not identical, neural correlates in striatum, insula, and medial orbitofrontal cortex. These results provide evidence for the hypothesis that responses in anterior insula can encode information about social norm violations that correlate with changes in overt behavior (changes in rejection rates). Together, these results demonstrate that the brain regions involved in reward prediction and risk prediction are also recruited in signaling social norm violations. PMID:23325247

Pyrene maleimide as a probe of microenvironmental and dynamics properties of protein binding sites

NASA Astrophysics Data System (ADS)

Benci, S.; Vaccari, S.; Schianchi, G.; Locatelli, Donata; Vaghi, P.; Bottiroli, Giovanni F.

1995-01-01

N-(1-Pyrene)maleimide is highly fluorescent upon covalent binding with sulfhydryl and amino groups of the proteins. Multiexponential fluorescence decays were observed for the dye bound to different proteins even when a single binding site is involved. The lack of information about the fluorescence decay of free dye does not allow to define the variations of fluorescence parameter following the conjugation and their correlation with the binding properties of the fluorophore. In this work, a study of the fluorescence of the probe, free in solution, bound to different antibodies and to the antigen-antibody complex both in solution and in cell, has been performed. The experimental results showed that chemico-physical properties of the medium influence the fluorescence decay of the probe in both the free and bound forms, although to a different extent. The variations of fluorescence decay and anisotropy of the bound probe are related to the electronic characteristics of microenvironment and show an increased stabilization of the probe binding site with the increasing complexity of the substrate. The sensitivity of the fluorescence properties of the probe to the binding site environment opens interesting perspectives concerning the application of Py- maleimide fluorochromization to assess the degree of specificity of immunocytochemical labelling.

A practical model for pressure probe system response estimation (with review of existing models)

NASA Astrophysics Data System (ADS)

Hall, B. F.; Povey, T.

2018-04-01

The accurate estimation of the unsteady response (bandwidth) of pneumatic pressure probe systems (probe, line and transducer volume) is a common practical problem encountered in the design of aerodynamic experiments. Understanding the bandwidth of the probe system is necessary to capture unsteady flow features accurately. Where traversing probes are used, the desired traverse speed and spatial gradients in the flow dictate the minimum probe system bandwidth required to resolve the flow. Existing approaches for bandwidth estimation are either complex or inaccurate in implementation, so probes are often designed based on experience. Where probe system bandwidth is characterized, it is often done experimentally, requiring careful experimental set-up and analysis. There is a need for a relatively simple but accurate model for estimation of probe system bandwidth. A new model is presented for the accurate estimation of pressure probe bandwidth for simple probes commonly used in wind tunnel environments; experimental validation is provided. An additional, simple graphical method for air is included for convenience.

Probing the structural dependency of photoinduced properties of colloidal quantum dots using metal-oxide photo-active substrates.

PubMed

Patty, Kira; Sadeghi, Seyed M; Campbell, Quinn; Hamilton, Nathan; West, Robert G; Mao, Chuanbin

2014-09-21

We used photoactive substrates consisting of about 1 nm coating of a metal oxide on glass substrates to investigate the impact of the structures of colloidal quantum dots on their photophysical and photochemical properties. We showed during irradiation these substrates can interact uniquely with such quantum dots, inducing distinct forms of photo-induced processes when they have different cores, shells, or ligands. In particular, our results showed that for certain types of core-shell quantum dot structures an ultrathin layer of a metal oxide can reduce suppression of quantum efficiency of the quantum dots happening when they undergo extensive photo-oxidation. This suggests the possibility of shrinking the sizes of quantum dots without significant enhancement of their non-radiative decay rates. We show that such quantum dots are not influenced significantly by Coulomb blockade or photoionization, while those without a shell can undergo a large amount of photo-induced fluorescence enhancement via such blockade when they are in touch with the metal oxide.

Probing the structural dependency of photoinduced properties of colloidal quantum dots using metal-oxide photo-active substrates

PubMed Central

Patty, Kira; Sadeghi, Seyed M.; Campbell, Quinn; Hamilton, Nathan; West, Robert G.; Mao, Chuanbin

2014-01-01

We used photoactive substrates consisting of about 1 nm coating of a metal oxide on glass substrates to investigate the impact of the structures of colloidal quantum dots on their photophysical and photochemical properties. We showed during irradiation these substrates can interact uniquely with such quantum dots, inducing distinct forms of photo-induced processes when they have different cores, shells, or ligands. In particular, our results showed that for certain types of core-shell quantum dot structures an ultrathin layer of a metal oxide can reduce suppression of quantum efficiency of the quantum dots happening when they undergo extensive photo-oxidation. This suggests the possibility of shrinking the sizes of quantum dots without significant enhancement of their non-radiative decay rates. We show that such quantum dots are not influenced significantly by Coulomb blockade or photoionization, while those without a shell can undergo a large amount of photo-induced fluorescence enhancement via such blockade when they are in touch with the metal oxide. PMID:25316953

X-ray probe of GaN thin films grown on InGaN compliant substrates

NASA Astrophysics Data System (ADS)

Xu, Xiaoqing; Li, Yang; Liu, Jianming; Wei, Hongyuan; Liu, Xianglin; Yang, Shaoyan; Wang, Zhanguo; Wang, Huanhua

2013-04-01

GaN thin films grown on InGaN compliant substrates were characterized by several X-ray technologies: X-ray reciprocal space mapping (RSM), grazing incidence X-ray diffraction (GIXRD), and X-ray photoemission spectrum (XPS). Narrow Lorentz broadening and stress free state were observed for GaN grown on InGaN compliant substrate, while mosaic structure and large tensile stress were observed at the presence of residual indium atoms. RSM disclosed the mosaicity, and the GIXRD was conducted to investigate the depth dependences of crystal quality and strain states. XPS depth profile of indium contents indicated that residual indium atoms deteriorated the crystal quality of GaN not only by producing lattice mismatch at the interface of InGaN and GaN but also by diffusing into GaN overlayers. Accordingly, two solutions were proposed to improve the efficiency of self-patterned lateral epitaxial overgrowth method. This research goes a further step in resolving the urgent substrate problem in GaN fabrication.

