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Sample records for label-free cell discrimination

  1. Raman spectroscopy as a tool for label-free lymphocyte cell line discrimination.

    PubMed

    Hobro, Alison J; Kumagai, Yutaro; Akira, Shizuo; Smith, Nicholas I

    2016-06-21

    Unactivated lymphocytes are morphologically identical and biochemically relatively similar, making them difficult to distinguish from one another with conventional light microscopy. Here, we use Raman spectroscopy to provide biochemical information on the composition of different lymphocyte cell lines. As could be expected, the biochemical differences measured with Raman spectroscopy between lymphocyte cell lines are small, but in combination with partial least squares discriminant analysis it is possible not only to distinguish between T- and B-cells, but also between individual T-cell and B-cell lines. PMID:27067644

  2. Label-free and non-invasive discrimination of HaCaT and melanoma cells in a co-culture model by hyperspectral confocal reflectance microscopy.

    PubMed

    Bertani, Francesca R; Botti, Elisabetta; Ferrari, Luisa; Mussi, Valentina; Costanzo, Antonio; D'Alessandro, Marco; Cilloco, Francesco; Selci, Stefano

    2016-06-01

    A novel hyperspectral confocal microscopy method to separate different cell populations in a co-culture model is presented here. The described methodological and instrumental approach allows discrimination of different cell types using a non-invasive, label free method with good accuracy with a single cell resolution. In particular, melanoma cells are discriminated from HaCaT cells by hyperspectral confocal imaging, principal component analysis and optical frequencies signing, as confirmed by fluorescence labelling cross check. The identification seems to be quite robust to be insensitive to the cellular shape within the studied samples, enabling to separate cells according to their cytotype down to a single cell sensitivity. Set of hyperspectral images of melanoma-keratinocytes co-culture model (left), score plot of principal component analysis and spectral analysis of principal components coefficients (center), label-free spectral identification of cell populations (right). PMID:26375607

  3. Functionalized nanopipettes: toward label-free, single cell biosensors

    PubMed Central

    Actis, Paolo; Mak, Andy C.

    2010-01-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms. PMID:20730113

  4. Label-free cell separation and sorting in microfluidic systems

    PubMed Central

    Gossett, Daniel R.; Weaver, Westbrook M.; Mach, Albert J.; Hur, Soojung Claire; Tse, Henry Tat Kwong; Lee, Wonhee; Amini, Hamed

    2010-01-01

    Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties PMID:20419490

  5. Deep Learning in Label-free Cell Classification

    DOE PAGESBeta

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-15

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less

  6. Deep Learning in Label-free Cell Classification.

    PubMed

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-01-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219

  7. Deep Learning in Label-free Cell Classification

    NASA Astrophysics Data System (ADS)

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  8. Deep Learning in Label-free Cell Classification

    PubMed Central

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-01-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219

  9. Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy.

    PubMed

    Wang, Panxue; Pang, Shintaro; Chen, Juhong; McLandsborough, Lynne; Nugen, Sam R; Fan, Mingtao; He, Lili

    2016-02-01

    Here we presented a simple, rapid and label-free surface-enhanced Raman spectroscopy (SERS) based mapping method for the detection and discrimination of Salmonella enterica and Escherichia coli on silver dendrites. The sample preparation was first optimized to maximize sensitivity. The mapping method was then used to scan through the bacterial cells adsorbed on the surface of silver dendrites. The intrinsic and distinct SERS signals of bacterial cells were used as the basis for label-free detection and discrimination. The results show the developed method is able to detect single bacterial cells adsorbed on the silver dendrites with a limit of detection as low as 10(4) CFU mL(-1), which is two orders of magnitude lower than the traditional SERS method under the same experimental condition. The time needed for collecting a 225 points map was approximately 24 minutes. Moreover, the developed SERS mapping method can realize simultaneous detection and identification of Salmonella enterica subsp. enterica BAA1045 and Escherichia coli BL21 from a mixture sample using principle component analysis. Our results demonstrate the great potential of the label-free SERS mapping method to detect, identify and quantify bacteria and bacterial mixtures simultaneously. PMID:26750611

  10. Label-free electronic detection of target cells

    NASA Astrophysics Data System (ADS)

    Esfandyarpour, Rahim; Javanmard, Mehdi; Harris, James; Davis, Ronald W.

    2014-03-01

    In this manuscript we describe an electronic label-free method for detection of target cells, which has potential applications ranging from pathogen detection for food safety all the way to detection of circulating tumor cells for cancer diagnosis. The nanoelectronic platform consists of a stack of electrodes separated by a 30nm thick insulating layer. Cells binding to the tip of the sensor result in a decrease in the impedance at the sensing tip due to an increase in the fringing capacitance between the electrodes. As a proof of concept we demonstrate the ability to detect Saccharomyces Cerevisae cells with high specificity using a sensor functionalized with Concanavalin A. Ultimately we envision using this sensor in conjunction with a technology for pre-concentration of target cells to develop a fully integrated micro total analysis system.

  11. Single-cell label-free photoacoustic flowoxigraphy in vivo

    PubMed Central

    Wang, Lidai; Maslov, Konstantin; Wang, Lihong V.

    2013-01-01

    Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (CHb), oxygen saturation (sO2), sO2 gradient (), flow speed (vf), and oxygen release rate (rO2), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO2, , vf, and rO2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications. PMID:23536296

  12. Noninvasive and label-free determination of virus infected cells by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Moor, Kamila; Ohtani, Kiyoshi; Myrzakozha, Diyas; Zhanserkenova, Orik; Andriana, Bibin. B.; Sato, Hidetoshi

    2014-06-01

    The present study demonstrates that Raman spectroscopy is a powerful tool for the detection of virus-infected cells. Adenovirus infection of human embryonic kidney 293 cells was successfully detected at 12, 24, and 48 h after initiating the infection. The score plot of principal component analysis discriminated the spectra of the infected cells from those of the control cells. The viral infection was confirmed by the conventional immunostaining method performed 24 h after the infection. The newly developed method provides a fast and label-free means for the detection of virus-infected cells.

  13. Label-free detection of immune complexes with myeloid cells.

    PubMed

    Szittner, Z; Bentlage, A E H; Rovero, P; Migliorini, P; Lóránd, V; Prechl, J; Vidarsson, G

    2016-07-01

    The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins

  14. Label-free imaging of goblet cells as a marker for differentiating colonic polyps by multiphoton microscopy Label-free imaging of goblet cells

    NASA Astrophysics Data System (ADS)

    Zhuo, S. M.; Wu, G. Z.; Chen, J. X.; Zhu, X. Q.; Xie, S. S.

    2012-06-01

    Discrimination of adenomas from hyperplastic polyps can reduce the risk of unnecessary complications and healthcare cost. However, it is challenging during colonoscopy screening, and histological analysis remains the ``gold standard'' for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), to the discrimination between adenomas and hyperplastic polyps. We find that multiphoton imaging provides cellular and subcellular details to the identification of adenomas from hyperplastic polyps. In particular, there is significant difference in the population density of goblet cells among normal colon, hyperplastic polyp, and adenoma, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early colonic lesions. To our knowledge, this is the first demonstration of the potential of MPM for differentiation of colonic polyps.

  15. Label-free high-throughput cell screening in flow

    PubMed Central

    Mahjoubfar, Ata; Chen, Claire; Niazi, Kayvan R.; Rabizadeh, Shahrooz; Jalali, Bahram

    2013-01-01

    Flow cytometry is a powerful tool for cell counting and biomarker detection in biotechnology and medicine especially with regards to blood analysis. Standard flow cytometers perform cell type classification both by estimating size and granularity of cells using forward- and side-scattered light signals and through the collection of emission spectra of fluorescently-labeled cells. However, cell surface labeling as a means of marking cells is often undesirable as many reagents negatively impact cellular viability or provide activating/inhibitory signals, which can alter the behavior of the desired cellular subtypes for downstream applications or analysis. To eliminate the need for labeling, we introduce a label-free imaging-based flow cytometer that measures size and cell protein concentration simultaneously either as a stand-alone instrument or as an add-on to conventional flow cytometers. Cell protein concentration adds a parameter to cell classification, which improves the specificity and sensitivity of flow cytometers without the requirement of cell labeling. This system uses coherent dispersive Fourier transform to perform phase imaging at flow speeds as high as a few meters per second. PMID:24049682

  16. Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

    NASA Astrophysics Data System (ADS)

    Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

    2014-03-01

    Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.

  17. Rapid and label-free single-nucleotide discrimination via an integrative nanoparticle-nanopore approach.

    PubMed

    Ang, Yan Shan; Yung, Lin-Yue Lanry

    2012-10-23

    Single-nucleotide polymorphism (SNP) is an important biomarker for disease diagnosis, treatment monitoring, and development of personalized medicine. Recent works focused primarily on ultrasensitive detection, while the need for rapid and label-free single-nucleotide discrimination techniques, which are crucial criteria for translation into clinical applications, remains relatively unexplored. In this work, we developed a novel SNP detection assay that integrates two complementary nanotechnology systems, namely, a highly selective nanoparticle-DNA detection system and a single-particle sensitive nanopore readout platform, for rapid detection of single-site mutations. Discrete nanoparticle-DNA structures formed in the presence of perfectly matched (PM) or single-mismatched (SM) targets exhibited distinct size differences, which were resolved on a size-tunable nanopore platform to generate corresponding "yes/no" readout signals. Leveraging the in situ reaction monitoring capability of the nanopore platform, we demonstrated that real-time single-nucleotide discrimination of a model G487A mutation, responsible for glucose-6-phosphate dehydrogenase deficiency, can be achieved within 30 min with no false positives. Semiquantification of DNA samples down to picomolar concentration was carried out using a simple parameter of particle count without the need for sample labeling or signal amplification. The unique combination of nanoparticle-based detection and nanopore readout presented in this work brings forth a rapid, specific, yet simple biosensing strategy that can potentially be developed for point-of-care application. PMID:22994459

  18. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  19. Label-free discrimination of different stage nasopharyngeal carcinoma tissue based on Raman spectroscopy

    PubMed Central

    QIU, SUFANG; HUANG, QINGTING; HUANG, LINGLING; LIN, JINYONG; LU, JUN; LIN, DUO; CAO, GANG; CHEN, CHAO; PAN, JIANJI; CHEN, RONG

    2016-01-01

    The present study aimed to evaluate a label-free tissue test for the detection of nasopharyngeal carcinoma (NPC) at early and advanced stages using Raman spectroscopy (RS). RS measurements were performed to acquire high quality Raman spectra on two groups of tissue samples: One group consists of 30 NPC patients at the early stages (I–II), and the other group is 46 NPC patients at the advanced stages (III–IV). Tentative assignment of Raman bands showed specific biomolecular changes associated with cancer development. Furthermore, effective diagnostic algorithms based on principal components analysis (PCA) and linear discriminant analysis (LDA) were applied for distinguishing Raman spectra of nasopharyngeal tissues from different stages, yielding a diagnostic sensitivity of 70% and a specificity of 78%. This exploratory work suggests that RS in conjunction with the PCA-LDA algorithms provides good diagnostic ability for the early and the advanced staged NPC tissues, and RS has enormous potential for the non-invasive detection of early and advanced stage NPC. PMID:27073522

  20. Label-free single cell analysis with a chip-based impedance flow cytometer

    NASA Astrophysics Data System (ADS)

    Pierzchalski, Arkadiusz; Hebeisen, Monika; Mittag, Anja; Di Berardino, Marco; Tarnok, Attila

    2010-02-01

    For description of cellular phenotypes and physiological states new developments are needed. Axetris' impedance flow cytometer (IFC) (Leister) is a new promising label-free alternative to fluorescence-based flow cytometry (FCM). IFC measures single cells at various frequencies simultaneously. The frequencies used for signal acquisition range from 0.1 to 20 MHz. The impedance signal provides information about cell volume (< 1 MHz), membrane capacitance (~1-4 MHz) and cytoplasmic conductivity (4-10 MHz), parameters directly related to the physiological conditions of single cells. In MCF-7 cell viability experiments, cells were treated with cytotoxic agents to induce cell death. Impedance analysis showed discrimination between viable and dead cells. This was clearly visible at 4 MHz suggesting that differentiation was possible based on cell membrane capacitance. Changes in cell membrane potential were also analysed by IFC. RN22 cells were loaded with membrane potential sensitive dye (DiBAC4). The cells were then treated with the ionophore valinomycin. Changes in membrane potential were detectable at the level of cytoplasm conductivity (>4 MHz) and membrane capacitance (1-4 MHz). Our data indicate that IFC can be a valuable alternative to conventional FCM for various applications in the field of cell death and physiology. The work will be extended to address further potential applications of IFC in biotechnology and biomedical cell analysis, as well as in cell sorting.

  1. Label-Free Detection and Discrimination of Bacterial Pathogens Based on Hemin Recognition.

    PubMed

    Maltais, Thora R; Adak, Avijit K; Younis, Waleed; Seleem, Mohamed N; Wei, Alexander

    2016-07-20

    Hemin linked to hexa(ethylene glycol)bishydrazide was patterned by inkjet printing into periodic microarrays, and evaluated for their ability to capture bacterial pathogens expressing various hemin receptors. Bacterial adhesion was imaged under darkfield conditions with Fourier analysis, supporting a label-free method of pathogen detection. Hemin microarrays were screened against a panel of 16 bacteria and found capable of capturing multiple species, some with limits of detection as low as 10(3) cfu/mL. Several Gram-positive strains including Staphylococcus aureus and Bacillus anthracis also exhibited rapid adhesion, enabling pattern recognition within minutes of exposure. This can be attributed to differences in hemin acquisition systems: aggressively adherent bacteria express cell-surface hemin receptors (CSHRs) that enable direct hemin binding and uptake, whereas other types of bacteria including most Gram-negative strains rely on the secretion and recapture of soluble proteins (hemophores) for hemin acquisition, with consequently longer times for ligand binding and detection. PMID:27337653

  2. Glycan heterogeneity on gold nanoparticles increases lectin discrimination capacity in label-free multiplexed bioassays.

    PubMed

    Otten, Lucienne; Vlachou, Denise; Richards, Sarah-Jane; Gibson, Matthew I

    2016-07-21

    The development of new analytical tools as point-of-care biosensors is crucial to combat the spread of infectious diseases, especially in the context of drug-resistant organisms, or to detect biological warfare agents. Glycan/lectin interactions drive a wide range of recognition and signal transduction processes within nature and are often the first site of adhesion/recognition during infection making them appealing targets for biosensors. Glycosylated gold nanoparticles have been developed that change colour from red to blue upon interaction with carbohydrate-binding proteins and may find use as biosensors, but are limited by the inherent promiscuity of some of these interactions. Here we mimic the natural heterogeneity of cell-surface glycans by displaying mixed monolayers of glycans on the surface of gold nanoparticles. These are then used in a multiplexed, label-free bioassay to create 'barcodes' which describe the lectin based on its binding profile. The increased information content encoded by using complex mixtures of a few sugars, rather than increased numbers of different sugars makes this approach both scalable and accessible. These nanoparticles show increased lectin identification power at a range of lectin concentrations, relative to single-channel sensors. It was also found that some information about the concentration of the lectins can be extracted, all from just a simple colour change, taking this technology closer to being a realistic biosensor. PMID:27181289

  3. Glycan heterogeneity on gold nanoparticles increases lectin discrimination capacity in label-free multiplexed bioassays†

    PubMed Central

    Otten, Lucienne; Vlachou, Denise; Richards, Sarah-Jane; Gibson, Matthew I.

    2016-01-01

    The development of new analytical tools as point-of-care biosensors is crucial to combat the spread of infectious diseases, especially in the context of drug-resistant organisms, or to detect biological warfare agents. Glycan/lectin interactions drive a wide range of recognition and signal transduction processes within nature and are often the first site of adhesion/recognition during infection making them appealing targets for biosensors. Glycosylated gold nanoparticles have been developed that change colour from red to blue upon interaction with carbohydrate-binding proteins and may find use as biosensors, but are limited by the inherent promiscuity of some of these interactions. Here we mimic the natural heterogeneity of cell-surface glycans by displaying mixed monolayers of glycans on the surface of gold nanoparticles. These are then used in a multiplexed, label-free bioassay to create ‘barcodes’ which describe the lectin based on its binding profile. The increased information content encoded by using complex mixtures of a few sugars, rather than increased numbers of different sugars makes this approach both scalable and accessible. These nanoparticles show increased lectin identification power at a range of lectin concentrations, relative to single-channel sensors. It was also found that some information about the concentration of the lectins can be extracted, all from just a simple colour change, taking this technology closer to being a realistic biosensor. PMID:27181289

  4. Scattering pulse of label free fine structure cells to determine the size scale of scattering structures

    NASA Astrophysics Data System (ADS)

    Zhang, Lu; Chen, Xingyu; Zhang, Zhenxi; Chen, Wei; Zhao, Hong; Zhao, Xin; Li, Kaixing; Yuan, Li

    2016-04-01

    Scattering pulse is sensitive to the morphology and components of each single label-free cell. The most direct detection result, label free cell's scattering pulse is studied in this paper as a novel trait to recognize large malignant cells from small normal cells. A set of intrinsic scattering pulse calculation method is figured out, which combines both hydraulic focusing theory and small particle's scattering principle. Based on the scattering detection angle ranges of widely used flow cytometry, the scattering pulses formed by cell scattering energy in forward scattering angle 2°-5° and side scattering angle 80°-110° are discussed. Combining the analysis of cell's illuminating light energy, the peak, area, and full width at half maximum (FWHM) of label free cells' scattering pulses for fine structure cells with diameter 1-20 μm are studied to extract the interrelations of scattering pulse's features and cell's morphology. The theoretical and experimental results show that cell's diameter and FWHM of its scattering pulse agree with approximate linear distribution; the peak and area of scattering pulse do not always increase with cell's diameter becoming larger, but when cell's diameter is less than about 16 μm the monotone increasing relation of scattering pulse peak or area with cell's diameter can be obtained. This relationship between the features of scattering pulse and cell's size is potentially a useful but very simple criterion to distinguishing malignant and normal cells by their sizes and morphologies in label free cells clinical examinations.

  5. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-08-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

  6. Single-cell printer: automated, on demand, and label free.

    PubMed

    Gross, Andre; Schöndube, Jonas; Niekrawitz, Sonja; Streule, Wolfgang; Riegger, Lutz; Zengerle, Roland; Koltay, Peter

    2013-12-01

    Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a "one cell per droplet" printing mode. To demonstrate the prototype instrument's suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved. PMID:24222537

  7. Label-free impedance detection of cancer cells.

    PubMed

    Venkatanarayanan, Anita; Keyes, Tia E; Forster, Robert J

    2013-02-19

    Ovarian cancer cells, SKOV3, have been immobilized onto platinum microelectrodes using anti-EPCAM capture antibodies and detected with high sensitivity using electrochemical impedance. The change in impedance following cell capture is strongly dependent on the supporting electrolyte concentration. By controlling the concentration of Dulbecco's phosphate buffered saline (DPBS) electrolyte, the double layer thickness can be manipulated so that the interfacial electric field interacts with the bound cells, rather than simply decaying across the antibody capture layer. Significantly, the impedance changes markedly upon cell capture over the frequency range from 3 Hz to 90 kHz. For example, using an alternating-current (ac) amplitude of 25 mV, a frequency of 81.3 kHz, and an open circuit potential (OCP) as the direct-current (dc) voltage, a detection limit of 4 captured cells was achieved. Assuming an average cell radius of 5 μm, the linear dynamic range is from 4 captured cells to 650 ± 2 captured cells, which is approximately equivalent to fractional coverages from 0.1% to 29%. An equivalent circuit that models the impedance response of the cell capture is discussed. PMID:23331159

  8. Tumor cell differentiation by label-free microscopy

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Weber, Petra; Wagner, Michael

    2013-05-01

    Autofluorescence and Raman measurements of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of the tumor (control) cells and the less malignant cells are similar, differences can be deduced from fluorescence spectra and nanosecond decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free coenzyme NADH depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. Slight differences are also observed in the Raman spectra of these cell lines, mainly originating from small granules (lysosomes) surrounding the cell nucleus. While larger numbers of fluorescence and Raman spectra are evaluated by multivariate statistical methods, additional information is obtained from spectral images and fluorescence lifetime images (FLIM).