Preparation of a micropatterned rigid-soft composite substrate for probing cellular rigidity sensing.

PubMed

Wong, Stephanie; Guo, Wei-hui; Hoffecker, Ian; Wang, Yu-li

2014-01-01

Substrate rigidity has been recognized as an important property that affects cellular physiology and functions. While the phenomenon has been well recognized, understanding the underlying mechanism may be greatly facilitated by creating a microenvironment with designed rigidity patterns. This chapter describes in detail an optimized method for preparing substrates with micropatterned rigidity, taking advantage of the ability to dehydrate polyacrylamide gels for micropatterning with photolithography, and subsequently rehydrate the gel to regain the original elastic state. While a wide range of micropatterns may be prepared, typical composite substrates consist of micron-sized islands of rigid photoresist grafted on the surface of polyacrylamide hydrogels of defined rigidity. These islands are displaced by cellular traction forces, for a distance determined by the size of the island, the rigidity of the underlying hydrogel, and the magnitude of traction forces. Domains of rigidity may be created using this composite material to allow systematic investigations of rigidity sensing and durotaxis. Copyright © 2014 Elsevier Inc. All rights reserved.

An investigation of Au/Ti multilayer thin-films: surface morphology, structure and interfacial/surface migration of constituents under applied thermal stress

NASA Astrophysics Data System (ADS)

Senevirathne, Indrajith; Kemble, Eric; Lavoie, John

2014-03-01

Multilayer thin films are ubiquitous in industry. Au/Ti/substrate is unique due to possible biological applications in proof of concept devices. Material used for substrates include borosilicate glass, and quartz. Typical Ti depositions on substrates give rise to Stanski-Krastonov (SK) like growth while Frank-van der Merwe (FM) like growth is preferred. Ti films with thickness of ~ 100nm were deposited onto varying substrates using a thermal evaporator. The additional Au layer is then deposited via magnetron sputter deposition at 100mtorr at low deposition rates (~ 1ML/min) onto the Ti thin film. These systems were annealed at varying temperatures and at different durations. Systems were investigated via AFM (Atomic Force Microscopy) probes to examine the surface morphology, and structure. Further, the ambient contamination and elemental distribution/diffusion at annealing was investigated via Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray spectroscopy (EDX). PASSHE FPDC Annual Grant (LOU # 2010-LHU-03)

A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

PubMed Central

Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

2015-01-01

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

Profilometry of thin films on rough substrates by Raman spectroscopy

PubMed Central

Ledinský, Martin; Paviet-Salomon, Bertrand; Vetushka, Aliaksei; Geissbühler, Jonas; Tomasi, Andrea; Despeisse, Matthieu; De Wolf , Stefaan; Ballif , Christophe; Fejfar, Antonín

2016-01-01

Thin, light-absorbing films attenuate the Raman signal of underlying substrates. In this article, we exploit this phenomenon to develop a contactless thickness profiling method for thin films deposited on rough substrates. We demonstrate this technique by probing profiles of thin amorphous silicon stripes deposited on rough crystalline silicon surfaces, which is a structure exploited in high-efficiency silicon heterojunction solar cells. Our spatially-resolved Raman measurements enable the thickness mapping of amorphous silicon over the whole active area of test solar cells with very high precision; the thickness detection limit is well below 1 nm and the spatial resolution is down to 500 nm, limited only by the optical resolution. We also discuss the wider applicability of this technique for the characterization of thin layers prepared on Raman/photoluminescence-active substrates, as well as its use for single-layer counting in multilayer 2D materials such as graphene, MoS2 and WS2. PMID:27922033

Mechanism of Contact between a Droplet and an Atomically Smooth Substrate

NASA Astrophysics Data System (ADS)

Lo, Hau Yung; Liu, Yuan; Xu, Lei

2017-04-01

When a droplet gently lands on an atomically smooth substrate, it will most likely contact the underlying surface in about 0.1 s. However, theoretical estimation from fluid mechanics predicts a contact time of 10-100 s. What causes this large discrepancy, and how does nature speed up contact by 2 orders of magnitude? To probe this fundamental question, we prepare atomically smooth substrates by either coating a liquid film on glass or using a freshly cleaved mica surface, and visualize the droplet contact dynamics with 30-nm resolution. Interestingly, we discover two distinct speed-up approaches: (1) droplet skidding due to even minute perturbations breaks rotational symmetry and produces early contact at the thinnest gap location, and (2) for the unperturbed situation with rotational symmetry, a previously unnoticed boundary flow around only 0.1 mm /s expedites air drainage by over 1 order of magnitude. Together, these two mechanisms universally explain general contact phenomena on smooth substrates. The fundamental discoveries shed new light on contact and drainage research.

High-performance SERS substrate based on hybrid structure of graphene oxide/AgNPs/Cu film@pyramid Si

NASA Astrophysics Data System (ADS)

Li, Zhe; Xu, Shi Cai; Zhang, Chao; Liu, Xiao Yun; Gao, Sai Sai; Hu, Li Tao; Guo, Jia; Ma, Yong; Jiang, Shou Zhen; Si, Hai Peng

2016-12-01

We present a novel surface-enhanced Raman scattering (SERS) substrate based on graphene oxide/silver nanoparticles/copper film covered silicon pyramid arrays (GO/AgNPs/PCu@Si) by a low-cost and simple method. The GO/AgNPs/PCu@Si substrate presents high sensitivity, good homogeneity and well stability with R6G molecules as a probe. The detected concentration of Rhodamine 6 G (R6G) is as low as 10-15 M. These sensitive SERS behaviors are also confirmed in theory via a commercial COMSOL software, the electric field enhancement is not only formed between the AgNPs, but also formed between the AgNPs and Cu film. And the GO/AgNPs/PCu@Si substrates also present good property on practical application for the detection of methylene blue (MB) and crystal violet (CV). This work may offer a novel and practical method to facilitate the SERS applications in areas of medicine, food safety and biotechnology.