  9. Tumor cell differentiation by label-free fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Weber, Petra; Wagner, Michael; Kioschis, Petra; Kessler, Waltraud; Schneckenburger, Herbert

    2012-10-01

    Autofluorescence spectra, images, and decay kinetics of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of tumor (control) cells and less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay kinetics. In particular, upon near UV excitation, the fluorescence ratio of the free and protein-bound coenzyme nicotinamid adenine dinucleotide depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. While larger numbers of fluorescence spectra are evaluated by principal component analysis, a multivariate data analysis method, additional information on cell metabolism is obtained from spectral imaging and fluorescence lifetime imaging microscopy.

  10. Label-Free Segmentation of Co-cultured Cells on a Nanotopographical Gradient

    PubMed Central

    2012-01-01

    The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents. PMID:23252684

  11. In situ label-free cell viability assessment of nucleus pulposus tissue.

    PubMed

    Dittmar, Roman; van Dijk, Bart G M; van Zandvoort, Marc A M J; Ito, Keita

    2014-04-01

    Regenerative medicine approaches aiming at treating degenerating intervertebral discs, a major cause of back pain, are increasingly tested in ex-vivo disc explant models mimicking in-vivo conditions. For assessing the efficacy of regenerative therapies, cell viability is commonly measured requiring specific labels to stain cells. Here, we demonstrate and evaluate how cellular auto-fluorescence can be utilized to non-invasively assess viability in disc tissue in-situ using label-free two-photon microscopy. Live and dead bovine disc cells (0% and 100% cell viability) from the nucleus pulposus were seeded into collagen gels and auto-fluorescence was characterized. Subsequently, nucleus pulposus explants were cultured for 6 days in media with different glucose supplementation (0, 0.25, 0.5, and 1 g/L) to induce different degrees of cell death. Then, samples were split and viability was assessed using label-free two-photon microscopy and conventional staining. Results show that live and dead nucleus pulposus cells systematically emit auto-fluorescent light with distinct characteristics. Cell viability values obtained with label-free microscopy did not significantly differ from those acquired with staining. In summary, monitoring auto-fluorescence facilitates accurate cell viability assessment in nucleus tissue requiring no additional dyes. Thus, this technique may be suitable for pre-clinical testing of regenerative therapies in nucleus pulposus cultures. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:545-550, 2014. PMID:24391094

  12. Label-free measurements on cell apoptosis using a terahertz metamaterial-based biosensor

    NASA Astrophysics Data System (ADS)

    Zhang, Caihong; Liang, Lanju; Ding, Liang; Jin, Biaobing; Hou, Yayi; Li, Chun; Jiang, Ling; Liu, Weiwei; Hu, Wei; Lu, Yanqing; Kang, Lin; Xu, Weiwei; Chen, Jian; Wu, Peiheng

    2016-06-01

    Label-free, real-time, and in-situ measurement on cell apoptosis is highly desirable in cell biology. We propose here a design of terahertz (THz) metamaterial-based biosensor for meeting this requirement. This metamaterial consists of a planar array of five concentric subwavelength gold ring resonators on a 10 μm-thick polyimide substrate, which can sense the change of dielectric environment above the metamaterial. We employ this sensor to an oral cancer cell (SCC4) with and without cisplatin, a chemotherapy drug for cancer treatment, and find a linear relation between cell apoptosis measured by Flow Cytometry and the relative change of resonant frequencies of the metamaterial measured by THz time-domain spectroscopy. This implies that we can determine the cell apoptosis in a label-free manner. We believe that this metamaterial-based biosensor can be developed into a cheap, label-free, real-time, and in-situ detection tool, which is of significant impact on the study of cell biology.

  13. A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

    PubMed Central

    Raghu, Deepa; Christodoulides, Joseph A.; Delehanty, James B.; Byers, Jeff M.; Raphael, Marc P.

    2015-01-01

    Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required. In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging. A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells. PMID:26650542

  14. Label-free hyperspectral microscopy for scatter imaging of biological processes in cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hwang, Jeeseong C.; Ray, Aniruddha; Cheney, Philip P.; Chon, Bonghwan; Lee, Ji Youn; Briggman, Kimberly A.

    2016-03-01

    We will present unique applications of a label-free, hyperspectral scatter imaging technique in different microscopy platforms including conventional wide-field, dark-field, and confocal. In different platforms, we conducted label-free imaging of cells undergoing biological processes such as nanoparticle uptake, apoptosis, and metabolic flux change in response to the variation of the osmotic pressure. Hyperspectral image analyses resolved spectral endmembers corresponding to unique scattering and absorption characteristics as a result of such processes at the single particle, single organelle, and single cell level, delineating the details of nanomaterial-cell interactions in a 2D cell culture, cell apoptotic characteristics in a 3D culture, and volumetric changes of single cells under the variation of osmotic pressure. Our label-free scatter imaging has the potential for a broad range of biological and biomedical applications such as the development of scatter-based imaging contrast agents and the measurement of scatter parameters of subcellular organelles to identify the sub-micron scale origins of scattering signals in tissue scattering measurements.

  15. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    PubMed Central

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2013-01-01

    Membrane proteins (MPs) mediate a variety of cellular responses to extracellular signals. While MPs are intensely studied for their values as disease biomarkers and therapeutic targets, in situ investigation of binding kinetics of MPs with their ligands has been a challenge. Traditional approaches isolate MPs and then study them ex situ, which does not accurately reflect their native structures and functions. We present here a label-free plasmonic microscopy method to map the local binding kinetics of MPs in their native environment. This new analytical method can perform simultaneous plasmonic and fluorescence imaging, thus making it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we have determined the distribution of MPs on the surface of single cells, and the local binding kinetic constants of different MPs. Furthermore, we have studied the polarization of the MPs on the cell surface during chemotaxis. PMID:23000999

  16. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  17. Suitability of Cell-Based Label-Free Detection for Cytotoxicity Screening of Carbon Nanotubes

    PubMed Central

    Meindl, Claudia; Absenger, Markus; Roblegg, Eva; Fröhlich, Eleonore

    2013-01-01

    Cytotoxicity testing of nanoparticles (NPs) by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs) are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA) and automated microscopy (Cell-IQ) compared to formazan bioreduction (MTS assay). For validation of the label-free systems different concentrations of ethanol and of amine (AMI) polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (<8 nm) CNTs as more cytotoxic than thick (>20 nm) CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs. PMID:24377092

  18. Uncovering stem-cell heterogeneity in the microniche with label-free microfluidics

    NASA Astrophysics Data System (ADS)

    Sohn, Lydia L.

    2013-03-01

    Better suited for large number of cells from bulk tissue, traditional cell-screening techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, they rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. We have developed a label-free, single-cell analysis microfluidic platform capable of quantifying cell-surface marker expression of functional organ stem cells directly isolated from their micro-anatomical niche. With this platform, we have screened single quiescent muscle stem (satellite) cells derived from single myofibers, and we have uncovered an important heterogeneity in the surface-marker expression of these cells. By sorting the screened cells with our microfluidic device, we have determined what this heterogeneity means in terms of muscle stem-cell functionality. For instance, we show that the levels of beta1-integrin can predict the differentiation capacity of quiescent satellite cells, and in contrast to recent literature, that some CXCR4 + cells are not myogenic. Our results provide the first direct demonstration of a microniche-specific variation in gene expression in stem cells of the same lineage. Overall, our label-free, single-cell analysis and cell-sorting platform could be extended to other systems involving rare-cell subsets. This work was funded by the W. M. Keck Foundation, NIH, and California Institute of Regenerative Medicine

  19. Discriminating unalike single nucleobase mismatches using a molecularly resolved, label-free, interfacial LNA-based assay.

    PubMed

    Lahiri, Hiya; Mishra, Sourav; Mana, Tanushree; Mukhopadhyay, Rupa

    2016-06-20

    A number of reports have been made in recent times on label-free detection of nucleic acid sequences. However, most of these studies deal with ensemble measurements, therefore lacking in molecular level resolution. These assays have usually employed ssDNA sensor probes, and often suffered from problems of irreproducibility and poor sequence-selectivity. Herein, the applicability of surface-anchored single stranded locked nucleic acid (ssLNA) probes has been assessed in the detection of target DNA sequences, as an alternative to the DNA-based assay. Importantly, the effectiveness of the LNA-based assay in identifying different types of single nucleobase mismatches has been tested. Since the duplex melting temperature is an indicator of duplex stability, the ensemble on-surface Tm values of the surface-confined LNA-DNA duplexes have been compared to the duplex unbinding force values obtained from atomic force spectroscopy (AFS) experiments. A common mismatch discrimination pattern elicited by both the ensemble and the molecular level AFS approach could be identified. Apart from quantitative delineation of the different types of mismatches, the label-free AFS analysis confirms different degrees of efficiency of the purine and pyrimidine bases, present on the LNA backbone, in discriminating different nucleobase mismatch types. Importantly, the LNA-based AFS analysis can distinguish between the disease-relevant gene fragments, e.g., multidrug-resistant Mycobacterium tuberculosis (MTB) mutation, and the wild type. Since LNA probes are nuclease-resistant, these findings could potentially pave way to diagnostic applications of the LNA-based AFS assay. PMID:27124266

  20. Computational cell analysis for label-free detection of cell properties in a microfluidic laminar flow.

    PubMed

    Zhang, Alex Ce; Gu, Yi; Han, Yuanyuan; Mei, Zhe; Chiu, Yu-Jui; Geng, Lina; Cho, Sung Hwan; Lo, Yu-Hwa

    2016-06-20

    Although a flow cytometer, being one of the most popular research and clinical tools for biomedicine, can analyze cells based on the cell size, internal structures such as granularity, and molecular markers, it provides little information about the physical properties of cells such as cell stiffness and physical interactions between the cell membrane and fluid. In this paper, we propose a computational cell analysis technique using cells' different equilibrium positions in a laminar flow. This method utilizes a spatial coding technique to acquire the spatial position of the cell in a microfluidic channel and then uses mathematical algorithms to calculate the ratio of cell mixtures. Most uniquely, the invented computational cell analysis technique can unequivocally detect the subpopulation of each cell type without labeling even when the cell type shows a substantial overlap in the distribution plot with other cell types, a scenario limiting the use of conventional flow cytometers and machine learning techniques. To prove this concept, we have applied the computation method to distinguish live and fixed cancer cells without labeling, count neutrophils from human blood, and distinguish drug treated cells from untreated cells. Our work paves the way for using computation algorithms and fluidic dynamic properties for cell classification, a label-free method that can potentially classify over 200 types of human cells. Being a highly cost-effective cell analysis method complementary to flow cytometers, our method can offer orthogonal tests in companion with flow cytometers to provide crucial information for biomedical samples. PMID:27163941

  1. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Derely, L.; Vegh, A.-G.; Durand, J.-C.; Gergely, C.; Larroque, C.; Fauroux, M.-A.; Cuisinier, F. J. G.

    2013-03-01

    Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

  2. Classification of blood cells and tumor cells using label-free ultrasound and photoacoustics.

    PubMed

    Strohm, Eric M; Kolios, Michael C

    2015-08-01

    A label-free method that can identify cells in a blood sample using high frequency photoacoustic and ultrasound signals is demonstrated. When the wavelength of the ultrasound or photoacoustic wave is similar to the size of a single cell (frequencies of 100-500 MHz), unique periodic features occur within the ultrasound and photoacoustic power spectrum that depend on the cell size, structure, and morphology. These spectral features can be used to identify different cell types present in blood, such as red blood cells (RBCs), white blood cells (WBCs), and circulating tumor cells. Circulating melanoma cells are ideal for photoacoustic detection due to their endogenous optical absorption properties. Using a 532 nm pulsed laser and a 375 MHz transducer, the ultrasound and photoacoustic signals from RBCs, WBCs, and melanoma cells were individually measured in an acoustic microscope to examine how the signals change between cell types. A photoacoustic and ultrasound signal was detected from RBCs and melanoma cells; only an ultrasound signal was detected from WBCs. The different cell types were distinctly separated using the ultrasound and photoacoustic signal amplitude and power spectral periodicity. The size of each cell was also estimated from the spectral periodicity. For the first time, sound waves generated using pulse-echo ultrasound and photoacoustics have been used to identify and size single cells, with applications toward counting and identifying cells, including circulating melanoma cells. PMID:26079610

  3. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    SciTech Connect

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  4. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-09-01

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  5. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    PubMed

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  6. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    PubMed Central

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  7. Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

    PubMed Central

    Zaytseva, Natalya; Lynn, Jeffery G.; Wu, Qi; Mudaliar, Deepti J.; Sun, Haiyan; Kuang, Patty Q.; Fang, Ye

    2013-01-01

    Cell adhesion to extracellular matrix (ECM) is fundamental to many distinct aspects of cell biology, and has been an active topic for label-free biosensors. However, little attention has been paid to study the impact of receptor signaling on the cell adhesion process. We here report the development of resonant waveguide grating biosensor-enabled label-free and fluorescent approaches, and their use for investigating the adhesion of an engineered HEK-293 cell line stably expressing green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2-AR) onto distinct surfaces under both ambient and physiological conditions. Results showed that cell adhesion is sensitive to both temperature and ECM coating, and distinct mechanisms govern the cell adhesion process under different conditions. The β2-AR agonists, but not its antagonists or partial agonists, were found to be capable of triggering signaling during the adhesion process, leading to an increase in the adhesion of the engineered cells onto fibronectin-coated biosensor surfaces. These results suggest that the dual approach presented is useful to investigate the mechanism of cell adhesion, and to identify drug molecules and receptor signaling that interfere with cell adhesion. PMID:24319319

  8. Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

    PubMed Central

    Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O’Toole, Peter; Chawla, Sangeeta

    2016-01-01

    Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator. PMID:26915695

  9. Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

    NASA Astrophysics Data System (ADS)

    Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

    2016-02-01

    Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

  10. Label-free protein profiling of adipose-derived human stem cells under hyperosmotic treatment.

    PubMed

    Oswald, Elizabeth S; Brown, Lewis M; Bulinski, J Chloë; Hung, Clark T

    2011-07-01

    Our previous work suggested that treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. Here, we used label-free proteomic profiling to evaluate the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. Spectra were recorded in a data-independent fashion at alternate low (precursor) and high (product) fragmentation voltages (MS(E)). This method was supplemented with data mining of accurate mass and retention time matches in precursor ion spectra across the experiment. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase and transgelin, have been previously linked to cell differentiation state. This study demonstrates that label-free profiling can serve as a useful tool in characterizing cellular responses to chondrogenic treatment regimes, recommending its use in optimization of cell priming protocols for cartilage tissue engineering. PMID:21604804

  11. 2D light scattering static cytometry for label-free single cell analysis with submicron resolution.

    PubMed

    Xie, Linyan; Yang, Yan; Sun, Xuming; Qiao, Xu; Liu, Qiao; Song, Kun; Kong, Beihua; Su, Xuantao

    2015-11-01

    Conventional optical cytometric techniques usually measure fluorescence or scattering signals at fixed angles from flowing cells in a liquid stream. Here we develop a novel cytometer that employs a scanning optical fiber to illuminate single static cells on a glass slide, which requires neither microfluidic fabrication nor flow control. This static cytometric technique measures two dimensional (2D) light scattering patterns via a small numerical aperture (0.25) microscope objective for label-free single cell analysis. Good agreement is obtained between the yeast cell experimental and Mie theory simulated patterns. It is demonstrated that the static cytometer with a microscope objective of a low resolution around 1.30 μm has the potential to perform high resolution analysis on yeast cells with distributed sizes. The capability of the static cytometer for size determination with submicron resolution is validated via measurements on standard microspheres with mean diameters of 3.87 and 4.19 μm. Our 2D light scattering static cytometric technique may provide an easy-to-use, label-free, and flow-free method for single cell diagnostics. PMID:26115102

  12. Planar Photonic Crystal Biosensor for Quantitative Label-Free Cell Attachment Microscopy

    PubMed Central

    Chen, Weili; Long, Kenneth D.; Kurniawan, Jonas; Hung, Margaret; Yu, Hojeong; Harley, Brendan A.

    2016-01-01

    In this study, a planar-surface photonic crystal (PC) biosensor for quantitative, kinetic, label-free imaging of cell–surface interactions is demonstrated. The planar biosensor surface eliminates external stimuli to the cells caused by substrate topography to more accurately reflect smooth surface environment encountered by many cell types in vitro. Here, a fabrication approach that combines nanoreplica molding and a horizontal dipping process is used to planarize the surface of the PC biosensor. The planar PC biosensor maintains a high detection sensitivity that enables the monitoring of live cell–substrate interactions with spatial resolution sufficient for observing intracellular attachment strength gradients and the extensions of filopodia from the cell body. The evolution of cell morphology during the attachment and spreading process of 3T3 fibroblast cells is compared between planar and grating-structured PC biosensors. The planar surface effectively eliminates the directionally biased cellular attachment behaviors that are observed on the grating-structured surface. This work represents an important step forward in the development of label-free techniques for observing cellular processes without unintended external environmental modulation. PMID:26877910

  13. Rapid and Label-Free Separation of Burkitt's Lymphoma Cells from Red Blood Cells by Optically-Induced Electrokinetics

    PubMed Central

    Liang, Wenfeng; Zhao, Yuliang; Liu, Lianqing; Wang, Yuechao; Dong, Zaili; Li, Wen Jung; Lee, Gwo-Bin; Xiao, Xiubin; Zhang, Weijing

    2014-01-01

    Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and

  14. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    PubMed

    Liang, Wenfeng; Zhao, Yuliang; Liu, Lianqing; Wang, Yuechao; Dong, Zaili; Li, Wen Jung; Lee, Gwo-Bin; Xiao, Xiubin; Zhang, Weijing

    2014-01-01

    Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and

  15. Label-free cell cycle analysis for high-throughput imaging flow cytometry

    PubMed Central

    Blasi, Thomas; Hennig, Holger; Summers, Huw D.; Theis, Fabian J.; Cerveira, Joana; Patterson, James O.; Davies, Derek; Filby, Andrew; Carpenter, Anne E.; Rees, Paul

    2016-01-01

    Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types. PMID:26739115

  16. Label-free Electrophysiological Cytometry for Stem Cell-Derived Cardiomyocyte Clusters

    PubMed Central

    Myers, Frank B.; Abilez, Oscar J.; Zarins, Christopher K.; Lee, Luke P.

    2012-01-01

    Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However, a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here, we describe a non-genetic, label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes, neurons, smooth muscle cells), this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 μVrms. Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals, the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry, in which a cell’s functionality is assessed rather than its expression profile or physical characteristics. PMID:23207961

  17. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    PubMed Central

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-01-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis. PMID:27225821

  18. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    NASA Astrophysics Data System (ADS)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43‑ symmetric stretch vibrations at 959 cm‑1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.

  19. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

    PubMed

    Hofemeier, Arne D; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F W; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-01-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis. PMID:27225821

  20. Hydrodynamic and label-free sorting of circulating tumor cells from whole blood

    NASA Astrophysics Data System (ADS)

    Geislinger, Thomas M.; Stamp, Melanie E. M.; Wixforth, Achim; Franke, Thomas

    2015-11-01

    We demonstrate continuous, passive, and label-free sorting of different in vitro cancer cell lines (MV3, MCF7, and HEPG2) as model systems for circulating tumor cells (CTCs) from undiluted whole blood employing the non-inertial lift effect as driving force. This purely viscous, repulsive cell-wall interaction is sensitive to cell size and deformability differences and yields highly efficient cell separation and high enrichment factors. We show that the performance of the device is robust over a large range of blood cell concentrations and flow rates as well as for the different cell lines. The collected samples usually contain more than 90% of the initially injected CTCs and exhibit average enrichment factors of more than 20 for sorting from whole blood samples.

  1. In vivo capture and label-free detection of early metastatic cells

    PubMed Central

    Azarin, Samira M.; Yi, Ji; Gower, Robert M.; Aguado, Brian A.; Sullivan, Megan E.; Goodman, Ashley G.; Jiang, Eric J.; Rao, Shreyas S.; Ren, Yinying; Tucker, Susan L.; Backman, Vadim; Jeruss, Jacqueline S.; Shea, Lonnie D.