High-performance SERS substrate based on hybrid structure of graphene oxide/AgNPs/Cu film@pyramid Si.

PubMed

Li, Zhe; Xu, Shi Cai; Zhang, Chao; Liu, Xiao Yun; Gao, Sai Sai; Hu, Li Tao; Guo, Jia; Ma, Yong; Jiang, Shou Zhen; Si, Hai Peng

2016-12-07

We present a novel surface-enhanced Raman scattering (SERS) substrate based on graphene oxide/silver nanoparticles/copper film covered silicon pyramid arrays (GO/AgNPs/PCu@Si) by a low-cost and simple method. The GO/AgNPs/PCu@Si substrate presents high sensitivity, good homogeneity and well stability with R6G molecules as a probe. The detected concentration of Rhodamine 6 G (R6G) is as low as 10 -15 M. These sensitive SERS behaviors are also confirmed in theory via a commercial COMSOL software, the electric field enhancement is not only formed between the AgNPs, but also formed between the AgNPs and Cu film. And the GO/AgNPs/PCu@Si substrates also present good property on practical application for the detection of methylene blue (MB) and crystal violet (CV). This work may offer a novel and practical method to facilitate the SERS applications in areas of medicine, food safety and biotechnology.

High-performance SERS substrate based on hybrid structure of graphene oxide/AgNPs/Cu film@pyramid Si

PubMed Central

Li, Zhe; Xu, Shi Cai; Zhang, Chao; Liu, Xiao Yun; Gao, Sai Sai; Hu, Li Tao; Guo, Jia; Ma, Yong; Jiang, Shou Zhen; Si, Hai Peng

2016-01-01

We present a novel surface-enhanced Raman scattering (SERS) substrate based on graphene oxide/silver nanoparticles/copper film covered silicon pyramid arrays (GO/AgNPs/PCu@Si) by a low-cost and simple method. The GO/AgNPs/PCu@Si substrate presents high sensitivity, good homogeneity and well stability with R6G molecules as a probe. The detected concentration of Rhodamine 6 G (R6G) is as low as 10−15 M. These sensitive SERS behaviors are also confirmed in theory via a commercial COMSOL software, the electric field enhancement is not only formed between the AgNPs, but also formed between the AgNPs and Cu film. And the GO/AgNPs/PCu@Si substrates also present good property on practical application for the detection of methylene blue (MB) and crystal violet (CV). This work may offer a novel and practical method to facilitate the SERS applications in areas of medicine, food safety and biotechnology. PMID:27924863

Effect of substrate rotation speed and off-center deposition on the structural, optical, and electrical properties of AZO thin films fabricated by DC magnetron sputtering

NASA Astrophysics Data System (ADS)

Turkoglu, F.; Koseoglu, H.; Zeybek, S.; Ozdemir, M.; Aygun, G.; Ozyuzer, L.

2018-04-01

In this study, aluminum-doped zinc oxide (AZO) thin films were deposited by DC magnetron sputtering at room temperature. The distance between the substrate and target axis, and substrate rotation speed were varied to get high quality AZO thin films. The influences of these deposition parameters on the structural, optical, and electrical properties of the fabricated films were investigated by X-ray diffraction (XRD), Raman spectroscopy, spectrophotometry, and four-point probe techniques. The overall analysis revealed that both sample position and substrate rotation speed are effective in changing the optical, structural, and electrical properties of the AZO thin films. We further observed that stress in the films can be significantly reduced by off-center deposition and rotating the sample holder during the deposition. An average transmittance above 85% in the visible range and a resistivity of 2.02 × 10-3 Ω cm were obtained for the AZO films.

Probing the Inelastic Interactions in Molecular Junctions by Scanning Tunneling Microscope

NASA Astrophysics Data System (ADS)

Xu, Chen

With a sub-Kelvin scanning tunneling microscope, the energy resolution of spectroscopy is improved dramatically. Detailed studies of finer features of spectrum become possible. The asymmetry in the line shape of carbon monoxide vibrational spectra is observed to correlate with the couplings of the molecule to the tip and substrates. The spin-vibronic coupling in the molecular junctions is revisited with two metal phthalocyanine molecules, unveiling sharp spin-vibronic peaks. Finally, thanks to the improved spectrum resolution, the bonding structure of the acyclic compounds molecules is surveyed with STM inelastic tunneling probe, expanding the capability of the innovative high resolution imaging technique.

Nanofabrication technique based on localized photocatalytic reactions using a TiO2-coated atomic force microscopy probe

NASA Astrophysics Data System (ADS)

Shibata, Takayuki; Iio, Naohiro; Furukawa, Hiromi; Nagai, Moeto

2017-02-01

We performed a fundamental study on the photocatalytic degradation of fluorescently labeled DNA molecules immobilized on titanium dioxide (TiO2) thin films under ultraviolet irradiation. The films were prepared by the electrochemical anodization of Ti thin films sputtered on silicon substrates. We also confirmed that the photocurrent arising from the photocatalytic oxidation of DNA molecules can be detected during this process. We then demonstrated an atomic force microscopy (AFM)-based nanofabrication technique by employing TiO2-coated AFM probes to penetrate living cell membranes under near-physiological conditions for minimally invasive intracellular delivery.

In-situ spectrophotometric probe

DOEpatents

Prather, William S.