    2015-01-01

    Breast cancer is a leading cause of death for women, with mortality resulting from metastasis. Metastases are often detected once tumor cells affect the function of solid organs, with a high disease burden limiting effective treatment. Here we report a method for the early detection of metastasis using an implanted scaffold to recruit and capture metastatic cells in vivo, which achieves high cell densities and reduces the tumor burden within solid organs 10-fold. Recruitment is associated with infiltration of immune cells, which include Gr1hiCD11b+ cells. We identify metastatic cells in the scaffold through a label-free detection system using inverse-spectroscopic optical coherence tomography, which identifies changes to nanoscale tissue architecture associated with the presence of tumor cells. For patients at risk of recurrence, scaffold implantation following completion of primary therapy has the potential to identify metastatic disease at the earliest stage, enabling initiation of therapy while the disease burden is low. PMID:26348915

  2. Probing Xylan-Specific Raman Bands for Label-Free Imaging Xylan in Plant Cell Wall

    SciTech Connect

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; Tucker, Melvin P.; Vinzant, Todd; Himmel, Michael E.

    2015-06-15

    Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike lignin and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.

  3. Label-free assessment of replicative senescence in mesenchymal stem cells by Raman microspectroscopy

    PubMed Central

    Bai, Hua; Li, Haiyu; Han, Zhibo; Zhang, Cheng; Zhao, Junfa; Miao, Changyun; Yan, Shulin; Mao, Aibin; Zhao, Hui; Han, Zhongchao

    2015-01-01

    Here, Raman microspectroscopy was employed to assess replicative senescence of mesenchymal stem cells (MSC). A regular spectral change related to the cell senescence was found in the ratio of two peaks at 1157 cm−1 and 1174 cm−1, which are assigned to C-C, C-N stretching vibrations in proteins and C-H bending vibrations in tyrosine and phenylalanine, respectively. With the cell aging, the ratio I1157 / I1174 exhibited a monotonic decline and showed small standard deviations, so that it can statistically distinguish between cells having slight changes in terms of aging. We propose that I1157 / I1174 can act as a characteristic spectral signature for label-free assessment of MSC senescence. PMID:26601012

  4. Label-free assessment of replicative senescence in mesenchymal stem cells by Raman microspectroscopy.

    PubMed

    Bai, Hua; Li, Haiyu; Han, Zhibo; Zhang, Cheng; Zhao, Junfa; Miao, Changyun; Yan, Shulin; Mao, Aibin; Zhao, Hui; Han, Zhongchao

    2015-11-01

    Here, Raman microspectroscopy was employed to assess replicative senescence of mesenchymal stem cells (MSC). A regular spectral change related to the cell senescence was found in the ratio of two peaks at 1157 cm(-1) and 1174 cm(-1), which are assigned to C-C, C-N stretching vibrations in proteins and C-H bending vibrations in tyrosine and phenylalanine, respectively. With the cell aging, the ratio I1157 / I1174 exhibited a monotonic decline and showed small standard deviations, so that it can statistically distinguish between cells having slight changes in terms of aging. We propose that I1157 / I1174 can act as a characteristic spectral signature for label-free assessment of MSC senescence. PMID:26601012

  5. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

    PubMed Central

    Stringari, Chiara; Sierra, Robert; Donovan, Peter J.

    2012-01-01

    Abstract. We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation. PMID:22559690

  6. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

    NASA Astrophysics Data System (ADS)

    Stringari, Chiara; Sierra, Robert; Donovan, Peter J.; Gratton, Enrico

    2012-04-01

    We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.

  7. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    PubMed Central

    Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

    2015-01-01

    A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

  8. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

    NASA Astrophysics Data System (ADS)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

  9. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns.

    PubMed

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis. PMID:27475572

  10. Node-Pore Sensing Enables Label-Free Surface-Marker Profiling of Single Cells

    PubMed Central

    2015-01-01

    Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside. PMID:25625182

  11. Node-pore sensing enables label-free surface-marker profiling of single cells.

    PubMed

    Balakrishnan, Karthik R; Whang, Jeremy C; Hwang, Richard; Hack, James H; Godley, Lucy A; Sohn, Lydia L

    2015-03-01

    Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside. PMID:25625182

  12. Label-free whole blood cell differentiation based on multiple frequency AC impedance and light scattering analysis in a micro flow cytometer.

    PubMed

    Simon, Peter; Frankowski, Marcin; Bock, Nicole; Neukammer, Jörg

    2016-06-21

    We developed a microfluidic sensor for label-free flow cytometric cell differentiation by combined multiple AC electrical impedance and light scattering analysis. The measured signals are correlated to cell volume, membrane capacity and optical properties of single cells. For an improved signal to noise ratio, the microfluidic sensor incorporates two electrode pairs for differential impedance detection. One-dimensional sheath flow focusing was implemented, which allows single particle analysis at kHz count rates. Various monodisperse particles and differentiation of leukocytes in haemolysed samples served to benchmark the microdevice applying combined AC impedance and side scatter analyses. In what follows, we demonstrate that AC impedance measurements at selected frequencies allow label-free discrimination of platelets, erythrocytes, monocytes, granulocytes and lymphocytes in whole blood samples involving dilution only. Immunofluorescence staining was applied to validate the results of the label-free cell analysis. Reliable differentiation and enumeration of cells in whole blood by AC impedance detection have the potential to support medical diagnosis for patients with haemolysis resistant erythrocytes or abnormally sensitive leucocytes, i.e. for patients suffering from anaemia or leukaemia. PMID:27229300

  13. Label-free characterization of white blood cells by measuring 3D refractive index maps

    PubMed Central

    Yoon, Jonghee; Kim, Kyoohyun; Park, HyunJoo; Choi, Chulhee; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs. PMID:26504637

  14. Fast Label-Free Cytoskeletal Network Imaging in Living Mammalian Cells

    PubMed Central

    Bon, Pierre; Lécart, Sandrine; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2014-01-01

    We present a full-field technique that allows label-free cytoskeletal network imaging inside living cells. This noninvasive technique allows monitoring of the cytoskeleton dynamics as well as interactions between the latter and organelles on any timescale. It is based on high-resolution quantitative phase imaging (modified Quadriwave lateral shearing interferometry) and can be directly implemented using any optical microscope without modification. We demonstrate the capability of our setup on fixed and living Chinese hamster ovary cells, showing the cytoskeleton dynamics in lamellipodia during protrusion and mitochondria displacement along the cytoskeletal network. In addition, using the quantitative function of the technique, along with simulation tools, we determined the refractive index of a single tubulin microtubule to be ntubu=2.36±0.6 at λ=527 nm. PMID:24739158

  15. Increasing label-free stem cell sorting capacity to reach transplantation-scale throughput

    PubMed Central

    Li, Ying; Arulmoli, Janahan; McDonnell, Lisa P.; Nourse, Jamison L.; Lee, Abraham P.; Flanagan, Lisa A.

    2014-01-01

    Dielectrophoresis (DEP) has proven an invaluable tool for the enrichment of populations of stem and progenitor cells owing to its ability to sort cells in a label-free manner and its biological safety. However, DEP separation devices have suffered from a low throughput preventing researchers from undertaking studies requiring large numbers of cells, such as needed for cell transplantation. We developed a microfluidic device designed for the enrichment of stem and progenitor cell populations that sorts cells at a rate of 150,000 cells/h, corresponding to an improvement in the throughput achieved with our previous device designs by over an order of magnitude. This advancement, coupled with data showing the DEP-sorted cells retain their enrichment and differentiation capacity when expanded in culture for periods of up to 2 weeks, provides sufficient throughput and cell numbers to enable a wider variety of experiments with enriched stem and progenitor cell populations. Furthermore, the sorting devices presented here provide ease of setup and operation, a simple fabrication process, and a low associated cost to use that makes them more amenable for use in common biological research laboratories. To our knowledge, this work represents the first to enrich stem cells and expand them in culture to generate transplantation-scale numbers of differentiation-competent cells using DEP. PMID:25553183

  16. Label-Free Cell Phenotypic Identification of D-Luciferin as an Agonist for GPR35.

    PubMed

    Hu, Heidi; Deng, Huayun; Fang, Ye

    2016-01-01

    D-Luciferin (also known as beetle or firefly luciferin) is one of the most widely used bioluminescent reporters for monitoring in vitro or in vivo luciferase activity. The identification of several natural phenols and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as agonists for GPR35, an orphan G protein-coupled receptor, had motivated us to examine the pharmacological activity of D-Luciferin, given that it also contains phenol and carboxylic acid moieties. Here, we describe label-free cell phenotypic assays that ascertain D-Luciferin as a partial agonist for GPR35. The agonistic activity of D-Luciferin at the GPR35 shall evoke careful interpretation of biological data when D-Luciferin or its analogues are used as probes. PMID:27424891

  17. High Throughput Label Free Measurement of Cancer Cell Adhesion Kinetics Under Hemodynamic Flow

    PubMed Central

    Spencer, Adrianne; Baker, Aaron B.

    2016-01-01

    The kinetics of receptor-mediated cell adhesion to extracellular matrix and adherent cell monolayers plays a key role in many physiological and pathological processes including cancer metastasis. Within this process the presence of fluidic shear forces is a key regulator of binding equilibrium and kinetics of cell adhesion. Current techniques to examine the kinetics of cell adhesion are either performed in the absence of flow or are low throughput, limiting their application to pharmacological compound screening or the high throughput investigation of biological mechanisms. We developed a high throughput flow device that applies flow in a multi-well format and interfaced this system with electric cell-substrate impedance sensing (ECIS) system to allow label free detection of cell adhesion. We demonstrate that this combined system is capable of making real time measurements of cancer cell adhesion to extracellular matrix and immobilized platelets. In addition, we examined the dependence of the kinetics of binding of cancer cells on the level of shear stress and in the presence of small molecule inhibitors to adhesion-related pathways. This versatile system is broadly adaptable to the high throughput study of cell adhesion kinetics for many applications including drug screening and the investigation of the mechanisms of cancer metastasis. PMID:26816215

  18. Label-free identification and characterization of living human primary and secondary tumour cells.

    PubMed

    Tsikritsis, Dimitrios; Richmond, Susanna; Stewart, Patrick; Elfick, Alistair; Downes, Andrew

    2015-08-01

    We used three label-free minimally invasive methods to characterize individual cells derived from primary and secondary tumours from the same patient, and of the same type – colorectal. Raman spectroscopy distinguished cells by their biochemical 'fingerprint' in a vibrational spectrum with 100% accuracy, and revealed that the primary cell line contains more lipids and alpha-helix proteins, whereas the secondary cell line contains more porphyrins and beta-sheet proteins. Stimulated Raman scattering (SRS) microscopy distinguished cells in chemically-specific images of CH2 bonds which revealed lipid droplets in secondary tumour cells. Atomic force microscopy (AFM) was used to distinguish cells with 80% accuracy by measuring their elasticity – secondary tumour cells (SW620) are around 3 times softer than primary ones (SW480). As well as characterizing the physical and biochemical differences between cell lines in vitro, these techniques offer three novel methods which could potentially be used for diagnosis – to assign a tumour as primary or secondary. PMID:26086957

  19. Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

    2015-03-01

    Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

  20. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.

    PubMed

    Riba, J; Gleichmann, T; Zimmermann, S; Zengerle, R; Koltay, P

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry. PMID:27596612

  1. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    PubMed Central

    Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry. PMID:27596612

  2. Label-free detection of liver cancer cells by aptamer-based microcantilever biosensor.

    PubMed

    Chen, Xuejuan; Pan, Yangang; Liu, Huiqing; Bai, Xiaojing; Wang, Nan; Zhang, Bailin

    2016-05-15

    Liver cancer is one of the most common and highly malignant cancers in the world. There are no effective therapeutic options if an early liver cancer diagnosis is not achieved. In this work, detection of HepG2 cells by label-free microcantilever array aptasensor was developed. The sensing microcantilevers were functionalized by HepG2 cells-specific aptamers. Meanwhile, to eliminate the interferences induced by the environment, the reference microcantilevers were modified with 6-mercapto-1-hexanol self-assembled monolayers. The aptasensor exhibits high specificity over not only human liver normal cells, but also other cancer cells of breast, bladder, and cervix tumors. The linear relation ranges from 1×10(3) to 1×10(5)cells/mL, with a detection limit of 300 cells/mL (S/N=3). Our work provides a simple method for detection of liver cancer cells with advantages in terms of simplicity and stability. PMID:26735868

  3. A silicon-based peptide biosensor for label-free detection of cancer cells

    NASA Astrophysics Data System (ADS)

    Martucci, Nicola M.; Rea, Ilaria; Ruggiero, Immacolata; Terracciano, Monica; De Stefano, Luca; Migliaccio, Nunzia; Dardano, Principia; Arcari, Paolo; Rendina, Ivo; Lamberti, Annalisa

    2015-05-01

    Sensitive and accurate detection of cancer cells plays a crucial role in diagnosis of cancer and minimal residual disease, so being one of the most hopeful approaches to reduce cancer death rates. In this paper, a strategy for highly selective and sensitive detection of lymphoma cells on planar silicon-based biosensor has been evaluated. In this setting an Idiotype peptide, able to specifically bind the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, has been covalently linked to the sensor active surface and used as molecular probe. The biochip here presented showed a coverage efficiency of 85% with a detection efficiency of 8.5×10-3 cells/μm2. The results obtained suggested an efficient way for specific label-free cell detection by using a silicon-based peptide biosensor. In addition, the present recognition strategy, besides being useful for the development of sensing devices capable of monitoring minimal residual disease, could be used to find and characterize new specific receptor-ligand interactions through the screening of a recombinant phage library.

  4. In situ label-free quantification of human pluripotent stem cells with electrochemical potential.

    PubMed

    Yea, Cheol-Heon; Jeong, Ho-Chang; Moon, Sung-Hwan; Lee, Mi-Ok; Kim, Kyeong-Jun; Choi, Jeong-Woo; Cha, Hyuk-Jin

    2016-01-01

    Conventional methods for quantification of undifferentiated pluripotent stem cells such as fluorescence-activated cell sorting and real-time PCR analysis have technical limitations in terms of their sensitivity and recyclability. Herein, we designed a real-time in situ label-free monitoring system on the basis of a specific electrochemical signature of human pluripotent stem cells in vitro. The intensity of the signal of hPSCs highly corresponded to the cell number and remained consistent in a mixed population with differentiated cells. The electrical charge used for monitoring did not markedly affect the proliferation rate or molecular characteristics of differentiated human aortic smooth muscle cells. After YM155 treatment to ablate undifferentiated hPSCs, their specific signal was significantly reduced. This suggests that detection of the specific electrochemical signature of hPSCs would be a valid approach to monitor potential contamination of undifferentiated hPSCs, which can assess the risk of teratoma formation efficiently and economically. PMID:26513417

  5. Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.

    PubMed

    Warkiani, Majid Ebrahimi; Khoo, Bee Luan; Wu, Lidan; Tay, Andy Kah Ping; Bhagat, Ali Asgar S; Han, Jongyoon; Lim, Chwee Teck

    2016-01-01

    Circulating tumor cells (CTCs) are rare cancer cells that are shed from primary or metastatic tumors into the peripheral blood circulation. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer staging and treatment, and thus further research into their characteristics and properties is an area of considerable interest. In this protocol, we describe detailed procedures for the production and use of a label-free spiral microfluidic device to allow size-based isolation of viable CTCs using hydrodynamic forces that are present in curvilinear microchannels. This spiral system enables us to achieve ≥ 85% recovery of spiked cells across multiple cancer cell lines and 99.99% depletion of white blood cells in whole blood. The described spiral microfluidic devices can be produced at an extremely low cost using standard microfabrication and soft lithography techniques (2-3 d), and they can be operated using two syringe pumps for lysed blood samples (7.5 ml in 12.5 min for a three-layered multiplexed chip). The fast processing time and the ability to collect CTCs from a large patient blood volume allows this technique to be used experimentally in a broad range of potential genomic and transcriptomic applications. PMID:26678083

  6. Label-free identification of white blood cell using optical diffraction tomography (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Yoon, Jonghee; Kim, Kyoohyun; Kim, Min-hyeok; Kang, Suk-Jo; Park, YongKeun

    2016-03-01

    White blood cells (WBC) have crucial roles in immune systems which defend the host against from disease conditions and harmful invaders. Various WBC subsets have been characterized and reported to be involved in many pathophysiologic conditions. It is crucial to isolate a specific WBC subset to study its pathophysiological roles in diseases. Identification methods for a specific WBC population are rely on invasive approaches, including Wright-Gimesa staining for observing cellular morphologies and fluorescence staining for specific protein markers. While these methods enable precise classification of WBC populations, they could disturb cellular viability or functions. In order to classify WBC populations in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a three-dimensional (3-D) quantitative phase imaging technique that measures 3-D refractive index (RI) distributions of individual WBCs. To test feasibility of label-free classification of WBC populations using ODT, we measured four subtypes of WBCs, including B cell, CD4 T cell, CD8 T cell, and natural killer (NK) cell. From measured 3-D RI tomograms of WBCs, we obtain quantitative structural and biochemical information and classify each WBC population using a machine learning algorithm.

  7. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    NASA Astrophysics Data System (ADS)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  8. Label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

    2016-03-01

    Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

  9. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  10. Label-free imaging of Schwann cell myelination by third harmonic generation microscopy.

    PubMed

    Lim, Hyungsik; Sharoukhov, Denis; Kassim, Imran; Zhang, Yanqing; Salzer, James L; Melendez-Vasquez, Carmen V

    2014-12-16

    Understanding the dynamic axon-glial cell interaction underlying myelination is hampered by the lack of suitable imaging techniques. Here we demonstrate third harmonic generation microscopy (THGM) for label-free imaging of myelinating Schwann cells in live culture and ex vivo and in vivo tissue. A 3D structure was acquired for a variety of compact and noncompact myelin domains, including juxtaparanodes, Schmidt-Lanterman incisures, and Cajal bands. Other subcellular features of Schwann cells that escape traditional optical microscopies were also visualized. We tested THGM for morphometry of compact myelin. Unlike current methods based on electron microscopy, g-ratio could be determined along an extended length of myelinated fiber in the physiological condition. The precision of THGM-based g-ratio estimation was corroborated in mouse models of hypomyelination. Finally, we demonstrated the feasibility of THGM to monitor morphological changes of myelin during postnatal development and degeneration. The outstanding capabilities of THGM may be useful for elucidation of the mechanism of myelin formation and pathogenesis. PMID:25453108

  11. Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information

    PubMed Central

    Marrison, Joanne; Räty, Lotta; Marriott, Poppy; O'Toole, Peter

    2013-01-01

    Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report on the biological application of a, label-free, high contrast microscopy technique known as ptychography, in which the image producing step is transferred from the microscope lens to a high-speed phase retrieval algorithm. We demonstrate that this technology is appropriate for label-free imaging of adherent cells and is particularly suitable for reporting cellular changes such as mitosis, apoptosis and cell differentiation. The high contrast, artefact-free, focus-free information rich images allow dividing cells to be distinguished from non-dividing cells by a greater than two-fold increase in cell contrast, and we demonstrate this technique is suitable for downstream automated cell segmentation and analysis. PMID:23917865

  12. Intrinsic Indicator of Photodamage during Label-Free Multiphoton Microscopy of Cells and Tissues

    PubMed Central

    Andresen, Elisabeth F.; Geiger, Kathrin D.; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2014-01-01

    Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments. PMID:25343251

  13. Label-free Screening of Multiple Cell-surface Antigens Using a Single Pore

    NASA Astrophysics Data System (ADS)

    Balakrishnan, Karthik; Chapman, Matthew; Kesavaraju, Anand; Sohn, Lydia

    2012-02-01

    Microfluidic pores have emerged as versatile tools for performing highly sensitive measurements. Pore functionalization can result in slower particle transit rates, thereby providing insight into the properties of particles that travel through a pore. While enhancing utility, functionalizing with only one species limits the broader applicability of pores for biosensing by restricting the insight gained in a single run. We have developed a method of using variable cross-section pores to create unique electronic signatures for reliable detection and automated data analysis. By defining a single pore into sections using common lithography techniques, we can detect when a cell passes through a given pore segment using resistive-pulse sensing. This offers such advantages as 1) the ability to functionalize each portion of a pore with a different antibody that corresponds to different cell surface receptors, enabling label-free multianalyte detection in a single run; and 2) a unique electronic signature that allows for both an accelerated real-time analysis and an additional level of precision to testing. This is particularly critical for clinical diagnostics where accuracy and reliability of results are crucial for healthcare professionals upon which to act.

  14. Identification of Novel GPR55 Modulators Using Cell-Impedance-Based Label-Free Technology.

    PubMed

    Morales, Paula; Whyte, Lauren S; Chicharro, Roberto; Gómez-Cañas, María; Pazos, M Ruth; Goya, Pilar; Irving, Andrew J; Fernández-Ruiz, Javier; Ross, Ruth A; Jagerovic, Nadine

    2016-03-10

    The orphan G protein-coupled receptor GPR55 has been proposed as a novel receptor of the endocannabinoid system. However, the validity of this categorization is still under debate mainly because of the lack of potent and selective agonists and antagonists of GPR55. Binding assays are not yet available for GPR55 screening, and discrepancies in GPR55 mediated signaling pathways have been reported. In this context, we have designed and synthesized novel GPR55 ligands based on a chromenopyrazole scaffold. Appraisal of GPR55 activity was accomplished using a label-free cell-impedance-based assay in hGPR55-HEK293 cells. The real-time impedance responses provided an integrative assessment of the cellular consequence to GPR55 stimulation taking into account the different possible signaling pathways. Potent GPR55 partial agonists (14b, 18b, 19b, 20b, and 21-24) have been identified; one of them (14b) being selective versus classical cannabinoid receptors. Upon antagonist treatment, chromenopyrazoles 21-24 inhibited lysophosphatidylinositol (LPI) effect. One of these GPR55 antagonists (21) is fully selective versus classic cannabinoid receptors. Compared to LPI, the predicted physicochemical parameters of the new compounds suggest a clear pharmacokinetic improvement. PMID:26789378

  15. Multimodal interferometric microscopy for label-free 3D imaging of live cells in flow (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Shaked, Natan Tzvi

    2016-03-01

    I present multimodal wide-field interferometric microscopy platform for label-free 3-D imaging of live cells during fast flow. Using holographic optical tweezers, multiple cells can be optically trapped and rapidity rotated on all axes, while acquired using an external off-axis wide-field interferometric module developed in our lab. The interferometric projections are rapidly processed into the 3-D refractive-index profile of the cells using a tomographic phase microscopy algorithms that take into consideration optical diffraction effects. The algorithms for the 3-D refractive-index reconstruction, and for calculating various morphological parameters that should serve for online sorting of cells, are efficiently implemented in a nearly real-time manner. The potential of this new high-throughput imaging technique is for label-free image analysis and sorting of cells during flow, to substitute current cell sorting devices, which are based on external labeling that eventually damages the cell sample.

  16. A novel self-powered and sensitive label-free DNA biosensor in microbial fuel cell.

    PubMed

    Asghary, Maryam; Raoof, Jahan Bakhsh; Rahimnejad, Mostafa; Ojani, Reza

    2016-08-15

    In this work, a novel self-powered, sensitive, low-cost, and label-free DNA biosensor is reported by applying a two-chambered microbial fuel cell (MFC) as a power supply. A graphite electrode and an Au nanoparticles modified graphite electrode (AuNP/graphite electrode) were used as anode and cathode in the MFC system, respectively. The active biocatalyst in the anodic chamber was a mixed culture of microorganisms. The sensing element of the biosensor was fabricated by the well-known Au-thiol binding the ssDNA probe on the surface of an AuNP/graphite cathode. Electrons produced by microorganisms were transported from the anode to the cathode through an external circuit, which could be detected by the terminal multi-meter detector. The difference between power densities of the ssDNA probe modified cathode in the absence and presence of complementary sequence served as the detection signal of the DNA hybridization with detection limit of 3.1nM. Thereafter, this biosensor was employed for diagnosis and determination of complementary sequence in a human serum sample. The hybridization specificity studies further revealed that the developed DNA biosensor could distinguish fully complementary sequences from one-base mismatched and non-complementary sequences. PMID:27085948

  17. Quantitative label-free redox proteomics of reversible cysteine oxidation in red blood cell membranes.

    PubMed

    Zaccarin, Mattia; Falda, Marco; Roveri, Antonella; Bosello-Travain, Valentina; Bordin, Luciana; Maiorino, Matilde; Ursini, Fulvio; Toppo, Stefano

    2014-06-01

    Reversible oxidation of cysteine residues is a relevant posttranslational modification of proteins. However, the low activation energy and transitory nature of the redox switch and the intrinsic complexity of the analysis render quite challenging the aim of a rigorous high-throughput screening of the redox status of redox-sensitive cysteine residues. We describe here a quantitative workflow for redox proteomics, where the ratio between the oxidized forms of proteins in the control vs treated samples is determined by a robust label-free approach. We critically present the convenience of the procedure by specifically addressing the following aspects: (i) the accurate ratio, calculated from the whole set of identified peptides rather than just isotope-tagged fragments; (ii) the application of a robust analytical pipeline to frame the most consistent data averaged over the biological variability; (iii) the relevance of using stringent criteria of analysis, even at the cost of losing potentially interesting but statistically uncertain data. The pipeline has been assessed on red blood cell membrane challenged with diamide as a model of a mild oxidative condition. The cluster of identified proteins encompassed components of the cytoskeleton more oxidized. Indirectly, our analysis confirmed the previous observation that oxidized hemoglobin binds to membranes while oxidized peroxiredoxin 2 loses affinity. PMID:24642086

  18. Label-free single-cell protein quantification using a drop-based mix-and-read system

    PubMed Central

    Abbaspourrad, Alireza; Zhang, Huidan; Tao, Ye; Cui, Naiwen; Asahara, Haruichi; Zhou, Ying; Yue, Dongxian; Koehler, Stephan A.; Ung, Lloyd W.; Heyman, John; Ren, Yukun; Ziblat, Roy; Chong, Shaorong; Weitz, David A.

    2015-01-01

    Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry. PMID:26234416

  19. Slanted spiral microfluidics for the ultra-fast, label-free isolation of circulating tumor cells.

    PubMed

    Warkiani, Majid Ebrahimi; Guan, Guofeng; Luan, Khoo Bee; Lee, Wong Cheng; Bhagat, Ali Asgar S; Chaudhuri, Parthiv Kant; Tan, Daniel Shao-Weng; Lim, Wan Teck; Lee, Soo Chin; Chen, Peter C Y; Lim, Chwee Teck; Han, Jongyoon

    2014-01-01

    The enumeration and characterization of circulating tumor cells (CTCs), found in the peripheral blood of cancer patients, provide a potentially accessible source for cancer diagnosis and prognosis. This work reports on a novel spiral microfluidic device with a trapezoidal cross-section for ultra-fast, label-free enrichment of CTCs from clinically relevant blood volumes. The technique utilizes the inherent Dean vortex flows present in curvilinear microchannels under continuous flow, along with inertial lift forces which focus larger CTCs against the inner wall. Using a trapezoidal cross-section as opposed to a traditional rectangular cross-section, the position of the Dean vortex core can be altered to achieve separation. Smaller hematologic components are trapped in the Dean vortices skewed towards the outer channel walls and eventually removed at the outer outlet, while the larger CTCs equilibrate near the inner channel wall and are collected from the inner outlet. By using a single spiral microchannel with one inlet and two outlets, we have successfully isolated and recovered more than 80% of the tested cancer cell line cells (MCF-7, T24 and MDA-MB-231) spiked in 7.5 mL of blood within 8 min with extremely high purity (400-680 WBCs mL(-1); ~4 log depletion of WBCs). Putative CTCs were detected and isolated from 100% of the patient samples (n = 10) with advanced stage metastatic breast and lung cancer using standard biomarkers (CK, CD45 and DAPI) with the frequencies ranging from 3-125 CTCs mL(-1). We expect this simple and elegant approach can surmount the shortcomings of traditional affinity-based CTC isolation techniques as well as enable fundamental studies on CTCs to guide treatment and enhance patient care. PMID:23949794

  20. CARS based label-free assay for assessment of drugs by monitoring lipid droplets in tumour cells.

    PubMed

    Steuwe, Christian; Patel, Imran I; Ul-Hasan, Mahmud; Schreiner, Alexander; Boren, Joan; Brindle, Kevin M; Reichelt, Stefanie; Mahajan, Sumeet

    2014-11-01

    Coherent anti-Stokes Raman scattering (CARS) is becoming an established tool for label-free multi-photon imaging based on molecule specific vibrations in the sample. The technique has proven to be particularly useful for imaging lipids, which are abundant in cells and tissues, including cytoplasmic lipid droplets (LD), which are recognized as dynamic organelles involved in many cellular functions. The increase in the number of lipid droplets in cells undergoing cell proliferation is a common feature in many neoplastic processes [1] and an increase in LD number also appears to be an early marker of drug-induced cell stress and subsequent apoptosis [3]. In this paper, a CARS-based label-free method is presented to monitor the increase in LD content in HCT116 colon tumour cells treated with the chemotherapeutic drugs Etoposide, Camptothecin and the protein kinase inhibitor Staurosporine. Using CARS, LDs can easily be distinguished from other cell components without the application of fluorescent dyes and provides a label-free non-invasive drug screening assay that could be used not only with cells and tissues ex vivo but potentially also in vivo. PMID:24343869

  1. Non-invasive and label-free detection of oral squamous cell carcinoma using saliva surface-enhanced Raman spectroscopy and multivariate analysis.

    PubMed

    Connolly, Jennifer M; Davies, Karen; Kazakeviciute, Agne; Wheatley, Antony M; Dockery, Peter; Keogh, Ivan; Olivo, Malini

    2016-08-01

    Reported here is the application of silver nanoparticle-based surface-enhanced Raman spectroscopy (SERS) as a label-free, non-invasive technique for detection of oral squamous cell cancer (OSCC) using saliva and desquamated oral cells. A total of 180 SERS spectra were acquired from saliva and 120 SERS spectra from oral cells collected from normal healthy individuals and from confirmed oropharyngeal cancer patients. Notable biochemical peaks in the SERS spectra were tentatively assigned to various components. Data were subjected to multivariate statistical techniques including principal component analysis, linear discriminate analysis (PCA-LDA) and logistic regression (LR) revealing a sensitivity of 89% and 68% and a diagnostic accuracy of 73% and 60% for saliva and oral cells, respectively. The results from this study demonstrate the potential of saliva and oral cell SERS combined with PCA-LDA or PCA-LR diagnostic algorithms as a promising clinical adjunct for the non-invasive detection of oral cancer. PMID:27015768

  2. Label-Free and Continuous-Flow Ferrohydrodynamic Separation of HeLa Cells and Blood Cells in Biocompatible Ferrofluids

    PubMed Central

    Zhao, Wujun; Zhu, Taotao; Cheng, Rui; Liu, Yufei; He, Jian; Qiu, Hong; Wang, Lianchun; Nagy, Tamas; Querec, Troy D.; Unger, Elizabeth R.

    2016-01-01

    In this study, a label-free, low-cost, and fast ferrohydrodynamic cell separation scheme is demonstrated using HeLa cells (an epithelial cell line) and red blood cells. The separation is based on cell size difference, and conducted in a custom-made biocompatible ferrofluid that retains the viability of cells during and after the assay for downstream analysis. The scheme offers moderate-throughput (≈106 cells h−1 for a single channel device) and extremely high recovery rate (>99%) without the use of any label. It is envisioned that this separation scheme will have clinical applications in settings where rapid cell enrichment and removal of contaminating blood will improve efficiency of screening and diagnosis such as cervical cancer screening based on mixed populations in exfoliated samples. PMID:27478429

  3. Toward label-free Raman-activated cell sorting of cardiomyocytes derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Pascut, Flavius C.; Goh, Huey T.; George, Vinoj; Denning, Chris; Notingher, Ioan

    2011-04-01

    Raman micro-spectroscopy (RMS) has been recently proposed for label-free phenotypic identification of human embryonic stem cells (hESC)-derived cardiomyocytes. However, the methods used for measuring the Raman spectra led to acquisition times of minutes per cell, which is prohibitive for rapid cell sorting applications. In this study we evaluated two measurement strategies that could reduce the measurement time by a factor of more than 100. We show that sampling individual cells with a laser beam focused to a line could eliminate the need of cell raster scanning and achieve high prediction accuracies (>95% specificity and >96% sensitivity) with acquisition times ~5 seconds per cell. However, the use of commercially-available higher power lasers could potentially lead to sorting speeds of ~10 cells per s. This would start to progress RMS to the field of cell sorting for applications such as enrichment and purification of hESC-derived cardiomyocytes.

  4. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

    NASA Astrophysics Data System (ADS)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-11-01

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

  5. When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis

    PubMed Central

    Hsu, Jen-Fang; Hsieh, Pei-Ying; Hsu, Hsin-Yun; Shigeto, Shinsuke

    2015-01-01

    In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo. PMID:26632877

  6. When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis

    NASA Astrophysics Data System (ADS)

    Hsu, Jen-Fang; Hsieh, Pei-Ying; Hsu, Hsin-Yun; Shigeto, Shinsuke

    2015-12-01

    In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo.

  7. Whole cell, label free protein quantitation with data independent acquisition: quantitation at the MS2 level.

    PubMed

    McQueen, Peter; Spicer, Vic; Schellenberg, John; Krokhin, Oleg; Sparling, Richard; Levin, David; Wilkins, John A

    2015-01-01

    Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R(2) ∼ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis. PMID:25348682

  8. Label-Free Detection of Rare Cell in Human Blood Using Gold Nano Slit Surface Plasmon Resonance

    PubMed Central

    Mousavi, Mansoureh Z.; Chen, Huai-Yi; Hou, Hsien-San; Chang, Chou-Yuan-Yuan; Roffler, Steve; Wei, Pei-Kuen; Cheng, Ji-Yen

    2015-01-01

    Label-free detection of rare cells in biological samples is an important and highly demanded task for clinical applications and various fields of research, such as detection of circulating tumor cells for cancer therapy and stem cells studies. Surface Plasmon Resonance (SPR) as a label-free method is a promising technology for detection of rare cells for diagnosis or research applications. Short detection depth of SPR (400 nm) provides a sensitive method with minimum interference of non-targets in the biological samples. In this work, we developed a novel microfluidic chip integrated with gold nanoslit SPR platform for highly efficient immunomagnetic capturing and detection of rare cells in human blood. Our method offers simple yet efficient detection of target cells with high purity. The approach for detection consists of two steps. Target cells are firs captured on functionalized magnetic nanoparticles (MNPs) with specific antibody I. The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film. MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area. The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits. PMID:25806834

  9. Label-free detection of rare cell in human blood using gold nano slit surface plasmon resonance.

    PubMed

    Mousavi, Mansoureh Z; Chen, Huai-Yi; Hou, Hsien-San; Chang, Chou-Yuan-Yuan; Roffler, Steve; Wei, Pei-Kuen; Cheng, Ji-Yen

    2015-03-01

    Label-free detection of rare cells in biological samples is an important and highly demanded task for clinical applications and various fields of research, such as detection of circulating tumor cells for cancer therapy and stem cells studies. Surface Plasmon Resonance (SPR) as a label-free method is a promising technology for detection of rare cells for diagnosis or research applications. Short detection depth of SPR (400 nm) provides a sensitive method with minimum interference of non-targets in the biological samples. In this work, we developed a novel microfluidic chip integrated with gold nanoslit SPR platform for highly efficient immunomagnetic capturing and detection of rare cells in human blood. Our method offers simple yet efficient detection of target cells with high purity. The approach for detection consists of two steps. Target cells are firs captured on functionalized magnetic nanoparticles (MNPs) with specific antibody I. The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film. MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area. The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits. PMID:25806834

  10. Label-free detection of multidrug resistance in K562 cells through isolated 3D-electrode dielectrophoresis.

    PubMed

    Demircan, Yağmur; Koyuncuoğlu, Aziz; Erdem, Murat; Özgür, Ebru; Gündüz, Ufuk; Külah, Haluk

    2015-05-01

    Dielectrophoresis (DEP), a technique used to separate particles based on different sizes and/or dielectric properties under nonuniform electric field, is a promising method to be applied in label-free, rapid, and effective cell manipulation and separation. In this study, a microelectromechanical systems-based, isolated 3D-electrode DEP device has been designed and implemented for the label-free detection of multidrug resistance in K562 leukemia cells, based on the differences in their cytoplasmic conductivities. Cells were hydrodynamically focused to the 3D-electrode arrays, placed on the side walls of the microchannel, through V-shaped parylene-C obstacles. 3D-electrodes extruded along the z-direction provide uniformly distributed DEP force through channel depth. Cell suspension containing resistant and sensitive cancer cells with 1:100 ratio was continuously flown through the channel at a rate of 10 μL/min. Detection was realized at 48.64 MHz, the cross-over frequency of sensitive K562 cells, at which sensitive cells flow with the fluid, while the resistant ones are trapped by positive DEP force. Device can be operated at considerably low voltages (<9 Vpp ). This is achieved by means of a very thin (0.5 μm) parylene coating on electrodes, providing the advantages offered by the isolation of electrodes from the sample, while the working voltage can still be kept low. Results prove that the presented DEP device can provide an efficient platform for the detection of multidrug resistance in leukemia, in a label-free manner. PMID:25781271

  11. The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue.

    PubMed

    Su, Ping-Jung; Chen, Wei-Liang; Li, Tsung-Hsien; Chou, Chen-Kuan; Chen, Te-Hsuen; Ho, Yi-Yun; Huang, Chi-Hsiu; Chang, Shwu-Jen; Huang, Yi-You; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2010-12-01

    Using excitation polarization-resolved second harmonic generation (SHG) microscopy, we measured SHG intensity as a function of the excitation polarization angle for type I and type II collagens. We determined the second order susceptibility (χ((2))) tensor ratios of type I and II collagens at each pixel, and displayed the results as images. We found that the χ((2)) tensor ratios can be used to distinguish the two types of collagen. In particular, we obtained χ(zzz)/χ(zxx) = 1.40 ± 0.04 and χ(xzx)/χ(zxx) = 0.53 ± 0.10 for type I collagen from rat tail tendon, and χ(zzz)/χ(zxx) = 1.14 ± 0.09 and χ(xzx)/χ(zxx) = 0.29 ± 0.11 for type II collagen from rat trachea cartilage. We also applied this methodology on the label-free imaging of engineered cartilage tissue which produces type I and II collagen simultaneously. By displaying the χ((2)) tensor ratios in the image format, the variation in the χ((2)) tensor ratios can be used as a contrast mechanism for distinguishing type I and II collagens. PMID:20875682

  12. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Datta, Rupsa; Heylman, Christopher; George, Steven C.; Gratton, Enrico

    2016-01-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  13. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Datta, Rupsa; Heylman, Christopher; George, Steven C; Gratton, Enrico

    2016-05-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  14. Label-free 3D refractive-index acquisition by micro-manipulations of cells in suspension (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.

    2016-03-01

    Our latest methods for non-invasive label-free acquisition of the three-dimensional (3-D) refractive-index maps of live cells in suspension are reviewed. These methods are based on the acquisition of off-axis interferograms of single or multiple cells in suspension from different angles using an external interferometric module, while fully rotating each cell using micro-manipulations. The interferometric projections are processed via computed tomographic phase microscopy reconstruction technique, which considers optical diffraction effects, into the 3-D refractive-index structure of the suspended cell. Till now, tomographic phase microscopy was obtained by acquiring a series of interferograms of the light transmitted through the sample in different angles by either using an entire sample rotation, or patch clamping a single cell, which is invasive to the cells, or alternatively, using various angles of illumination, which causes a limited acceptance angle, and an incomplete 3-D Fourier spectrum. In contrast, our methods allow fast acquisition with full angular range, and thus obtain an accurate 3-D refractive-index map of the imaged cell. By inspection of the 3-D refractive-index distribution of cells in suspension, the proposed methods can be useful for high-throughput, label-free characterization of biological processes and cellular transformations from healthy to pathological conditions.

  15. Label-free and dynamic evaluation of cell-surface epidermal growth factor receptor expression via an electrochemiluminescence cytosensor.