1992-01-01

A spectrophotometric probe for in situ absorption spectra measurements comprising a first optical fiber carrying light from a remote light source, a second optical fiber carrying light to a remote spectrophotometer, the proximal ends of the first and second optical fibers parallel and coterminal, a planoconvex lens to collimate light from the first optical fiber, a reflecting grid positioned a short distance from the lens to reflect the collimated light back to the lens for focussing on the second optical fiber. The lens is positioned with the convex side toward the optical fibers. A substrate for absorbing analyte or an analyte and reagent mixture may be positioned between the lens and the reflecting grid.

Silver Eco-Solvent Ink for Reactive Printing of Polychromatic SERS and SPR Substrates

PubMed Central

Dustov, Mavlavi; Goldt, Anastasia E.; Sukhorukova, Irina V.; Grünert, Wolfgang; Grigorieva, Anastasia V.

2018-01-01

A new reactive ink based on a silver citrate complex is proposed for a photochemical route to surface-enhanced Raman spectroscopy active substrates with controllable extinction spectra. The drop-cast test of the ink reveals homogeneous nucleation of silver and colloid particle growth originating directly from photochemical in situ reduction in droplets, while the following evaporation of the deposited ink produces small nano- and micron-size particles. The prepared nanostructures and substrates were accurately characterized by electron microscopy methods and optical extinction spectroscopy. Varying the duration of UV irradiation allows tuning the morphology of individual silver nanoparticles forming hierarchical ring structures with numerous “hot spots” for most efficient Raman enhancement. Raman measurements of probe molecules of rhodamine 6G and methylene blue reached the largest signal enhancement of 106 by the resonance effects. PMID:29425119

Silver Eco-Solvent Ink for Reactive Printing of Polychromatic SERS and SPR Substrates.

PubMed

Dustov, Mavlavi; Golovina, Diana I; Polyakov, Alexander Yu; Goldt, Anastasia E; Eliseev, Andrei A; Kolesnikov, Efim A; Sukhorukova, Irina V; Shtansky, Dmitry V; Grünert, Wolfgang; Grigorieva, Anastasia V

2018-02-09

A new reactive ink based on a silver citrate complex is proposed for a photochemical route to surface-enhanced Raman spectroscopy active substrates with controllable extinction spectra. The drop-cast test of the ink reveals homogeneous nucleation of silver and colloid particle growth originating directly from photochemical in situ reduction in droplets, while the following evaporation of the deposited ink produces small nano- and micron-size particles. The prepared nanostructures and substrates were accurately characterized by electron microscopy methods and optical extinction spectroscopy. Varying the duration of UV irradiation allows tuning the morphology of individual silver nanoparticles forming hierarchical ring structures with numerous "hot spots" for most efficient Raman enhancement. Raman measurements of probe molecules of rhodamine 6G and methylene blue reached the largest signal enhancement of 10⁶ by the resonance effects.

Christmas-tree Derived Amplification Immuno-strategy for Sensitive Visual Detection of Vibrio parahaemolyticus Based on Gold Label Silver Stain Technology.

PubMed

Song, Xinxin; Wu, Yanjie; Wu, Lin; Hu, Yufang; Li, Wenrou; Guo, Zhiyong; Su, Xiurong; Jiang, Xiaohua

2017-01-01

A developed Christmas-tree derived immunosensor based on a gold label silver stain (GLSS) technique was fabricated for a highly sensitive analysis of Vibrio parahaemolyticu (VP). In this strategy, captured VP antibody (cAb) was immobilized on a solid substrate; then, the VPs were sequentially tagged with a signal probe by incubating the assay with a detection VP antibody (dAb) conjugated gold nanoparticles (AuNPs)-labeled graphite-like carbon nitride (g-C 3 N 4 ). Finally, the attached signal probe could harvest a visible signal by the silver meal deposition, and then followed by homebrew Matlab 6.0 as a grey value acquisition. In addition, the overall design of the biosensor was established in abundant AuNPs and g-C 3 N 4 with a two-dimensional structure, affording a bulb-decorated Christmas-tree model. Moreover, with the optimized conditions, the detection limit of the as-proposed biosensor is as low as 10 2 CFU (Colony-Forming Units) mL -1 , exhibiting an increase of two orders of magnitude compared with the traditional immune-gold method. Additionally, the developed visible immunosensor was also successfully applied to the analysis of complicated samples.

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study†

PubMed Central

Peng, Dungeng; Satterlee, James D.; Ma, Li-Hua; Dallas, Jerry L.; Smith, Kevin M.; Zhang, Xuhong; Sato, Michihiko; La Mar, Gerd N.

2011-01-01

Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which secures host iron, shares many properties with mammalian HOs, but also exhibits some key differences. The crystal structure appears more compact and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D 1H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~102 increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed. PMID:21870860

Atmospheric Probe Model: Construction and Wind Tunnel Tests

NASA Technical Reports Server (NTRS)

Vogel, Jerald M.

1998-01-01

The material contained in this document represents a summary of the results of a low speed wind tunnel test program to determine the performance of an atmospheric probe at low speed. The probe configuration tested consists of a 2/3 scale model constructed from a combination of hard maple wood and aluminum stock. The model design includes approximately 130 surface static pressure taps. Additional hardware incorporated in the baseline model provides a mechanism for simulating external and internal trailing edge split flaps for probe flow control. Test matrix parameters include probe side slip angle, external/internal split flap deflection angle, and trip strip applications. Test output database includes surface pressure distributions on both inner and outer annular wings and probe center line velocity distributions from forward probe to aft probe locations.

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

Photoinduced Br Desorption from CsBr Thin Films Grown on Cu(100)

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

Halliday, Matthew T.; Joly, Alan G.; Hess, Wayne P.