    PubMed

    Qiu, Youyi; Wen, Qingqing; Zhang, Lin; Yang, Peihui

    2016-04-01

    A label-free electrochemiluminescence (ECL) cytosensor was developed for dynamically evaluating of epidermal growth factor receptor (EGFR) expression on MCF-7 cancer cells based on the specific recognition of epidermal growth factor (EGF) with its receptor (EGFR). EGF-cytosensor was fabricated by in-situ electro-polymerization of polyaniline as substrate, using CdS quantum dots (CdS QDs) as ECL probe and gold nanoparticles (AuNPs) as a carrier for loading of EGF. AuNPs and CdS QDs were jointly attached on polyaniline surface to provide a sensitive and stable sensing interface, as well as a simple and label-free mode for ECL assay. Electron microscopy, atomic force microscopy (AFM) and electrochemical methods were employed to characterize the multilayer construction process of the sensing interface. The proposed EGF-cytosensor exhibited excellent analytical performance for MCF-7 cancer cells, ranging from 12 to 1.2 × 10(6) cells mL(-1), with a low detection limit of 12 cells mL(-1). Also, it was successfully applied in evaluating EGFR expression of cells surface, which was stimulated by some inhibitors or activator, and the results were confirmed by using flow cytometry and laser scanning confocal microscopy analysis. The proposed ECL cytosensor has potential applications in monitoring the dynamic variation of receptor molecules expression on cell surfaces in response to external stimulation by drugs and screening anti-cancer therapeutic agents. PMID:26838410

  16. Label-free selection and enrichment of liver cancer stem cells by surface niches build up with polyelectrolyte multilayer films.

    PubMed

    Lee, I-Chi; Chang, Jen-Fu

    2015-01-01

    Recent studies indicate that a small population of cancer cells exhibits stem cell properties and are referred to as cancer-initiating or cancer stem cells (CSCs). The selection and identification of cancer stem cells through methods require well-defined biomarkers and immunolabeling procedures are complicated and often unreliable. Herein, we fabricated a series of microenviroment by using polyelectrolyte multilayers (PEM) nanofilms to program and mimic hepatocellular carcinoma CSCs niches for CSCs selection with a label-free method. When cultured on PEM substrates, human cancer cell lines-Huh7 cells grew into individual round colonies and these cells displayed high marker expression of CSCs. Especially, these selected cells demonstrated significant chemo-resistant property in comparison with normal population. Therefore, we believed that niches selection and colony formation method may provide a new strategy on CSCs selection and drug evaluation for cancer therapy. PMID:25461919

  17. Longitudinal label-free tracking of cell death dynamics in living engineered human skin tissue with a multimodal microscope

    PubMed Central

    Zhao, Youbo; Marjanovic, Marina; Chaney, Eric J.; Graf, Benedikt W.; Mahmassani, Ziad; Boppart, Marni D.; Boppart, Stephen A.

    2014-01-01

    We demonstrate real-time, longitudinal, label-free tracking of apoptotic and necrotic cells in living tissue using a multimodal microscope. The integrated imaging platform combines multi-photon microscopy (MPM, based on two-photon excitation fluorescence), optical coherence microscopy (OCM), and fluorescence lifetime imaging microscopy (FLIM). Three-dimensional (3-D) co-registered images are captured that carry comprehensive information of the sample, including structural, molecular, and metabolic properties, based on light scattering, autofluorescence intensity, and autofluorescence lifetime, respectively. Different cell death processes, namely, apoptosis and necrosis, of keratinocytes from different epidermal layers are longitudinally monitored and investigated. Differentiation of the two cell death processes in a complex living tissue environment is enabled by quantitative image analysis and high-confidence classification processing based on the multidimensional, cross-validating imaging data. These results suggest that despite the limitations of each individual label-free modality, this multimodal imaging approach holds the promise for studies of different cell death processes in living tissue and in vivo organs. PMID:25360383

  18. Flexible Bioimpedance Sensor for Label-Free Detection of Cell Viability and Biomass.

    PubMed

    Fernandez, Renny Edwin; Lebiga, Elise; Koklu, Anil; Sabuncu, Ahmet Can; Beskok, Ali

    2015-10-01

    We introduce a flexible microfluidic bioimpedance sensor that is capable of detecting biomass and cell viability variations in a cell suspension. The sensor is developed on indium tin oxide (ITO) coated polyethylene terephthalate (PET) substrate and is devoid of gold, silicon, PDMS, or glass. In conjugation with a custom built PCB read-out module, the impedance characteristics of a cell suspension can be measured within one minute of sample introduction using liquid volumes less than 5 μL. The portable sensor system occupies very little bench space and has the potential to be developed as a disposable electrical bioimpedance probe for rapid detection of dielectric variations in a biological suspension. The sensor is designed to generate a differential impedance spectra exclusive to a cell suspension with a dual-electrode-pair system. The potential of the sensor to discriminate between live and heat treated Saccharomyces cerevisiae is demonstrated in this study. The disposable sensor along with the distance variation technique is touted to be an inexpensive alternative to some of the existing online disposable biomass detection probes and electrochemical sensors. PMID:26415205

  19. Selective Label-free Electrokinetic Cell Tracker (SELECT): a novel liquid platform for cell characterization

    NASA Astrophysics Data System (ADS)

    Taruvai Kalyana Kumar, Rajeshwari; de Mello Gindri, Izabelle; Kinnamon, David; Kanchustambham, Pradyotha; Rodrigues, Danieli; Prasad, Shalini; BiomaterialsOsseointegration; Novel Engineering Lab Collaboration

    2015-03-01

    Characterization and analysis of rare cells provide critical cues for early diagnosis of diseases. Electrokinetic cell separation has been previously established to have greater efficiency when compared to traditional flow cytometry methods. It has been shown by many researchers that buffer solutions in which cells are suspended in, have enormous effects on producing required dielectrophoretic (DEP) forces to characterize cells. Most commonly used suspension buffers used are deionized water and cell media. However, these solutions exhibit high level of intrinsic noise, which greatly masks the electrokinetic signals from cells under study. Ionic liquids (ILs) show promise towards the creation of conductive fluids with required electrical properties. The goal of this project is to design and test ILs for enhancing DEP forces on cells while creating an environment for preserving their integrity. We analyzed two methylimidazolium based ILs as suspension medium for cell separation. These dicationic ILs possess slight electrical and structural differences with high thermal stability. The two ILs were tested for cytotoxicity using HeLa and bone cells. The effects of electrical neutrality, free charge screening due to ILs towards enhanced electrokinetic signals from cells were studied with improved system resolution and no harmful effects.

  20. Label-free separation of human embryonic stem cells (hESCs) and their cardiac derivatives using Raman spectroscopy

    SciTech Connect

    Chan, J W; Lieu, D K; Huser, T R; Li, R A

    2008-09-08

    Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and non-recoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a non-destructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combining the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98% and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the non-invasive and label-free sorting of (i) high-quality hESCs for expansion, and (ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

  1. Label-Free Digital Quantification of Lipid Droplets in Single Cells by Stimulated Raman Microscopy on a Microfluidic Platform.

    PubMed

    Cao, Chen; Zhou, Dong; Chen, Tao; Streets, Aaron M; Huang, Yanyi

    2016-05-01

    Quantitative characterization of a single-cell phenotype remains challenging. We combined a scalable microfluidic array of parallel cell culture chambers and stimulated Raman scattering (SRS) microscopy to quantitatively characterize the response of lipid droplet (LD) formation to free-fatty-acid stimuli with single-LD resolution at the single-cell level. By enabling the systematic live-cell imaging with SRS microscopy in a microfluidic device, we were able to quantify the morphology of over a thousand live cells in 10 different chemical environments and with 8 replicates for each culture condition, in a single experiment, and without relying on fluorescent labeling. We developed an image processing pipeline for cell segmentation and LD morphology quantification using dual-channel SRS images. This allows us to construct distributions of the morphological parameters of LDs in the cellular population and expose the vast phenotypic heterogeneity among genetically similar cells. Specifically, this approach provides an analytical tool for quantitatively investigating LD morphology in live cells in situ. With this high-throughput, high-resolution, and label-free method, we found that LD growth dynamics showed considerable cell to cell variation. Lipid accumulation in nonadipocyte cells is mainly reflected in the increase of LD number, as opposed to an increase in their size or lipid concentration. Our method allows statistical single-cell quantification of the LD distribution for further investigation of lipid metabolism and dynamic behavior, and also extends the possibility to couple with other "omics" technologies in the future. PMID:27041129

  2. Nutrient depletion and metabolic profiles in breast carcinoma cell lines measured with a label-free platform.

    PubMed

    Demmel, F; Brischwein, M; Wolf, P; Huber, F; Pfister, C; Wolf, B

    2015-07-01

    The response of two well-characterized human breast cancer cell lines (MCF-7 and MDA-MB-231) to a series of nutrient deficiencies is investigated with a label-free cell assay platform. The motivation of the research is to analyze adaptive responses of tumor cell metabolism and to find limiting conditions for cell survival. The platform measures extracellular values of pH and dissolved oxygen saturation to provide data of extracellular acidification rates and oxygen uptake rates. Additional electric cell substrate impedance sensing and bright-field cell imaging supports the data interpretation by providing information about cell morphological parameters. A sequential administration of nutrient depletions does not cause metabolic reprogramming, since the ratios of oxygen uptake to acidification return to their basal values. While the extracellular acidification drops sharply upon reduction of glucose and glutamine, the oxygen uptake is not affected. In contrast to other published data, cell death is not observed when both glucose and glutamine are depleted and cell proliferation is not inhibited, at least in MCF-7 cultures. It is assumed that residual concentrations of nutrients from the serum component are able to maintain cell viability when delivered regularly by active flow like in the cell assay platform, and, in a similar way, under physiological conditions. PMID:26015442

  3. Comparative Label-free LC-MS/MS Analysis of Colorectal Adenocarcinoma and Metastatic Cells Treated with 5-Fluorouracil

    PubMed Central

    Bauer, Kerry M.; Lambert, Paul A.; Hummon, Amanda B.

    2013-01-01

    A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC50 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increasedwith treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells. PMID:22623418

  4. Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques.

    PubMed

    Xavier, Miguel; Oreffo, Richard O C; Morgan, Hywel

    2016-01-01

    Skeletal stem cells (SSC) are a sub-population of bone marrow stromal cells that reside in postnatal bone marrow with osteogenic, chondrogenic and adipogenic differentiation potential. SSCs reside only in the bone marrow and have organisational and regulatory functions in the bone marrow microenvironment and give rise to the haematopoiesis-supportive stroma. Their differentiation capacity is restricted to skeletal lineages and therefore the term SSC should be clearly distinguished from mesenchymal stem cells which are reported to exist in extra-skeletal tissues and, critically, do not contribute to skeletal development. SSCs are responsible for the unique regeneration capacity of bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge is the isolation of homogeneous populations of SSCs, in vitro, with homogeneous regeneration and differentiation capacities. Challenges that limit SSC isolation include a) the scarcity of SSCs in bone marrow aspirates, estimated at between 1 in 10-100,000 mononuclear cells; b) the absence of specific markers and thus the phenotypic ambiguity of the SSC and c) the complexity of bone marrow tissue. Microfluidics provides innovative approaches for cell separation based on bio-physical features of single cells. Here we review the physical principles underlying label-free microfluidic sorting techniques and review their capacity for stem cell selection/sorting from complex (heterogeneous) samples. PMID:27236022

  5. Label-free drug discovery

    PubMed Central

    Fang, Ye

    2014-01-01

    Current drug discovery is dominated by label-dependent molecular approaches, which screen drugs in the context of a predefined and target-based hypothesis in vitro. Given that target-based discovery has not transformed the industry, phenotypic screen that identifies drugs based on a specific phenotype of cells, tissues, or animals has gained renewed interest. However, owing to the intrinsic complexity in drug–target interactions, there is often a significant gap between the phenotype screened and the ultimate molecular mechanism of action sought. This paper presents a label-free strategy for early drug discovery. This strategy combines label-free cell phenotypic profiling with computational approaches, and holds promise to bridge the gap by offering a kinetic and holistic representation of the functional consequences of drugs in disease relevant cells that is amenable to mechanistic deconvolution. PMID:24723889

  6. Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines

    PubMed Central

    2013-01-01

    Background Reliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated. Methods Cytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells. Results Interference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner. Conclusions Using the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies. PMID:24103467

  7. A label-free and high-throughput separation of neuron and glial cells using an inertial microfluidic platform.

    PubMed

    Jin, Tiantian; Yan, Sheng; Zhang, Jun; Yuan, Dan; Huang, Xu-Feng; Li, Weihua

    2016-05-01

    While neurons and glial cells both play significant roles in the development and therapy of schizophrenia, their specific contributions are difficult to differentiate because the methods used to separate neurons and glial cells are ineffective and inefficient. In this study, we reported a high-throughput microfluidic platform based on the inertial microfluidic technique to rapidly and continuously separate neurons and glial cells from dissected brain tissues. The optimal working condition for an inertial biochip was investigated and evaluated by measuring its separation under different flow rates. Purified and enriched neurons in a primary neuron culture were verified by confocal immunofluorescence imaging, and neurons performed neurite growth after separation, indicating the feasibility and biocompatibility of an inertial separation. Phencyclidine disturbed the neuroplasticity and neuron metabolism in the separated and the unseparated neurons, with no significant difference. Apart from isolating the neurons, purified and enriched viable glial cells were collected simultaneously. This work demonstrates that an inertial microchip can provide a label-free, high throughput, and harmless tool to separate neurological primary cells. PMID:27190569

  8. Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry.

    PubMed

    Kim, Sang Hoon; Pajarillo, Edward Alain B; Balolong, Marilen P; Lee, Ji Yoon; Kang, Dae-Kyung

    2016-06-28

    In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications. PMID:26975772

  9. HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

    NASA Astrophysics Data System (ADS)

    Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

    2016-03-01

    Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

  10. xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies

    PubMed Central

    Martinez-Serra, Jordi; Gutierrez, Antonio; Muñoz-Capó, Saúl; Navarro-Palou, María; Ros, Teresa; Amat, Juan Carlos; Lopez, Bernardo; Marcus, Toni F; Fueyo, Laura; Suquia, Angela G; Gines, Jordi; Rubio, Francisco; Ramos, Rafael; Besalduch, Joan

    2014-01-01

    The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs. PMID:24959085

  11. Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumors in vivo by label-free third-harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Weng, Wei-Hung; Liao, Yi-Hua; Tsai, Ming-Rung; Wei, Ming-Liang; Huang, Hsin-Yi; Sun, Chi-Kuang

    2016-07-01

    Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of -0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.

  12. Cancer-cells on a chip for label-free optic detection of secreted molecules

    NASA Astrophysics Data System (ADS)

    Berthuy, Ophélie I.; Blum, Loïc. J.; Marquette, Christophe A.

    2015-05-01

    To unravel cell complexity, living-cell chips have been developed that allow delivery of experimental stimuli but also measurement of the resulting cellular responses. We have been developing a new concept for multiplexed detection of biomolecules secreted by different cancer cells. In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. For that purpose, antibodies and different cell lines were spotted using a piezoelectric spotter. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and β-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip. There is no doubt that our chip will, in a near future, be applied to more multiplexed and complex biological secretion systems for which kinetic data are at the moment not reachable using standard cellular biology tools.

  13. Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.

    PubMed

    Hill, Jennifer J; Moreno, Maria J; Lam, Jean C Y; Haqqani, Arsalan S; Kelly, John F

    2009-02-01

    Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N-linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to approximately 2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine-protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments. PMID:19137551

  14. [Label-free monitoring 5-FU induced SW 620 cells apoptosis using FTIR microspectroscopy].

    PubMed

    Liu, Dong; Sun, Xue-Jun; Zhang, Chao; He, Sai; Du, Jun-Kai; Huo, Xiong-Wei; Zheng, Jian-Bao; Zhang, Shi-Yun; Zhang, Yuan-Fuz; Xu, Yi-Zhuang; Wu, Jin-Guang

    2013-12-01

    The aim of the present study was to evaluate Fourier transform infrared spectroscopy (FTIR) monitoring of biochemical changes in apoptosis cells. Different concentrations of 5-fluorouracil (5-FU) treated colon cancer cell lines SW620 were used to determine the optimum concentration of 5-FU IC50 by means of MTT assay. Cell starvation and 5-Fu synergistic cell cycle arrest was in G1 and S phase. FTIR combined with flow cytometry was applied to analysis of SW 620 cells and SW620 cells treated with 5-FU for 12h, 24h (early apoptosis) and 48 h (late apoptosis) respectively. The peak position and the intensity of all bands were measured and comparison was made between the SW620 and apoptotic SW620 cells. Apoptosis cells have following characteristics compared with SW620 cells (1) The band at 1 740 cm-1 is an C=O stretching vibration. Changes in these bands can reflect lipid changes, and relative peak intensity ratio 11740/11460 significantly increased (p<0. 05), indicating that the relative contents of lipid in apoptosis cells increased. (2) The band at the 1 410 cm-1 peak represents that C-H stretching related was increased to amino acid residues and shifted to higher wave numbers compared to other groups. I1410o/I 460 at early and late death phase was significantly increased, which suggests that the relative contents of amino acid residues in apoptosis cells increased (p <0. 05). New vibrational bands at 1 120 cm-1 appeared at 24 h and increased at 48 h compared with other groups. The 1 120 cm-1 absorption band is mainly due to ser, serine and threonine C-O(H) stretching vibration, and I1120/I 1460 significantly increased (p<0. 05), indicating that the relative quantity of amino acid residues in apoptosis cells increased due to that DNA unwinds the double helix. (3) 1 240 cm-1 is mainly due to the asymmetric stretching modes of phosphodiester groups shifting to higher wave number, illustrating that nucleic acid conformation was changed in apoptosis cells. (4) The band

  15. Cancer-Cells on Chip for Label-Free Detection of Secreted Molecules

    PubMed Central

    Berthuy, Ophélie I.; Blum, Loïc J.; Marquette, Christophe A.

    2016-01-01

    In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24 h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and β-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip. PMID:26784243

  16. Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

    NASA Astrophysics Data System (ADS)

    Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

    2014-02-01

    Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

  17. Label-free magnetic resonance imaging to locate live cells in three-dimensional porous scaffolds

    PubMed Central

    Abarrategi, A.; Fernandez-Valle, M. E.; Desmet, T.; Castejón, D.; Civantos, A.; Moreno-Vicente, C.; Ramos, V.; Sanz-Casado, J. V.; Martínez-Vázquez, F. J.; Dubruel, P.; Miranda, P.; López-Lacomba, J. L.

    2012-01-01

    Porous scaffolds are widely tested materials used for various purposes in tissue engineering. A critical feature of a porous scaffold is its ability to allow cell migration and growth on its inner surface. Up to now, there has not been a method to locate live cells deep inside a material, or in an entire structure, using real-time imaging and a non-destructive technique. Herein, we seek to demonstrate the feasibility of the magnetic resonance imaging (MRI) technique as a method to detect and locate in vitro non-labelled live cells in an entire porous material. Our results show that the use of optimized MRI parameters (4.7 T; repetition time = 3000 ms; echo time = 20 ms; resolution 39 × 39 µm) makes it possible to obtain images of the scaffold structure and to locate live non-labelled cells in the entire material, with a signal intensity higher than that obtained in the culture medium. In the current study, cells are visualized and located in different kinds of porous scaffolds. Moreover, further development of this MRI method might be useful in several three-dimensional biomaterial tests such as cell distribution studies, routine qualitative testing methods and in situ monitoring of cells inside scaffolds. PMID:22442095

  18. Metal plasmon-coupled fluorescence imaging and label free coenzyme detection in cells

    SciTech Connect

    Zhang, Jian; Fu, Yi; Li, Ge; Zhao, Richard Y.

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer Metal nanoparticle for fluorescence cell imaging. Black-Right-Pointing-Pointer Non-invasive emission detection of coenzyme in cell on time-resolved confocal microscope. Black-Right-Pointing-Pointer Near-field interaction of flavin adenine dinucleotide with silver substrate. Black-Right-Pointing-Pointer Isolation of emissions by coenzymes from cellular autofluorescence on fluorescence cell imaging. -- Abstract: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.

  19. Label-free biochemical characterization of bovine sperm cells using Raman microscopy

    NASA Astrophysics Data System (ADS)

    De Luca, A. C.; Managò, S.; Ferrara, M. A.; Sirleto, L.; Puglisi, R.; Balduzzi, D.; Galli, A.; Rendina, I.; Ferraro, P.; Coppola, G.

    2014-02-01

    The current study relates to a Raman spectroscopy-based method for addressing the problem of sex assessment in mammals. A direct method for sex predetermination in animals is based on the X- and Y-bearing sperm cells sorting before insemination. Our Raman spectroscope allows distinguishing and characterizing the difference between X- and Y-bearing sperm cells by detecting and analyzing their Raman spectra in a non-invasive and non-destructive way.

  20. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

    PubMed Central

    Jünger, Felix; Olshausen, Philipp v.; Rohrbach, Alexander

    2016-01-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

  1. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy.

    PubMed

    Jünger, Felix; Olshausen, Philipp V; Rohrbach, Alexander

    2016-01-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

  2. A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects.

    PubMed

    Cheng, Xuanhong; Irimia, Daniel; Dixon, Meredith; Sekine, Kazuhiko; Demirci, Utkan; Zamir, Lee; Tompkins, Ronald G; Rodriguez, William; Toner, Mehmet

    2007-02-01

    Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes with high efficiency directly from 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R(2) = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings. PMID:17268618

  3. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

    NASA Astrophysics Data System (ADS)

    Jünger, Felix; Olshausen, Philipp V.; Rohrbach, Alexander

    2016-07-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

  4. Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

    PubMed Central

    Zhou, Ming-Da; Hao, Sijie; Williams, Anthony J.; Harouaka, Ramdane A.; Schrand, Brett; Rawal, Siddarth; Ao, Zheng; Brennaman, Randall; Gilboa, Eli; Lu, Bo; Wang, Shuwen; Zhu, Jiyue; Datar, Ram; Cote, Richard; Tai, Yu-Chong; Zheng, Si-Yang

    2014-01-01

    The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research. PMID:25487434

  5. Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

    NASA Astrophysics Data System (ADS)

    Zhou, Ming-Da; Hao, Sijie; Williams, Anthony J.; Harouaka, Ramdane A.; Schrand, Brett; Rawal, Siddarth; Ao, Zheng; Brennaman, Randall; Gilboa, Eli; Lu, Bo; Wang, Shuwen; Zhu, Jiyue; Datar, Ram; Cote, Richard; Tai, Yu-Chong; Zheng, Si-Yang

    2014-12-01

    The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

  6. Label-free multiphoton imaging and photoablation of preinvasive cancer cells

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Wu, Guizhu; Zhu, Xiaoqin; Jiang, Xingshan; Xie, Shusen

    2012-01-01

    Detection and treatment of early lesions in epithelial tissue offer several possibilities for curing cancer, but it is challenging. Here, we present an optical technique, the combination of multiphoton imaging and absorption, to label-freely detect and ablate preinvasive cancer cells in epithelial tissue. We find that multiphoton imaging can label-freely visualize the principal features of nuclear atypia associated with epithelial precancerous lesions, and the spatial localization of multiphoton absorption can perform targeted ablation of preinvasive cancer cells with micrometer-sized volume precision. These results indicate that this optical technique has the capability to label-freely visualize and remove preinvasive cancer cells in epithelial tissue. This study highlights the potential of this technique as a "seek-and-treat" tool for early lesions in epithelial tissue.

  7. A microfluidic device for label-free, physical capture of circulating tumor cell clusters.

    PubMed

    Sarioglu, A Fatih; Aceto, Nicola; Kojic, Nikola; Donaldson, Maria C; Zeinali, Mahnaz; Hamza, Bashar; Engstrom, Amanda; Zhu, Huili; Sundaresan, Tilak K; Miyamoto, David T; Luo, Xi; Bardia, Aditya; Wittner, Ben S; Ramaswamy, Sridhar; Shioda, Toshi; Ting, David T; Stott, Shannon L; Kapur, Ravi; Maheswaran, Shyamala; Haber, Daniel A; Toner, Mehmet

    2015-07-01

    Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30-40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis. PMID:25984697

  8. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays

    NASA Astrophysics Data System (ADS)

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N.; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji

    2016-04-01

    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ˜100% sensitivity, ˜91% specificity and ˜96% accuracy. In the blinded test, the signals were classified with ˜91% sensitivity, ˜82% specificity and ˜86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ˜1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

  9. Label-free impedance detection of cancer cells from whole blood on an integrated centrifugal microfluidic platform.

    PubMed

    Nwankire, Charles E; Venkatanarayanan, Anita; Glennon, Thomas; Keyes, Tia E; Forster, Robert J; Ducrée, Jens

    2015-06-15

    An electrochemical Lab-on-a-Disc (eLoaD) platform for the automated quantification of ovarian cancer cells (SKOV3) from whole blood is reported. This centrifugal microfluidic system combines complex sample handling, i.e., blood separation and cancer cell extraction from plasma, with specific capture and sensitive detection using label-free electrochemical impedance. Flow control is facilitated using rotationally actuated valving strategies including siphoning, capillary and centrifugo-pneumatic dissolvable-film (DF) valves. For the detection systems, the thiol-containing amino acid, L-Cysteine, was self-assembled onto smooth gold electrodes and functionalized with anti-EpCAM. By adjusting the concentration of buffer electrolyte, the thickness of the electrical double layer was extended so the interfacial electric field interacts with the bound cells. Significant impedance changes were recorded at 117.2 Hz and 46.5 Hz upon cell capture. Applying AC amplitude of 50 mV at 117.2 Hz and open circuit potential, a minimum of 214 captured cells/mm(2) and 87% capture efficiency could be recorded. The eLoaD platform can perform five different assays in parallel with linear dynamic range between 16,400 and (2.6±0.0003)×10(6) cancer cells/mL of blood, i.e. covering nearly three orders of magnitude. Using the electrode area of 15.3 mm(2) and an SKOV3 cell radius of 5 µm, the lower detection limit is equivalent to a fractional surface coverage of approximately 2%, thus making eLoaD a highly sensitive and efficient prognostic tool that can be developed for clinical settings where ease of handling and minimal sample preparation are paramount. PMID:25613813

  10. A microfluidic device for label-free, physical capture of circulating tumor cell-clusters

    PubMed Central

    Sarioglu, A. Fatih; Aceto, Nicola; Kojic, Nikola; Donaldson, Maria C.; Zeinali, Mahnaz; Hamza, Bashar; Engstrom, Amanda; Zhu, Huili; Sundaresan, Tilak K.; Miyamoto, David T.; Luo, Xi; Bardia, Aditya; Wittner, Ben S.; Ramaswamy, Sridhar; Shioda, Toshi; Ting, David T.; Stott, Shannon L.; Kapur, Ravi; Maheswaran, Shyamala; Haber, Daniel A.; Toner, Mehmet

    2015-01-01

    Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identify CTC-clusters in 30–40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. PMID:25984697

  11. Development of a Microfluidic-Based Optical Sensing Device for Label-Free Detection of Circulating Tumor Cells (CTCs) Through Their Lactic Acid Metabolism

    PubMed Central

    Chiu, Tzu-Keng; Lei, Kin-Fong; Hsieh, Chia-Hsun; Hsiao, Hung-Bo; Wang, Hung-Ming; Wu, Min-Hsien

    2015-01-01

    This study reports a microfluidic-based optical sensing device for label-free detection of circulating tumor cells (CTCs), a rare cell species in blood circulation. Based on the metabolic features of cancer cells, live CTCs can be quantified indirectly through their lactic acid production. Compared with the conventional schemes for CTC detection, this label-free approach could prevent the biological bias due to the heterogeneity of the surface antigens on cancer cells. In this study, a microfluidic device was proposed to generate uniform water-in-oil cell-encapsulating micro-droplets, followed by the fluorescence-based optical detection of lactic acid produced within the micro-droplets. To test its feasibility to quantify cancer cells, experiments were carried out. Results showed that the detection signals were proportional to the number of cancer cells within the micro-droplets, whereas such signals were insensitive to the existence and number of leukocytes within. To further demonstrate its feasibility for cancer cell detection, the cancer cells with known cell number in a cell suspension was detected based on the method. Results revealed that there was no significant difference between the detected number and the real number of cancer cells. As a whole, the proposed method opens up a new route to detect live CTCs in a label-free manner. PMID:25808775

  12. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT.

    PubMed

    Jung, Yookyung; Klein, Oliver J; Wang, Hequn; Evans, Conor L

    2016-01-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation. PMID:27248849

  13. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    PubMed Central

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-01-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation. PMID:27248849

  14. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    NASA Astrophysics Data System (ADS)

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-06-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

  15. Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

    PubMed Central

    Brusnichkin, Anton V.; Nedosekin, Dmitry A.; Galanzha, Ekaterina I.; Vladimirov, Yuri A.; Shevtsova, Elena F.; Proskurnin, Mikhail A.; Zharov, Vladimir P.

    2012-01-01

    Light-absorbing endogenous cellular proteins, in particular cytochrome c, are used as intrinsic biomarkers for studies of cell biology and environment impacts. To sense cytochrome c against real biological backgrounds, we combined photothermal (PT) thermal-lens single channel schematic in a back-synchronized measurement mode and a multiplex thermal-lens schematic in a transient high resolution (ca. 350 nm) imaging mode. These multifunctional PT techniques using continuous-wave (cw) Ar+ laser and a nanosecond pulsed optical parametric oscillator in the visible range demonstrated the capability for label-free spectral identification and quantification of trace amounts of cytochrome c in a single mitochondrion alone or within a single live cell. PT imaging data were verified in parallel by molecular targeting and fluorescent imaging of cellular cytochrome c. The detection limit of cytochrome c in a cw mode was 5 × 10−9 mol/L (80 attomols in the signal-generation zone); that is ca. 103 lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 × 10−21 mol) in the ultra-small irradiated volumes limited by optical diffraction effects. For the first time, we demonstrate a combination of high resolution PT imaging with PT spectral identification and ultrasensitive quantitative PT characterization of cytochrome c within individual mitochondria in single live cells. A potential of far-field PT microscopy to sub-zeptomol detection thresholds, resolution beyond diffraction limit, PT Raman spectroscopy, and 3D imaging are further highlighted. PMID:20572284

  16. Possible target-related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ-based and label-free proteomic analysis.

    PubMed

    Zhang, Dong-Mei; Feng, Li-Xing; Liu, Miao; Jin, Wen-Hai; Luo, Ji; Nie, Ai-Ying; Zhou, Yue; Li, Yin; Wu, Wan-Ying; Jiang, Bao-Hong; Yang, Min; Hu, Li-Hong; Guo, De-An; Liu, Xuan

    2016-03-01

    Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target-related proteins, protein expression profiles of BF-treated and control cells were compared using two quantitative proteomic methods, iTRAQ-based and label-free proteomic analysis. A total of 5428 proteins were identified in iTRAQ-based analysis while 6632 proteins were identified in label-free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20-fold for upregulated and 0.83-fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ-based and label-free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore(TM) showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin-related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF-induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target-related proteins and signal network of BF. PMID:26787099

  17. Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Mouras, Rabah; Bagnaninchi, Pierre O.; Downes, Andrew R.; Elfick, Alistair P. D.

    2012-11-01

    Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell's morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.

  18. Sci—Fri AM: Mountain — 04: Label-free Raman spectroscopy of single tumour cells detects early radiation-induced glycogen synthesis associated with increased radiation resistance

    SciTech Connect

    Matthews, Q; Lum, JJ; Isabelle, M; Harder, S; Jirasek, A; Brolo, AG

    2014-08-15

    Purpose: To use label-free Raman spectroscopy (RS) for early treatment monitoring of tumour cell radioresistance. Methods: Three human tumour cell lines, two radioresistant (H460, SF{sub 2} = 0.57 and MCF7, SF{sub 2} = 0.70) and one radiosensitive (LNCaP, SF{sub 2} = 0.36), were irradiated with single fractions of 2, 4, 6, 8 or 10 Gy. In additional experiments, H460 and MCF7 cells were irradiated under co-treatment with the anti-diabetic drug metformin, a known radiosensitizing agent. Treated and control cultures were analyzed with RS daily for 3 days post-treatment. Single-cell Raman spectra were acquired from 20 live cells per sample, and experiments were repeated in triplicate. The combined data sets were analyzed with principal component analysis using standard algorithms. Cells from each culture were also subjected to standard assays for viability, proliferation, cell cycle, and radiation clonogenic survival. Results: The radioresistant cells (H460, MCF7) exhibited a RS molecular radiation response signature, detectable as early as 1 day post-treatment, of which radiation-induced glycogen synthesis is a significant contributor. The radiosensitive cells (LNCaP) exhibited negligible glycogen synthesis. Co-treatment with metformin in MCF7 cells blocked glycogen synthesis, reduced viability and proliferation, and increased radiosensitivity. Conversely, metformin co-treatment in H460 cells did not produce these same effects; importantly, both radiation-induced synthesis of glycogen and radiosensitivity were unaffected. Conclusions: Label-free RS can detect early glycogen synthesis post-irradiation, a previously undocumented metabolic mechanism associated with tumour cell radioresistance that can be targeted to increase radiosensitivity. RS monitoring of intratumoral glycogen may provide new opportunities for personalized combined modality radiotherapy treatments.

  19. Cell-Sorting System with On-Chip Imaging for Label-Free Shape-Based Selection of Cells

    NASA Astrophysics Data System (ADS)

    Terazono, Hideyuki; Hayashi, Masahito; Kim, Hyonchol; Hattori, Akihiro; Yasuda, Kenji

    2012-06-01

    We have developed a novel cell-sorting system involving microscopic imaging using a poly(methyl methacrylate) (PMMA)-based microfluidic chip with a pair of gel electrodes and real-time image-processing procedures for the quantification of cell shapes. The features of this system are as follows. 1) It can recognize cells both by microscopic cell imaging with a 10,000 event/s high-speed camera and by the photodetection of fluorescence. 2) Multistage sorting is used to reduce errors to an infinitesimally low level by using a pair of wide agarose-gel electrodes. 3) Carry-over-free analysis can be performed using a disposable microfluidic chip. 4) An field programmable gate array (FPGA) 10,000 event/s real-time image analysis unit for quantifying the cell images in cell sorting. To separate the target cells from other cells on the basis of the cell shape, we adopted an index of roughness for the cell surface R, which compares the actual perimeter of cell surface and the estimated perimeter of cross-sectional view of cell shape by approximating the cell as a sphere. Sample cells flowing through microchannels on the chip were distinguished by the dual recognition system involving optical analysis and a fluorescence detector, and then separated. Target cells could be sorted automatically by applying an electrophoretic force, and the sorting ability depended on the precision with which cells were shifted within the laminar flow. These results indicate that the cell-sorting system with on-chip imaging is practically applicable for biological research and clinical diagnostics.

  20. Biopatterning for label-free detection.

    PubMed

    Goddard, Julie M; Mandal, Sudeep; Nugen, Sam R; Baeumner, Antje J; Erickson, David

    2010-03-01

    We present a biopatterning technique suitable for applications which demand a high degree of surface cleanliness, such as immobilization of biological recognition elements onto label-free biosensors. In the case of label-free biosensing, the mechanism of signal transduction is based on surface bound matter, making them highly sensitive to surface contamination including residues left during the biopatterning process. In this communication we introduce a simple, rapid processing step that removes 98% of the residues that often remain after standard parylene lift-off patterning. Residue-free parylene biopatterning is combined with microfluidics to localize biomolecule immobilization onto the sensing region and to enable multiplexed biopatterning. We demonstrate the applicability of this method to multiplexed label-free detection platforms by patterning nucleic acid capture probes corresponding to the four different serotypes of Dengue virus onto parallel 1D photonic crystal resonator sensors. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to quantify surface cleanliness and uniformity. In addition to label-free biosensors, this technique is well suited to other nanobiotechnology patterning applications which demand a pristine, residue-free surface, such as immobilization of enzymes, antibodies, growth factors, or cell cultures. PMID:19939644

  1. Persistent GnRH receptor activation in pituitary αT3-1 cells analyzed with a label-free technology.

    PubMed

    Nederpelt, I; Vergroesen, R D; IJzerman, A P; Heitman, L H

    2016-05-15

    The gonadotropin-releasing hormone (GnRH) receptor is a drug target for certain hormone-dependent diseases such as prostate cancer. In this study, we examined the activation profiles of the endogenous ligand, GnRH and a well-known marketed analog, buserelin using a label-free assay in pituitary αT3-1 cells with endogenous GnRH receptor expression. This whole cell impedance-based technology allows for the real-time measurement of morphological cellular changes. Both agonists dose-dependently decreased the impedance as a result of GnRH receptor activation with potencies of 9.3 ± 0.1 (pEC50 value, buserelin) and 7.8 ± 0.06 (pEC50 value, GnRH). Subsequently, GnRH receptor activation was completely abolished with a selective Gαq inhibitor, thereby confirming the Gαq-coupling of the GnRH receptor in pituitary αT3-1 cells. Additionally, we observed continued responses after agonist stimulation of αT3-1 cells indicating long-lasting cellular effects. Wash-out experiments demonstrated that the long-lasting effects induced by GnRH were most likely caused by rebinding since over 70% of the original response was abolished after wash-out. In contrast, a long receptor residence time was responsible for the prolonged effects caused by buserelin, with over 70% of the original response remaining after wash-out. In summary, we validated that impedance-based label-free technology is suited for studying receptor-mediated activation in cell lines endogenously expressing the target of interest. Moreover, this real-time monitoring allows the examination of binding kinetics and its influence on receptor activation at a cellular level. PMID:26774084

  2. Label-free, live optical imaging of reprogrammed bipolar disorder patient-derived cells reveals a functional correlate of lithium responsiveness.

    PubMed

    Wang, J L; Shamah, S M; Sun, A X; Waldman, I D; Haggarty, S J; Perlis, R H

    2014-01-01

    Development of novel treatments and diagnostic tools for psychiatric illness has been hindered by the absence of cellular models of disease. With the advent of cellular reprogramming, it may be possible to recapitulate the disease biology of psychiatric disorders using patient skin cells transdifferentiated to neurons. However, efficiently identifying and characterizing relevant neuronal phenotypes in the absence of well-defined pathophysiology remains a challenge. In this study, we collected fibroblast samples from patients with bipolar 1 disorder, characterized by their lithium response (n=12), and healthy control subjects (n=6). We identified a cellular phenotype in reprogrammed neurons using a label-free imaging assay based on a nanostructured photonic crystal biosensor and found that an optical measure of cell adhesion was associated with clinical response to lithium treatment. This cellular phenotype may represent a useful biomarker to evaluate drug response and screen for novel therapeutics. PMID:25158003

  3. A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration.

    PubMed

    Fukano, Yasufumi; Okino, Nozomu; Furuya, Shigeki; Ito, Makoto

    2016-09-16

    We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (PTX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the Gαi pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of Gαi. PMID:27480930

  4. Label-Free Detection of Neuronal Differentiation in Cell Populations Using High-Throughput Live-Cell Imaging of PC12 Cells

    PubMed Central

    Nascimento, Juliana M.; Knauer, Steffen; Offermann, Barbara; Murphy, Robert F.

    2013-01-01

    Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation. PMID:23451069

  5. Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living.

    PubMed

    Baday, Murat; Calamak, Semih; Durmus, Naside Gozde; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

    2016-03-01

    There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. PMID:26523938

  6. Evaluating the Equilibrium Association Constant between ArtinM Lectin and Myeloid Leukemia Cells by Impedimetric and Piezoelectric Label Free Approaches

    PubMed Central

    Carvalho, Fernanda C.; Martins, Denise C.; Santos, Adriano; Roque-Barreira, Maria-Cristina; Bueno, Paulo R.

    2014-01-01

    Label-free methods for evaluating lectin–cell binding have been developed to determine the lectin–carbohydrate interactions in the context of cell-surface oligosaccharides. In the present study, mass loading and electrochemical transducer signals were compared to characterize the interaction between lectin and cellular membranes by measuring the equilibrium association constant, Ka, between ArtinM lectin and the carbohydrate sites of NB4 leukemia cells. By functionalizing sensor interfaces with ArtinM, it was possible to determine Ka over a range of leukemia cell concentrations to construct analytical curves from impedimetric and/or mass-associated frequency shifts with analytical signals following a Langmuir pattern. Using the Langmuir isotherm-binding model, the Ka obtained were (8.9 ± 1.0) × 10−5 mL/cell and (1.05 ± 0.09) × 10−6 mL/cell with the electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM) methods, respectively. The observed differences were attributed to the intrinsic characteristic sensitivity of each method in following Langmuir isotherm premises. PMID:25587428

  7. Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique

    NASA Astrophysics Data System (ADS)

    Lamprecht, C.; Gierlinger, N.; Heister, E.; Unterauer, B.; Plochberger, B.; Brameshuber, M.; Hinterdorfer, P.; Hild, S.; Ebner, A.

    2012-04-01

    The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs’ G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized. The authors declare no conflict of interest.