2015-10-22

Thin films of CsBr deposited onto metals such as copper are potential photocathode materials for light sources and other applications. We investigate desorption dynamics of Br atoms from CsBr films grown on insulator (KBr, LiF) and metal (Cu) substrates induced by sub-bandgap 6.4 eV laser pulses. The experimental results demonstrate that the peak kinetic energy of Br atoms desorbed from CsBr/Cu films is much lower than that for the hyperthermal desorption from CsBr/LiF films. Kelvin probe measurements indicate negative charge at the surface following Br desorption from CsBr/Cu films. Our ab initio calculations of excitons at CsBr surfaces demonstrate thatmore » this behavior can be explained by an exciton model of desorption including electron trapping at the CsBr surface. Trapped negative charges reduce the energy of surface excitons available for Br desorption. We examine the electron-trapping characteristics of low-coordinated sites at the surface, in particular, divacancies and kink sites. We also provide a model of cation desorption caused by Franck-Hertz excitation of F centers at the surface in the course of irradiation of CsBr/Cu films. These results provide new insights into the mechanisms of photoinduced structural evolution of alkali halide films on metal substrates and activation of metal photocathodes coated with CsBr.« less

Computational modeling of ion transport through nanopores.

PubMed

Modi, Niraj; Winterhalter, Mathias; Kleinekathöfer, Ulrich

2012-10-21

Nanoscale pores are ubiquitous in biological systems while artificial nanopores are being fabricated for an increasing number of applications. Biological pores are responsible for the transport of various ions and substrates between the different compartments of biological systems separated by membranes while artificial pores are aimed at emulating such transport properties. As an experimental method, electrophysiology has proven to be an important nano-analytical tool for the study of substrate transport through nanopores utilizing ion current measurements as a probe for the detection. Independent of the pore type, i.e., biological or synthetic, and objective of the study, i.e., to model cellular processes of ion transport or electrophysiological experiments, it has become increasingly important to understand the dynamics of ions in nanoscale confinements. To this end, numerical simulations have established themselves as an indispensable tool to decipher ion transport processes through biological as well as artificial nanopores. This article provides an overview of different theoretical and computational methods to study ion transport in general and to calculate ion conductance in particular. Potential new improvements in the existing methods and their applications are highlighted wherever applicable. Moreover, representative examples are given describing the ion transport through biological and synthetic nanopores as well as the high selectivity of ion channels. Special emphasis is placed on the usage of molecular dynamics simulations which already have demonstrated their potential to unravel ion transport properties at an atomic level.

Nanostructured carbon films with oriented graphitic planes

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

Teo, E. H. T.; Kalish, R.; Kulik, J.

2011-03-21

Nanostructured carbon films with oriented graphitic planes can be deposited by applying energetic carbon bombardment. The present work shows the possibility of structuring graphitic planes perpendicular to the substrate in following two distinct ways: (i) applying sufficiently large carbon energies for deposition at room temperature (E>10 keV), (ii) utilizing much lower energies for deposition at elevated substrate temperatures (T>200 deg. C). High resolution transmission electron microscopy is used to probe the graphitic planes. The alignment achieved at elevated temperatures does not depend on the deposition angle. The data provides insight into the mechanisms leading to the growth of oriented graphiticmore » planes under different conditions.« less

Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis.

PubMed

Kim, Young-Woo; Grossmann, Tom N; Verdine, Gregory L

2011-06-01

This protocol provides a detailed procedure for the preparation of stapled α-helical peptides, which have proven their potential as useful molecular probes and as next-generation therapeutics. Two crucial features of this protocol are (i) the construction of peptide substrates containing hindered α-methyl, α-alkenyl amino acids and (ii) the ring-closing olefin metathesis (RCM) of the resulting resin-bound peptide substrates. The stapling systems described in this protocol, namely bridging one or two turns of an α-helix, are highly adaptable to most peptide sequences, resulting in favorable RCM kinetics, helix stabilization and promotion of cellular uptake.

Focused analyte spray emission apparatus and process for mass spectrometric analysis

DOEpatents

Roach, Patrick J [Kennewick, WA; Laskin, Julia [Richland, WA; Laskin, Alexander [Richland, WA

2012-01-17

An apparatus and process are disclosed that deliver an analyte deposited on a substrate to a mass spectrometer that provides for trace analysis of complex organic analytes. Analytes are probed using a small droplet of solvent that is formed at the junction between two capillaries. A supply capillary maintains the droplet of solvent on the substrate; a collection capillary collects analyte desorbed from the surface and emits analyte ions as a focused spray to the inlet of a mass spectrometer for analysis. The invention enables efficient separation of desorption and ionization events, providing enhanced control over transport and ionization of the analyte.

Wavelength modulated surface enhanced (resonance) Raman scattering for background-free detection.

PubMed

Praveen, Bavishna B; Steuwe, Christian; Mazilu, Michael; Dholakia, Kishan; Mahajan, Sumeet

2013-05-21

Spectra in surface-enhanced Raman scattering (SERS) are always accompanied by a continuum emission called the 'background' which complicates analysis and is especially problematic for quantification and automation. Here, we implement a wavelength modulation technique to eliminate the background in SERS and its resonant version, surface-enhanced resonance Raman scattering (SERRS). This is demonstrated on various nanostructured substrates used for SER(R)S. An enhancement in the signal to noise ratio for the Raman bands of the probe molecules is also observed. This technique helps to improve the analytical ability of SERS by alleviating the problem due to the accompanying background and thus making observations substrate independent.

Strategies for Determining Correct Cytochrome P450 Contributions in Hepatic Clearance Predictions: In Vitro-In Vivo Extrapolation as Modelling Approach and Tramadol as Proof-of Concept Compound.

PubMed

T'jollyn, Huybrecht; Snoeys, Jan; Van Bocxlaer, Jan; De Bock, Lies; Annaert, Pieter; Van Peer, Achiel; Allegaert, Karel; Mannens, Geert; Vermeulen, An; Boussery, Koen

2017-06-01

Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes. Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity. In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities. This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound, obtained in a particular batch, should be corrected with the AAF for the respective enzymes. In the current study, especially the CYP2D6 contribution was found, to better reflect the average in vivo situation. It is recommended that this novel approach is further evaluated using a broader range of compounds.