  8. Label-free concentration of viable neurons, hESCs and cancer cells by means of acoustophoresis.

    PubMed

    Zalis, Marina C; Reyes, Juan F; Augustsson, Per; Holmqvist, Staffan; Roybon, Laurent; Laurell, Thomas; Deierborg, Tomas

    2016-03-14

    Concentration of viable cell populations in suspension is of interest for several clinical and pre-clinical applications. Here, we report that microfluidic acoustophoresis is an effective method to efficiently concentrate live and viable cells with high target purity without any need for protein fluorescent labeling using antibodies or over-expression. We explored the effect of the acoustic field acoustic energy density and systematically used different protocols to induce apoptosis or cell death and then determined the efficiency of live and dead cell separation. We used the breast cancer cell line MCF-7, the mouse neuroblastoma N2a as well as human embryonic stem cells (hESCs) to demonstrate that this method is gentle and can be applied to different cell populations. First, we induced cell death by means of high osmotic shock using a high concentration of PBS (10×), the protein kinase inhibitor staurosporine, high concentrations of dimethyl sulfoxide (DMSO, 10%), and finally, cell starvation. In all the methods employed, we successfully induced cell death and were able to purify and concentrate the remaining live cells using acoustophoresis. Importantly, the concentration of viable cells was not dependent on a specific cell type. Further, we demonstrate that different death inducing stimuli have different effects on the intrinsic cell properties and therefore affect the efficiency of the acoustophoretic separation. PMID:26915333

  9. Label-free surface-enhanced Raman scattering imaging to monitor the metabolism of antitumor drug 6-mercaptopurine in living cells.

    PubMed

    Han, Guangmei; Liu, Renyong; Han, Ming-Yong; Jiang, Changlong; Wang, Jianping; Du, Shuhu; Liu, Bianhua; Zhang, Zhongping

    2014-12-01

    The molecular processes of drugs from cellular uptake to intracellular distribution as well as the intracellular interaction with the target molecule are critically important for the development of new antitumor drugs. In this work, we have successfully developed a label-free surface-enhanced Raman scattering (SERS) technique to monitor and visualize the metabolism of antitumor drug 6-mercaptopurine in living cells. It has been clearly demonstrated that Au@Ag NPs exhibit an excellent Raman enhancement effect to both 6-mercaptopurine and its metabolic product 6-mercaptopurine-ribose. Their different ways to absorb at the surface of Au@Ag NPs lead to the obvious spectral difference for distinguishing the antitumor drug and its metabolite by SERS spectra. The Au@Ag NPs can easily pass through cell membranes in a large amount and sensitively respond to the biological conversion of 6-mercaptopurine in tumor cells. The Raman imaging can visualize the real-time distribution of 6-mercaptopurine and its biotransformation with the concentrations in tumor cells. The SERS-based method reported here is simple and efficient for the assessments of drug efficacy and the understanding of the molecular therapeutic mechanism of antitumor drugs at the cellular level. PMID:25372629

  10. Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor

    PubMed Central

    Orgovan, Norbert; Peter, Beatrix; Bősze, Szilvia; Ramsden, Jeremy J.; Szabó, Bálint; Horvath, Robert

    2014-01-01

    A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (vRGD) of integrin ligand RGD-motifs. vRGD was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm−2 (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels. PMID:24503534

  11. The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

    PubMed Central

    Liu, Qun; Peng, Yong-Bo; Qi, Lian-Wen; Cheng, Xiao-Lan; Xu, Xiao-Jun; Liu, Le-Le; Liu, E-Hu; Li, Ping

    2012-01-01

    Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale). In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA) suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism. PMID:23243437

  12. Silicon photonic crystal microarrays for high throughput label-free detection of lung cancer cell line lysates with sensitivity and specificity

    NASA Astrophysics Data System (ADS)

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Gemmill, Robert M.; Chen, Ray T.

    2013-03-01

    Detection of biomolecules on microarrays based on label-free on-chip optical biosensors is very attractive since this format avoids complex chemistries caused by steric hindrance of labels. Application areas include the detection of cancers and allergens, and food-borne pathogens to name a few. We have demonstrated photonic crystal microcavity biosensors with high sensitivity down to 1pM concentrations (67pg/ml). High sensitivities were achieved by slow light engineering which reduced the radiation loss and increased the stored energy in the photonic crystal microcavity resonance mode. Resonances with high quality factor Q~26,760 in liquid ambient, coupled with larger optical mode volumes allowed enhanced interaction with the analyte biomolecules which resulted in sensitivities down to 10 cells per micro-liter to lung cancer cell lysates. The specificity of detection was ensured by multiplexed detections from multiple photonic crystal microcavities arrayed on the arms of a multimode interference power splitter. Specific binding interactions and control experiments were performed simultaneously at the same instant of time with the same 60 microliter sample volume. Specificity is further ensured by sandwich assay methods in the multiplexed experiment. Sandwich assay based amplification increased the sensitivity further resulting in the detection of lung cancer cell lysates down to concentrations of 2 cells per micro-liter. The miniaturization enabled by photonic crystal biosensors coupled with waveguide interconnected layout thus offers the potential of high throughput proteomics with high sensitivity and specificity.

  13. Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor

    NASA Astrophysics Data System (ADS)

    Orgovan, Norbert; Peter, Beatrix; Bősze, Szilvia; Ramsden, Jeremy J.; Szabó, Bálint; Horvath, Robert

    2014-02-01

    A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (vRGD) of integrin ligand RGD-motifs. vRGD was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 +/- 243 μm-2 (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels.

  14. Label-free and high-sensitive detection of human breast cancer cells by aptamer-based leaky surface acoustic wave biosensor array.

    PubMed

    Chang, Kai; Pi, Yan; Lu, Weiping; Wang, Feng; Pan, Feng; Li, Fake; Jia, Shuangrong; Shi, Jianfeng; Deng, Shaoli; Chen, Ming

    2014-10-15

    A label-free and high-sensitive sensing technology for tumor cell recognition and detection was developed based on a novel 2 × 3 model of leaky surface acoustic wave (LSAW) aptasensor array. In this methodology, every resonator crystal unit of the LSAW aptasensor array had an individual oscillator circuit to work without mutual interference, and could oscillate independently with the phase shift stability of ± 0.15° in air phase and ± 0.3° in liquid phase. The aptamer was firstly assembled to the gold electrode surface of 100 MHz LiTaO3 piezoelectric crystal, which could effectively captured target cells (MCF-7 cells) based on the specific interaction between aptamer and the overexpression of MUC1 protein on tumor cell surface. The aptamer-cell complexes increased the mass loading of LSAW aptasensor and led to phase shifts of LSAW. The plot of phase shift against the logarithm of concentration of MCF-7 cells was linear over the range from 1 × 10(2) cells mL(-1) to 1 × 10(7) cells mL(-1) with a correlation coefficient of 0.994. The detection limit as low as 32 cells mL(-1) was achieved for MCF-7 cells. The LSAW aptasensor also exhibited excellent specificity and stability. In addition, this aptasensor could be regenerated for ten times without irreversible loss of activity. Therefore, the LSAW aptasensor may offer a promising approach for tumor cell detection and have great potential in clinical applications. PMID:24836014

  15. Label-free multimodal microspectroscopic differentiation of glioblastoma tumor model cell lines combined with multivariate data analysis

    NASA Astrophysics Data System (ADS)

    Ostertag, Edwin; Boldrini, Barbara; Luckow, Sabrina; Kessler, Rudolf W.

    2012-06-01

    Glioblastoma multiforme represents a highly lethal brain tumor. A tumor model has been developed based on the U-251 MG cell line from a human explant. The tumor model simulates different malignancies by controlled expression of the tumor suppressor proteins PTEN and TP53 within the cell lines derived from the wild type. The cells from each different malignant cell line are grown on slides, followed by a paraformaldehyde fixation. UV / VIS and IR spectra are recorded in the cell nuclei. For the differentiation of the cell lines a principal component analysis (PCA) is performed. The PCA demonstrates a good separation of the tumor model cell lines both with UV / VIS spectroscopy and with IR spectroscopy.

  16. High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.

    PubMed

    Abdiche, Yasmina Noubia; Miles, Adam; Eckman, Josh; Foletti, Davide; Van Blarcom, Thomas J; Yeung, Yik Andy; Pons, Jaume; Rajpal, Arvind

    2014-01-01

    Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending

  17. PTRF/Cavin-1 and MIF Proteins Are Identified as Non-Small Cell Lung Cancer Biomarkers by Label-Free Proteomics

    PubMed Central

    Gámez-Pozo, Angelo; Sánchez-Navarro, Iker; Calvo, Enrique; Agulló-Ortuño, María Teresa; López-Vacas, Rocío; Díaz, Esther; Camafeita, Emilio; Nistal, Manuel; Madero, Rosario; Espinosa, Enrique; López, Juan Antonio; Vara, Juan Ángel Fresno

    2012-01-01

    With the completion of the human genome sequence, biomedical sciences have entered in the “omics” era, mainly due to high-throughput genomics techniques and the recent application of mass spectrometry to proteomics analyses. However, there is still a time lag between these technological advances and their application in the clinical setting. Our work is designed to build bridges between high-performance proteomics and clinical routine. Protein extracts were obtained from fresh frozen normal lung and non-small cell lung cancer samples. We applied a phosphopeptide enrichment followed by LC-MS/MS. Subsequent label-free quantification and bioinformatics analyses were performed. We assessed protein patterns on these samples, showing dozens of differential markers between normal and tumor tissue. Gene ontology and interactome analyses identified signaling pathways altered on tumor tissue. We have identified two proteins, PTRF/cavin-1 and MIF, which are differentially expressed between normal lung and non-small cell lung cancer. These potential biomarkers were validated using western blot and immunohistochemistry. The application of discovery-based proteomics analyses in clinical samples allowed us to identify new potential biomarkers and therapeutic targets in non-small cell lung cancer. PMID:22461895

  18. A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

    PubMed Central

    Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

    2013-01-01

    Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis. PMID:24287532

  19. Label-Free and Sensitive Detection of Thrombomodulin, a Marker of Endothelial Cell Injury, Using Quartz Crystal Microbalance.

    PubMed

    Luo, Yiqun; Liu, Tong; Zhu, Jiaming; Kong, Liyan; Wang, Wen; Tan, Liang

    2015-11-17

    Thrombomodulin (TM), an integral glycoprotein on the surface of endothelial cells, can be released during endothelial cell injury and the levels of serum TM are regarded as an important parameter of activity in vasculitides in vivo. Quantitative detection of TM and investigation on the release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells using quartz crystal microbalance (QCM) were achieved in this work. Anti-antibody (AAb) and bovine serum albumin (BSA) were bound on gold nanoparticles (GNPs) to construct BSA-GNPs-AAb nanocomposites and they were characterized by transmission electron microscope, UV-vis, and infrared spectrophotometry, respectively. The capture of the nanocomposites on the TM antibody modified electrode, which was tested by scanning electron microscope, could result in a great decrease of the resonant frequency (f0). This binding was effectively inhibited by the beforehand immobilized TM proteins on the electrode surface due to the strong steric hindrance effect. It led to the decrease of the frequency changing extent. The relative frequency-shift was found to be proportional to the logarithm of the TM concentration from 10 to 5000 ng mL(-1) with a detection limit of 2 ng mL(-1). By analyzing the growth medium used for cell incubation, the release of sTM by the injured HUVEC-C cells in the presence of H2O2 was confirmed. The sTM amount in the growth medium was increased with the enhancement of contact time of the cells with H2O2, proving that sTM may serve as a specific marker of endothelial cell injury. PMID:26507327

  20. Label-free and quantitative evaluation of cytotoxicity based on surface nanostructure and biophysical property of cells utilizing AFM.

    PubMed

    Lee, Young Ju; Lee, Gi-Ja; Kang, Sung Wook; Cheong, Youjin; Park, Hun-Kuk

    2013-06-01

    In this study, the four commonly used cytotoxicity assays and the mechanical properties as evaluated by atomic force microscopy (AFM) were compared in a cellular system. A cytotoxicity assay is the first and most essential test to evaluate biocompatibility of various toxic substances. Many of the cytotoxicity methods require complicated and labor-intensive process, as well as introduce experimental error. In addition, these methods cannot provide instantaneous and quantitative cell viability information. AFM has become an exciting analytical tool in medical, biological, and biophysical research due to its unique abilities. AFM-based force-distance curve measurements precisely measure the changes in the biophysical properties of the cell. Therefore, we observed the morphological changes and mechanical property changes in L929 cells following sodium lauryl sulfate (SLS) treatment utilizing AFM. AFM imaging showed that the toxic effects of SLS changed not only the spindle-like shape of L929 cells into a round shape, but also made a rough cell surface. As the concentration of SLS was increased, the surface roughness of L929 cell was increased, and stiffness decreased. We confirmed that inhibition of proliferation clearly increased with increases in SLS concentration based on results from MTT, WST, neutral red uptake, and LIVE/DEAD viability/cytotoxicity assays. The estimated IC₅₀ value by AFM analysis was similar to those of other conventional assays and was included within the 95% confidence interval range. We suggest that an AFM quantitative analysis of the morphological and biophysical changes in cells can be utilized as a new method for evaluating cytotoxicity. PMID:23582483

  1. Label-free electrochemical aptasensor constructed by layer-by-layer technology for sensitive and selective detection of cancer cells.

    PubMed

    Wang, Tianshu; Liu, Jiyang; Gu, Xiaoxiao; Li, Dan; Wang, Jin; Wang, Erkang

    2015-07-01

    Here, a cytosensor was constructed with ferrocene-appended poly(allylamine hydrochloride) (Fc-PAH) functionalized graphene (Fc-PAH-G), poly(sodium-p-styrenesulfonate) (PSS) and aptamer (AS1411) by layer-by-layer assembly technology. The hybrid nanocomposite Fc-PAH-G not only brings probes on the electrode and also promotes electron transfer between the probes and the substrate electrode. Meanwhile, LBL technology provides more effective probes to enhance amplified signal for improving the sensitivity of the detection. While AS1411 forming G-quardruplex structure and binding cancer cells, the current response of the sensing electrode decreased due to the insulating properties of cellular membrane. Differential pulse voltammetry (DPV) was performed to investigate the electrochemical detection of HeLa cells attributing to its sensitivity of the current signal change. The as-prepared aptasensor showed a high sensitivity and good stability, a widely detection range from 10 to 10(6) cells/mL with a detection limit as low as 10 cells/mL for the detection of cancer cells. PMID:26043089

  2. Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

    PubMed

    Zhao, Peng; Fu, Juanling; Yao, Biyun; Jia, Yongrui; Zhang, Hongtao; Li, Xuehui; Dong, Lisha; Gao, Ya; Liu, Wenli; Chen, Wen; Zhou, Zongcan

    2016-02-01

    To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis. PMID:26748308

  3. Label-free and turn-on aptamer strategy for cancer cells detection based on a DNA-silver nanocluster fluorescence upon recognition-induced hybridization.

    PubMed

    Yin, Jinjin; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Shangguan, Jingfang; He, Dinggeng; Shi, Hui

    2013-12-17

    We present here a label-free and turn-on aptamer strategy for cancer cell detection based on the recognition-induced conformation alteration of aptamer and hybridization-induced fluorescence enhancement effect of DNA-silver nanoclusters (DNA-Ag NCs) in proximity of guanine-rich DNA sequences. In this strategy, two tailored DNA probes were involved. One is designed as a hairpin-shaped structure consisting of a target specific aptamer sequence at the 3'-end, a guanine-rich DNA sequence, and an arm segment at the 5'-end (denote as recognition probe). The other, serving as a signal probe, contains a sequence for Ag NCs templated synthesis and a link sequence complementary to the arm segment of the recognition probe. Recognizing and binding of the aptamer to cancer cells enforces the recognition probe to undergo a conformational alteration and then initiates hybridization between the arm segment of the recognition probe and the link sequence of the signal probe. The Ag NCs are then close to the guanine-rich DNA, leading to an enhanced fluorescence readout. As proof-of-concept, the CCRF-CEM cancer cell detection were performed by using the specific aptamer, sgc8c. It was demonstrated that this strategy could specially image the CCRF-CEM cells. Determination by flow cytometry allowed for detection of as low as 150 CCRF-CEM cells in 200 μL binding buffer. The general applicability of the strategy is also achieved in the successful detection of Ramos cells. These results implied that this strategy holds considerable potential for simple, sensitive, universal, and specific cancer cell detection with no required washing and separation steps. PMID:24266455

  4. PCR-free and label-free fluorescent detection of telomerase activity at single-cell level based on triple amplification.

    PubMed

    Gao, Yanfang; Xu, Jing; Li, Baoxin; Jin, Yan

    2016-07-15

    As a universal biomarker for cancer diagnostics and cancer therapeutics, telomerase has attracted extensive attention concerning its detection and discovery of its inhibitors. Herein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase elongation product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3'-azido-3'-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability. PMID:26999622

  5. Study of acetowhitening mechanisms in live mammalian cells with label-free subcellular-level multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Jian; Teh, Sengkhoon; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2015-03-01

    The tissue acetowhitening effect in acetic acid instillation procedure is a simple and economic method for neoplasia detection and has been clinically utilized since 1925. It is suspected that the optical property (e.g. scattering) change in acetowhitening is due to coagulation of intracellular proteins, but no experimental proof has been reported yet. In this work, we use third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) to investigate the acetowhitening phenomenon induced by acidic acid in live mammalian cells without labeling. We studied the acetowhitening effect with different acetic acid concentrations and the co-localized TPEF and THG imaging on tryptophan and NADH at subcellular-level reveals that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

  6. Label-free characterization of vitrification-induced morphology changes in single-cell embryos with full-field optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Zarnescu, Livia; Leung, Michael C.; Abeyta, Michael; Sudkamp, Helge; Baer, Thomas; Behr, Barry; Ellerbee, Audrey K.

    2015-09-01

    Vitrification is an increasingly popular method of embryo cryopreservation that is used in assisted reproductive technology. Although vitrification has high post-thaw survival rates compared to other freezing techniques, its long-term effects on embryo development are still poorly understood. We demonstrate an application of full-field optical coherence tomography (FF-OCT) to visualize the effects of vitrification on live single-cell (2 pronuclear) mouse embryos without harmful labels. Using FF-OCT, we observed that vitrification causes a significant increase in the aggregation of structures within the embryo cytoplasm, consistent with reports in literature based on fluorescence techniques. We quantify the degree of aggregation with an objective metric, the cytoplasmic aggregation (CA) score, and observe a high degree of correlation between the CA scores of FF-OCT images of embryos and of fluorescence images of their mitochondria. Our results indicate that FF-OCT shows promise as a label-free assessment of the effects of vitrification on embryo mitochondria distribution. The CA score provides a quantitative metric to describe the degree to which embryos have been affected by vitrification and could aid clinicians in selecting embryos for transfer.

  7. Real-Time Detection of Telomerase Activity in Cancer Cells using a Label-Free Electrochemical Impedimetric Biosensing Microchip

    PubMed Central

    Cunci, Lisandro; Vargas, Marina Martinez; Cunci, Roman; Gomez-Moreno, Ramon; Perez, Ivan; Baerga-Ortiz, Abel; Gonzalez, Carlos I.; Cabrera, Carlos R.