Ultra-fast movies of thin-film laser ablation

NASA Astrophysics Data System (ADS)

Domke, Matthias; Rapp, Stephan; Schmidt, Michael; Huber, Heinz P.

2012-11-01

Ultra-short-pulse laser irradiation of thin molybdenum films from the glass substrate side initiates an intact Mo disk lift off free from thermal effects. For the investigation of the underlying physical effects, ultra-fast pump-probe microscopy is used to produce stop-motion movies of the single-pulse ablation process, initiated by a 660-fs laser pulse. The ultra-fast dynamics in the femtosecond and picosecond ranges are captured by stroboscopic illumination of the sample with an optically delayed probe pulse of 510-fs duration. The nanosecond and microsecond delay ranges of the probe pulse are covered by an electronically triggered 600-ps laser. Thus, the setup enables an observation of general laser ablation processes from the femtosecond delay range up to the final state. A comparison of time- and space-resolved observations of film and glass substrate side irradiation of a 470-nm molybdenum layer reveals the driving mechanisms of the Mo disk lift off initiated by glass-side irradiation. Observations suggest that a phase explosion generates a liquid-gas mixture in the molybdenum/glass interface about 10 ps after the impact of the pump laser pulse. Then, a shock wave and gas expansion cause the molybdenum layer to bulge, while the enclosed liquid-gas mixture cools and condenses at delay times in the 100-ps range. The bulging continues for approximately 20 ns, when an intact Mo disk shears and lifts off at a velocity of above 70 m/s. As a result, the remaining hole is free from thermal effects.

Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

PubMed

Kallmyer, Nathaniel E; Musielewicz, Joseph; Sutter, Joel; Reuel, Nigel F

2018-04-17

Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

Solution-Phase Photochemical Nanopatterning Enabled by High-Refractive-Index Beam Pen Arrays.

PubMed

Xie, Zhuang; Gordiichuk, Pavlo; Lin, Qing-Yuan; Meckes, Brian; Chen, Peng-Cheng; Sun, Lin; Du, Jingshan S; Zhu, Jinghan; Liu, Yuan; Dravid, Vinayak P; Mirkin, Chad A

2017-08-22

A high-throughput, solution-based, scanning-probe photochemical nanopatterning approach, which does not require the use of probes with subwavelength apertures, is reported. Specifically, pyramid arrays made from high-refractive-index polymeric materials were constructed and studied as patterning tools in a conventional liquid-phase beam pen lithography experiment. Two versions of the arrays were explored with either metal-coated or metal-free tips. Importantly, light can be channeled through both types of tips and the appropriate solution phase (e.g., H 2 O or CH 3 OH) and focused on subwavelength regions of a substrate to effect a photoreaction in solution that results in localized patterning of a self-assembled monolayer (SAM)-coated Au thin film substrate. Arrays with as many as 4500 pyramid-shaped probes were used to simultaneously initiate thousands of localized free-radical photoreactions (decomposition of a lithium acylphosphinate photoinitiator in an aqueous solution) that result in oxidative removal of the SAM. The technique is attractive since it allows one to rapidly generate features less than 200 nm in diameter, and the metal-free tips afford more than 10-fold higher intensity than the tips with nanoapertures over a micrometer propagation length. In principle, this mask-free method can be utilized as a versatile tool for performing a wide variety of photochemistries across multiple scales that may be important in high-throughput combinatorial screening applications related to chemistry, biology, and materials science.

Exploring the quantitative relationship between metabolism and enzymatic phenotype by physiological modeling of glucose metabolism and lactate oxidation in solid tumors

NASA Astrophysics Data System (ADS)

Wang, Qian; Vaupel, Peter; Ziegler, Sibylle I.; Shi, Kuangyu

2015-03-01

Molecular imaging using PET or hyperpolarized MRI can characterize tumor phenotypes by assessing the related metabolism of certain substrates. However, the interpretation of the substrate turnover in terms of a pathophysiological understanding is not straightforward and only semiquantitative. The metabolism of imaging probes is influenced by a number of factors, such as the microvascular structure or the expression of key enzymes. This study aims to use computational simulation to investigate the relationship between the metabolism behind molecular imaging and the underlying tumor phenotype. The study focused on the pathways of glucose metabolism and lactate oxidation in order to establish the quantitative relationship between the expression of several transporters (GLUT, MCT1 and MCT4), expression of the enzyme hexokinase (HK), microvasculature and the metabolism of glucose or lactate and the extracellular pH distribution. A computational model for a 2D tumor tissue phantom was constructed and the spatio-temporal evolution of related species (e.g. oxygen, glucose, lactate, protons, bicarbonate ions) was estimated by solving reaction-diffusion equations. The proposed model was tested by the verification of the simulation results using in vivo and in vitro literature data. The influences of different expression levels of GLUT, MCT1, MCT4, HK and microvessel distribution on substrate concentrations were analyzed. The major results are consistent with experimental data (e.g. GLUT is more influential to glycolytic flux than HK; extracellular pH is not correlated with MCT expressions) and provide theoretical interpretation of the co-influence of multiple factors of the tumor microenvironment. This computational simulation may assist the generation of hypotheses to bridge the discrepancy between tumor metabolism and the functions of transporters and enzymes. It has the potential to accelerate the development of multi-modal imaging strategies for assessment of tumor phenotypes.

A model system to mimic environmentally active surface film roughness and hydrophobicity.