    2014-01-01

    The enzyme telomerase is present in about 85% of human cancers which makes it not only a good target for cancer treatment but also an excellent marker for cancer detection. Using a single stranded DNA probe specific for telomerase binding and reverse transcription tethered to an interdigital gold electrode array surface, the chromosome protection provided by the telomerase was replicated and followed by Electrochemical Impedance Spectroscopy as an unlabeled biosensor. Using this system designed in-house, easy and affordable, impedance measurements were taken while incubating at 37 °C and promoting the probe elongation. This resulted in up to 14-fold increase in the charge transfer resistance when testing a telomerase-positive nuclear extract from Jurkat cells compared to the heat-inactivated telomerase-negative nuclear extract. The electron transfer process at the Au electrodes was studied before the elongation, at different times after the elongation, and after desorption of non-specific binding. PMID:25598969

  8. Label-free real-time acoustic sensing of microvesicle release from prostate cancer (PC3) cells using a Quartz Crystal Microbalance

    SciTech Connect

    Stratton, Dan; Lange, Sigrun; Kholia, Sharad; Jorfi, Samireh; Antwi-Baffour, Samuel; Inal, Jameel

    2014-10-24

    Highlights: • Microvesiculating cells record loss of mass on a Quartz Crystal Microbalance. • Using the Quartz Crystal Microbalance microvesicles are measured at 0.24 pg. • The QCM-D reveals loss in viscoelastic properties in microvesiculating cells. - Abstract: Using a Quartz Crystal Microbalance with dissipation monitoring, QCM-D (label-free system) measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, we showed the attachment, over a 60 min period, of a monolayer of PC3 cells to the gold electrodes of the quartz crystal sensor, which had been rendered hydrophilic. That MVs were released upon BzATP stimulation of cells was confirmed by NTA analysis (average 250 nm diameter), flow cytometry, showing high phosphatidylserine exposition and by fluorescent (Annexin V Alexa Fluor® 488-positive) and electron microscopy. Over a period of 1000s (16.7 min) during which early apoptosis increased from 4% plateauing at 10% and late apoptosis rose to 2%, the Δf increased 20 Hz, thereupon remaining constant for the last 1000s of the experiment. Using the Sauerbrey equation, the loss in mass, which corresponded to the release of 2.36 × 10{sup 6} MVs, was calculated to be 23 ng. We therefore estimated the mass of an MV to be 0.24 pg. With the deposition on the QCM-D of 3.5 × 10{sup 7} MVs over 200s, the decrease in Δf (Hz) gave an estimate of 0.235 pg per MV.

  9. Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE.

    PubMed

    Kalayou, Shewit; Granum, Cesilie; Berntsen, Hanne Friis; Groseth, Per Kristian; Verhaegen, Steven; Connolly, Lisa; Brandt, Ingvar; de Souza, Gustavo Antonio; Ropstad, Erik

    2016-03-30

    Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions

  10. Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes.

    PubMed

    Shamsipur, Mojtaba; Molaabasi, Fatemeh; Hosseinkhani, Saman; Rahmati, Fereshteh

    2016-02-16

    Cytochrome c (Cyt c) is an important biomarker in cell lysates for the early stage of apoptosis or anticancer agents. Here, two novel label-free fluorescence assays based on hemoglobin-stabilized gold nanoclusters (Hb/AuNCs) and aptamer-stabilized silver nanoclusters (DNA/AgNCs) for analysis of Cyt c are presented. The heme group of the protein induces sensitive sensing platforms accompanied by the decreased fluorescence of both metal nanoclusters. The quenching processes observed found to be based on the fluorescence resonance energy transfer mechanism from Hb/AuNCs to Cyt c and photoinduced electron transfer from DNA/AgNCs to the aptamer-Cyt c complex. The linear range for Cyt c was found to be 0-10 μM for Hb/AuNCs and from 0 to 1 μM for DNA/AgNCs, with limits of detection of ∼15 nM. On the basis of strong binding affinity of DNA aptamers for their target proteins, the DNA/AgNCs probe was successfully applied to the quantitative determination of Cyt c in cell lysates, which opens a new avenue to early diagnostics and drug screening with high sensitivity. Compared to the conventional Western blot method, the presented assays are low cost, easy to prepare the fluorescent probes, and sensitive, while overall time for the detection and quantitation of Cyt c from isolated mitochondria is only 20 min. The proposed method for Cyt c detection may also be useful for the study of those materials that cause mitochondrial dysfunction and apoptotic cell death. PMID:26812937

  11. A novel graphene-based label-free fluorescence `turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells

    NASA Astrophysics Data System (ADS)

    Ke, Yaotang; Garg, Bhaskar; Ling, Yong-Chien

    2016-02-01

    A novel label-free fluorescence `turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti4+. The as-prepared rGO@PDA-Ti4+-FMNs (nanosensor), fluoresce only weakly due to the ineffective Förster resonance energy transfer between the FMNs and rGO@PDA-Ti4+. The experimental findings revealed that the microwave-assisted interaction of the nanosensor with α-, β-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti4+), leading to an excellent fluorescence `turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of α-, β-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future.A novel label-free fluorescence `turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions

  12. Label free redox capacitive biosensing.

    PubMed

    Fernandes, Flávio C Bedatty; Góes, Márcio S; Davis, Jason J; Bueno, Paulo R

    2013-12-15

    A surface confined redox group contributes to an interfacial charging (quantifiable by redox capacitance) that can be sensitively probed by impedance derived capacitance spectroscopy. In generating mixed molecular films comprising such redox groups, together with specific recognition elements (here antibodies), this charging signal is able to sensitively transduce the recognition and binding of specific analytes. This novel transduction method, exemplified here with C-reactive protein, an important biomarker of cardiac status and general trauma, is equally applicable to any suitably prepared interfacial combination of redox reporter and receptor. The assays are label free, ultrasensitive, highly specific and accompanied by a good linear range. PMID:23896524

  13. A novel graphene-based label-free fluorescence 'turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells.

    PubMed

    Ke, Yaotang; Garg, Bhaskar; Ling, Yong-Chien

    2016-02-28

    A novel label-free fluorescence 'turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti(4+)-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti(4+)) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti(4+). The as-prepared rGO@PDA-Ti(4+)-FMNs (nanosensor), fluoresce only weakly due to the ineffective Förster resonance energy transfer between the FMNs and rGO@PDA-Ti(4+). The experimental findings revealed that the microwave-assisted interaction of the nanosensor with α-, β-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti(4+)), leading to an excellent fluorescence 'turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of α-, β-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future. PMID:26758942

  14. Label-free molecular imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Junqi; Li, Qi; Fu, Rongxin; Wang, Tongzhou; Wang, Ruliang; Huang, Guoliang

    2014-03-01

    Optical microscopy technology has achieved great improvements in the 20th century. The detection limit has reached about twenty nanometers (with near-field optics, STED, PALM and STORM). But in the application areas such as life science, medical science, clinical treatment and especially in vivo dynamic measurement, mutual restrictions still exist between numeric aperture/magnification and working distance, fluorescent dependent, and between resolution and frame rate/field size, etc. This paper explores a hyperspectral scanning super-resolution label free molecules imaging method based on the white light interferometry. The vertical detection resolution was approximate to 1 nm which is the thickness of a single molecular layer and dynamic measuring range of thickness reaches to 10 μm. The spectrum-shifting algorithm is developed for robust restructure of images when the pixels are overlapped. Micro-biochip with protein binding and DNA amplification could be detected by using this spectral scanning super-resolution molecules imaging in label free. This method has several advantages as following: Firstly, the decoding and detecting steps are combined into one step. It makes tests faster and easier. Secondly, we used thickness-coded, minimized chips instead of a large microarray chip to carry the probes. This accelerates the interaction of the biomolecules. Thirdly, since only one kind of probes are attached to our thickness-coded, minimized chip, users can only pick out the probes they are interested in for a test without wasting unnecessary probes and chips.

  15. Label-free cytotoxicity screening assay by digital holographic microscopy.

    PubMed

    Kühn, Jonas; Shaffer, Etienne; Mena, Julien; Breton, Billy; Parent, Jérôme; Rappaz, Benjamin; Chambon, Marc; Emery, Yves; Magistretti, Pierre; Depeursinge, Christian; Marquet, Pierre; Turcatti, Gerardo

    2013-03-01

    We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy. PMID:23062077

  16. Label-free photoacoustic nanoscopy

    PubMed Central

    Danielli, Amos; Maslov, Konstantin; Garcia-Uribe, Alejandro; Winkler, Amy M.; Li, Chiye; Wang, Lidai; Chen, Yun; Dorn, Gerald W.; Wang, Lihong V.

    2014-01-01

    Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules. PMID:25104412

  17. Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

    PubMed Central

    Rappaz, Benjamin; Breton, Billy; Shaffer, Etienne; Turcatti, Gerardo

    2014-01-01

    Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

  18. Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features

    PubMed Central

    Gosnell, Martin E.; Anwer, Ayad G.; Mahbub, Saabah B.; Menon Perinchery, Sandeep; Inglis, David W.; Adhikary, Partho P.; Jazayeri, Jalal A.; Cahill, Michael A.; Saad, Sonia; Pollock, Carol A.; Sutton-McDowall, Melanie L.; Thompson, Jeremy G.; Goldys, Ewa M.

    2016-01-01

    Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent. Label-free classifications are validated by the analysis of Classification Determinant (CD) antigen expression. The versatility of our method is illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. PMID:27029742

  19. Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features.

    PubMed

    Gosnell, Martin E; Anwer, Ayad G; Mahbub, Saabah B; Menon Perinchery, Sandeep; Inglis, David W; Adhikary, Partho P; Jazayeri, Jalal A; Cahill, Michael A; Saad, Sonia; Pollock, Carol A; Sutton-McDowall, Melanie L; Thompson, Jeremy G; Goldys, Ewa M

    2016-01-01

    Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent. Label-free classifications are validated by the analysis of Classification Determinant (CD) antigen expression. The versatility of our method is illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. PMID:27029742

  20. Quantitative proteomic analysis of the effects of a GalNAc/Man-specific lectin CSL on yeast cells by label-free LC-MS.

    PubMed

    Liu, Shuai; Li, Linge; Tong, Changqing; Zhao, Qiancheng; Lukyanov, Pavel A; Chernikov, Oleg V; Li, Wei

    2016-04-01

    A Ca(2+)-dependent GalNAc/Man-specific lectin (CSL) from Cyclina sinensis was isolated, and its stimulatory action was characterized in yeast. CSL showed a potent effect on the production of ethanol by Saccharomyces cerevisiae. In this work, the changes in the protein expression profiles of S. cerevisiae after 24h of incubation with CSL were analyzed using label-free quantitative proteomics. A total of 1410 proteins were identified, but only 117 proteins showed significant differences in normalized volume (p<0.05). Among the latter proteins, 24 proteins were up-regulated, and 93 were down-regulated. Analysis of the proteome revealed that CSL triggered changes in the concentrations of some enzymes, such as increased expression of hexokinase, glyceraldehyde 3-phosphate dehydrogenase and enolase and decreased expression of dihydrolipoamide dehydrogenase and aldehyde dehydrogenase. These results indicate that CSL can cause some changes in the metabolic pathway involved in ethanol synthesis in S. cerevisiae. These data may help us understand the stimulatory mechanism of lectin in the fermentation process. PMID:26794310

  1. Carbon dots as a fluorescent probe for label-free detection of physiological potassium level in human serum and red blood cells.

    PubMed

    Zhang, Lingyang; Chen, Shenna; Zhao, Qian; Huang, Haowen

    2015-06-23

    A unique photoluminescence carbon dots (CDs) with larger size were prepared by microwave-assisted method. Complex functional groups on the surface of the CDs facilitate the nanoparticles to form affinity with some metal ions. Taking advantage of the effective fluorescence quenching effect of K(+), a highly sensitive CD-based fluorescence analytical system for label-free detection of K(+) with limit of detection (LOD) 1.0×10(-12) M was established. The concentrations of potassium ion in biological samples such as human serum are usually found at millimolar levels or even higher. The proposed method begins with a substantial dilution of the sample to place the K(+) concentration in the dynamic range for quantification, which covers 3 orders of magnitude. This offers some advantages: the detection of K(+) only needs very small quantities of biological samples, and the dilution of samples such as serum may effectively eliminate the potential interferences that often originate from the background matrix. The determined potassium levels were satisfactory and closely comparable with the results given by the hospital, indicating that this fluorescent probe is applicable to detection of physiological potassium level with high accuracy. Compared with other relative biosensors requiring modified design, bio-molecular modification or/and sophisticated instruments, this CD-based sensor is very simple, cost-effective and easy detection, suggesting great potential applications for successively monitoring physiological potassium level and the change in biological system. PMID:26092345

  2. Top-down label-free LC-MALDI analysis of the peptidome during neural progenitor cell differentiation reveals complexity in cytoskeletal protein dynamics and identifies progenitor cell markers.

    PubMed

    Maltman, Daniel J; Brand, Sven; Belau, Eckhard; Paape, Rainer; Suckau, Detlev; Przyborski, Stefan A

    2011-10-01

    In the field of stem cell research, there is a strong requirement for the discovery of new biomarkers that more accurately define stem and progenitor cell populations, as well as their differentiated derivatives. The very-low-molecular-weight (<5 kDa) proteome/peptidome remains a poorly investigated but potentially rich source of cellular biomarkers. Here we describe a label-free LC-MALDI-TOF/TOF quantification approach to screen the very-low-molecular-weight proteome, i.e. the peptidome, of neural progenitor cells and derivative populations to identify potential neural stem/progenitor cell biomarkers. Twelve different proteins were identified on the basis of MS/MS analysis of peptides, which displayed differential abundance between undifferentiated and differentiated cultures. These proteins included major cytoskeletal components such as nestin, vimentin, and glial fibrillary acidic protein, which are all associated with neural development. Other cytoskeletal proteins identified were dihydropyrimidinase-related protein 2, prothymosin (thymosin α-1), and thymosin β-10. These findings highlight novel stem cell/progenitor cell marker candidates and demonstrate proteomic complexity, which underlies the limitations of major intermediate filament proteins long established as neural markers. PMID:21761558

  3. Quantitative, Label-Free Characterization of Stem Cell Differentiation at the Single-Cell Level by Broadband Coherent Anti-Stokes Raman Scattering Microscopy

    PubMed Central

    Lee, Young Jong; Vega, Sebastián L.; Patel, Parth J.; Aamer, Khaled A.; Moghe, Prabhas V.

    2014-01-01

    We use broadband coherent anti-Stokes Raman scattering (BCARS) microscopy to characterize lineage commitment of individual human mesenchymal stem cells cultured in adipogenic, osteogenic, and basal culture media. We treat hyperspectral images obtained by BCARS in two independent ways, obtaining robust metrics for differentiation. In one approach, pixel counts corresponding to functional markers, lipids, and minerals, are used to classify individual cells as belonging to one of the three lineage groups: adipocytes, osteoblasts, and undifferentiated stem cells. In the second approach, we use multivariate analysis of Raman spectra averaged exclusively over cytosol regions of individual cells to classify the cells into the same three groups, with consistent results. The exceptionally high speed of spectral imaging with BCARS allows us to chemically map a large number of cells with high spatial resolution, revealing not only the phenotype of individual cells, but also population heterogeneity in the degree of phenotype commitment. PMID:24224876

  4. Dielectrophoretic discrimination of cancer cells on a microchip

    NASA Astrophysics Data System (ADS)

    Huang, Chengjun; Liu, Chengxun; Minne, Bart; Ramirez Hernandez, Juan Enrique; Stakenborg, Tim; Lagae, Liesbet

    2014-10-01

    The analysis of single cell type typically requires expensive equipments in combination with labeling techniques. As a label-free alternative, in this letter, the characteristic dielectric properties of various cancer cell lines (MCF-7, SKOV-3, MDA-MB-231, and LnCap) and healthy peripheral blood mononuclear cells were examined and compared using the dielectrophoretic (DEP) crossover frequency technique. We found that each type of the cancer cells shows a distinct DEP crossover frequency with an order of SKOV-3, MDA-MB-231, MCF-7, and LnCap from low to high frequency, from which the specific cell membrane capacitance and membrane conductance could be derived. Cell fixation and antibody coupling were found to have minimal or no effects on the cell dielectric properties while cell permeabilization significantly changed the DEP crossover frequency. These findings suggested that the DEP crossover frequency is promising to be used as a "dielectric finger print" to discriminate different cell types and may even enable the specific manipulation of certain cell types, for example, to isolate cancer cells from blood.

  5. Absolute quantification of UGT1A1 in various tissues and cell lines using isotope label-free UPLC-MS/MS method determines its turnover number and correlates with its glucuronidation activities.

    PubMed

    Xu, Beibei; Gao, Song; Wu, Baojian; Yin, Taijun; Hu, Ming

    2014-01-01

    Uridine 5'-diphosphate-glucuronosyltransferase (UGT)1A1 is a major phase II metabolism enzyme responsible for glucuronidation of drugs and endogenous compounds. The purpose of this study was to determine the expression level of UGT1A1 in human liver microsomes and human cell lines by using an isotope label-free LC-MS/MS method. A Waters Ultra performance liquid chromatography (UPLC) system coupled with an API 5500Qtrap mass spectrometer was used for the analysis. Two signature peptides (Pep-1, and Pep-2) were employed to quantify UGT1A1 by multiple reaction monitoring (MRM) approach. Standard addition method was used to validate the assay to account for the matrix effect. 17β-Estradiol was used as the marker substrate to determine UGT1A1 activities. The validated method has a linear range of 200-0.0195nM for both signature peptides. The precision, accuracy, and matrix effect were in acceptable ranges. UGT1A1 expression levels were then determined using 8 individual human liver microsomes, a pooled human liver microsomes, three UGT1A1 genotyped human liver microsomes, and four cell lines (Caco-2, MCF-7, Hela, and HepG2). The correlations study showed that the UGT1A1 protein levels were strongly correlated with its glucuronidation activities in human liver microsomes (R(2)=0.85) and in microsomes prepared from cell lines (R(2)=0.95). Isotope-labeled peptides were not necessary for LC-MS/MS quantitation of proteins. The isotope label-free absolute quantification method used here had good accuracy, sensitivity, linear range, and reproducibility, and were used successfully for the accurate determination of UGT1A1 from tissues and cell lines. PMID:24055854

  6. Label-free morphology-based prediction of multiple differentiation potentials of human mesenchymal stem cells for early evaluation of intact cells.

    PubMed

    Sasaki, Hiroto; Takeuchi, Ichiro; Okada, Mai; Sawada, Rumi; Kanie, Kei; Kiyota, Yasujiro; Honda, Hiroyuki; Kato, Ryuji

    2014-01-01

    Precise quantification of cellular potential of stem cells, such as human bone marrow-derived mesenchymal stem cells (hBMSCs), is important for achieving stable and effective outcomes in clinical stem cell therapy. Here, we report a method for image-based prediction of the multiple differentiation potentials of hBMSCs. This method has four major advantages: (1) the cells used for potential prediction are fully intact, and therefore directly usable for clinical applications; (2) predictions of potentials are generated before differentiation cultures are initiated; (3) prediction of multiple potentials can be provided simultaneously for each sample; and (4) predictions of potentials yield quantitative values that correlate strongly with the experimental data. Our results show that the collapse of hBMSC differentiation potentials, triggered by in vitro expansion, can be quantitatively predicted far in advance by predicting multiple potentials, multi-lineage differentiation potentials (osteogenic, adipogenic, and chondrogenic) and population doubling potential using morphological features apparent during the first 4 days of expansion culture. In order to understand how such morphological features can be effective for advance predictions, we measured gene-expression profiles of the same early undifferentiated cells. Both senescence-related genes (p16 and p21) and cytoskeleton-related genes (PTK2, CD146, and CD49) already correlated to the decrease of potentials at this stage. To objectively compare the performance of morphology and gene expression for such early prediction, we tested a range of models using various combinations of features. Such comparison of predictive performances revealed that morphological features performed better overall than gene-expression profiles, balancing the predictive accuracy with the effort required for model construction. This benchmark list of various prediction models not only identifies the best morphological feature conversion

  7. Label-Free Morphology-Based Prediction of Multiple Differentiation Potentials of Human Mesenchymal Stem Cells for Early Evaluation of Intact Cells

    PubMed Central

    Sasaki, Hiroto; Takeuchi, Ichiro; Okada, Mai; Sawada, Rumi; Kanie, Kei; Kiyota, Yasujiro; Honda, Hiroyuki; Kato, Ryuji

    2014-01-01

    Precise quantification of cellular potential of stem cells, such as human bone marrow–derived mesenchymal stem cells (hBMSCs), is important for achieving stable and effective outcomes in clinical stem cell therapy. Here, we report a method for image-based prediction of the multiple differentiation potentials of hBMSCs. This method has four major advantages: (1) the cells used for potential prediction are fully intact, and therefore directly usable for clinical applications; (2) predictions of potentials are generated before differentiation cultures are initiated; (3) prediction of multiple potentials can be provided simultaneously for each sample; and (4) predictions of potentials yield quantitative values that correlate strongly with the experimental data. Our results show that the collapse of hBMSC differentiation potentials, triggered by in vitro expansion, can be quantitatively predicted far in advance by predicting multiple potentials, multi-lineage differentiation potentials (osteogenic, adipogenic, and chondrogenic) and population doubling potential using morphological features apparent during the first 4 days of expansion culture. In order to understand how such morphological features can be effective for advance predictions, we measured gene-expression profiles of the same early undifferentiated cells. Both