PubMed

Grant, Jacob S; Shaw, Scott K

2017-10-01

This work presents the development and initial assessment of a laboratory platform to allow quantitative studies on model urban films. The platform consists of stearic acid and eicosane mixtures that are solution deposited from hexanes onto smooth, solid substrates. We show that this model has distinctive capabilities to better mimic a naturally occurring film's morphology and hydrophobicity, two important parameters that have not previously been incorporated into model film systems. The physical and chemical properties of the model films are assessed using a variety of analytical instruments. The film thickness and roughness are probed via atomic force microscopy while the film composition, wettability, and water uptake are analyzed by Fourier transform infrared spectroscopy, contact angle goniometry, and quartz crystal microbalance, respectively. Simulated environmental maturation is achieved by exposing the film to regulated amounts of UV/ozone. Ultimately, oxidation of the film is monitored by the analytical techniques mentioned above and proceeds as expected to produce a utile model film system. Including variable roughness and tunable surface coverage results in several key advantages over prior model systems, and will more accurately represent native urban film behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

Axisymmetric Lattice Boltzmann Model of Droplet Impact on Solid Surfaces

NASA Astrophysics Data System (ADS)

Dalgamoni, Hussein; Yong, Xin

2017-11-01

Droplet impact is a ubiquitous fluid phenomena encountered in scientific and engineering applications such as ink-jet printing, coating, electronics manufacturing, and many others. It is of great technological importance to understand the detailed dynamics of drop impact on various surfaces. The lattice Boltzmann method (LBM) emerges as an efficient method for modeling complex fluid systems involving rapidly evolving fluid-fluid and fluid-solid interfaces with complex geometries. In this work, we model droplet impact on flat solid substrates with well-defined wetting behavior using a two-phase axisymmetric LBM with high density and viscosity contrasts. We extend the two-dimensional Lee and Liu model to capture axisymmetric effect in the normal impact. First we compare the 2D axisymmetric results with the 2D and 3D results reported by Lee and Liu to probe the effect of axisymmetric terms. Then, we explore the effects of Weber number, Ohnesorge number, and droplet-surface equilibrium contact angle on the impact. The dynamic contact angle and spreading factor of the droplet during impact are investigated to qualitatively characterize the impact dynamics.

Nanoscale Probing of Electrical Signals in Biological Systems

DTIC Science & Technology

2012-03-18

Membranes Anodized aluminum oxide ( AAO ) is an ideal prototype substrate for studying ion transport through nanoporous membranes . For optimal...electrochemical microscopy, scanning ion conductance microscopy, nanoporous membranes , anodized aluminum oxide , atomic layer deposition, focused ion beam...capacity. This approach utilizes atomic layer deposition (ALD) of a thin conformal Ir film into a nanoporous anodized aluminum oxide (

Probing photo-carrier collection efficiencies of individual silicon nanowire diodes on a wafer substrate.

PubMed

Schmitt, S W; Brönstrup, G; Shalev, G; Srivastava, S K; Bashouti, M Y; Döhler, G H; Christiansen, S H

2014-07-21

Vertically aligned silicon nanowire (SiNW) diodes are promising candidates for the integration into various opto-electronic device concepts for e.g. sensing or solar energy conversion. Individual SiNW p-n diodes have intensively been studied, but to date an assessment of their device performance once integrated on a silicon substrate has not been made. We show that using a scanning electron microscope (SEM) equipped with a nano-manipulator and an optical fiber feed-through for tunable (wavelength, power using a tunable laser source) sample illumination, the dark and illuminated current-voltage (I-V) curve of individual SiNW diodes on the substrate wafer can be measured. Surprisingly, the I-V-curve of the serially coupled system composed of SiNW/wafers is accurately described by an equivalent circuit model of a single diode and diode parameters like series and shunting resistivity, diode ideality factor and photocurrent can be retrieved from a fit. We show that the photo-carrier collection efficiency (PCE) of the integrated diode illuminated with variable wavelength and intensity light directly gives insight into the quality of the device design at the nanoscale. We find that the PCE decreases for high light intensities and photocurrent densities, due to the fact that considerable amounts of photo-excited carriers generated within the substrate lead to a decrease in shunting resistivity of the SiNW diode and deteriorate its rectification. The PCE decreases systematically for smaller wavelengths of visible light, showing the possibility of monitoring the effectiveness of the SiNW device surface passivation using the shown measurement technique. The integrated device was pre-characterized using secondary ion mass spectrometry (SIMS), TCAD simulations and electron beam induced current (EBIC) measurements to validate the properties of the characterized material at the single SiNW diode level.

Toward the Atomic-Level Mass Analysis of Biomolecules by the Scanning Atom Probe.

PubMed

Nishikawa, Osamu; Taniguchi, Masahiro

2017-04-01

In 1994, a new type of atom probe instrument, named the scanning atom probe (SAP), was proposed. The unique feature of the SAP is the introduction of a small extraction electrode, which scans over a specimen surface and confines the high field, required for field evaporation of surface atoms in a small space, between the specimen and the electrode. Thus, the SAP does not require a sharp specimen tip. This indicates that the SAP can mass analyze the specimens which are difficult to form in a sharp tip, such as organic materials and biomolecules. Clean single wall carbon nanotubes (CNT), made by high-pressure carbon monoxide process are found to be the best substrates for biomolecules. Various amino acids and dipeptide biomolecules were successfully mass analyzed, revealing characteristic clusters formed by strongly bound atoms in the specimens. The mass analysis indicates that SAP analysis of biomolecules is not only qualitative, but also quantitative.

Spin label studies of micellar and pre-micellar aggregates.

PubMed

Ernades, J R; Schreier, S; Chaimovich, H

1976-02-01

Micelles of hexadecyl trimethyl ammonium bromide (CTABr) have been investigated with the use of a faty acid spin label and its methyl ester derivative. The esr * spectra provided information about the degree of motion of the probes in the micelles as evaluated from calculation of rotational correlation times. Evidence is presented for the formation of pre-micellar aggregates at concentrations below the cmc. The effect of addition of thiophenoxide on the structure of CTABr micelles was to decrease the rate of motion of the spin probes, probably due to a tighter packing of the hydrophobic core as a consequence of charge neutralization at the micelle surface by the substrate. Decreasing values of the isotropic hyperfine splitting of the spin probe with increasing concentration of thiophenoxide were taken as indicating that the latter causes a decrease of the degree of hydration of the polar head region of the detergent.

Fabrication of near-field optical apertures in aluminium by a highly selective corrosion process in the evanescent field.

PubMed

Haefliger, D; Stemmer, A

2003-03-01

A simple, one-step process to fabricate high-quality apertures for scanning near-field optical microscope probes based on aluminium-coated silicon nitride cantilevers is presented. A thin evanescent optical field at a glass-water interface was used to heat the aluminium at the tip apex due to light absorption. The heat induced a breakdown of the passivating oxide layer and local corrosion of the metal, which selectively exposed the front-most part of the probe tip from the aluminium. Apertures with a protruding silicon nitride tip up to 72 nm in height were fabricated. The height of the protrusion was controlled by the extent of the evanescent field, whereas the diameter depended on the geometry of the probe substrate. The corrosion process proved to be self-terminating, yielding highly reproducible tip heights. Near-field optical resolution in a transmission mode of 85 nm was demonstrated.

In Situ Hot-Spot Assembly as a General Strategy for Probing Single Biomolecules.

PubMed

Liu, Huiqiao; Li, Qiang; Li, Mingmin; Ma, Sisi; Liu, Dingbin

2017-05-02

Single-molecule detection using surface-enhanced Raman spectroscopy (SERS) has attracted increasing attention in chemical and biomedical analysis. However, it remains a major challenge to probe single biomolecules by means of SERS hot spots owing to the small volume of hot spots and their random distribution on substrates. We here report an in situ hot-spot assembly method as a general strategy for probing single biomolecules. As a proof-of-concept, this proposed strategy was successfully used for the detection of single microRNA-21 (miRNA-21, a potential cancer biomarker) at the single-cell level, showing great capability in differentiating the expression of miRNA-21 in single cancer cells from normal cells. This approach was further extended to single-protein detection. The versatility of the strategy opens an exciting avenue for single-molecule detection of biomarkers of interest and thus holds great promise in a variety of biological and biomedical applications.

Nanoscale infrared absorption spectroscopy of individual nanoparticles enabled by scattering-type near-field microscopy.

PubMed

Stiegler, Johannes M; Abate, Yohannes; Cvitkovic, Antonija; Romanyuk, Yaroslav E; Huber, Andreas J; Leone, Stephen R; Hillenbrand, Rainer

2011-08-23

Infrared absorption spectroscopy is a powerful and widely used tool for analyzing the chemical composition and structure of materials. Because of the diffraction limit, however, it cannot be applied for studying individual nanostructures. Here we demonstrate that the phase contrast in substrate-enhanced scattering-type scanning near-field optical microscopy (s-SNOM) provides a map of the infrared absorption spectrum of individual nanoparticles with nanometer-scale spatial resolution. We succeeded in the chemical identification of silicon nitride nanoislands with heights well below 10 nm, by infrared near-field fingerprint spectroscopy of the Si-N stretching bond. Employing a novel theoretical model, we show that the near-field phase spectra of small particles correlate well with their far-field absorption spectra. On the other hand, the spectral near-field contrast does not scale with the volume of the particles. We find a nearly linear scaling law, which we can attribute to the near-field coupling between the near-field probe and the substrate. Our results provide fundamental insights into the spectral near-field contrast of nanoparticles and clearly demonstrate the capability of s-SNOM for nanoscale chemical mapping based on local infrared absorption. © 2011 American Chemical Society

N-mustard analogs of S-adenosyl-L-methionine as biochemical probes of protein arginine methylation.

PubMed

Hymbaugh Bergman, Sarah J; Comstock, Lindsay R

2015-08-01

Nucleosomes, the fundamental building blocks of eukaryotic chromatin, undergo post-synthetic modifications and play a major role in the regulation of transcriptional processes. Combinations of these modifications, including methylation, regulate chromatin structure, determining its different functional states and playing a central role in differentiation. The biological significance of cellular methylation, particularly on chromatin, is widely recognized, yet we know little about the mechanisms that link biological methylation events. To characterize and fully understand protein methylation, we describe here novel N-mustard analogs of S-adenosyl-l-methionine (SAM) as biochemical tools to better understand protein arginine methylation events using protein arginine methyltransferase 1 (PRMT1). Specifically, azide- and alkyne-functionalized N-mustard analogs serve as cofactor mimics of SAM and are enzymatically transferred to a model peptide substrate in a PRMT1-dependent fashion. Once incorporated, the resulting alkynes and azides can be modified through chemoselective ligations, including click chemistry and the Staudinger ligation. These results readily demonstrate the feasibility of utilizing N-mustard analogs as biochemical tools to site-specifically label substrates of PRMT1 and serve as an alternative approach to study protein methylation events. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

PubMed

Moreno-Cencerrado, Alberto; Iturri, Jagoba; Pecorari, Ilaria; D M Vivanco, Maria; Sbaizero, Orfeo; Toca-Herrera, José L

2017-01-01

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates

PubMed Central

Versaevel, Marie; Riaz, Maryam; Corne, Tobias; Grevesse, Thomas; Lantoine, Joséphine; Mohammed, Danahe; Bruyère, Céline; Alaimo, Laura; De Vos, Winnok H.; Gabriele, Sylvain

2017-01-01

ABSTRACT The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures. PMID:27111836

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates.

PubMed

Versaevel, Marie; Riaz, Maryam; Corne, Tobias; Grevesse, Thomas; Lantoine, Joséphine; Mohammed, Danahe; Bruyère, Céline; Alaimo, Laura; De Vos, Winnok H; Gabriele, Sylvain

2017-01-02

The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures.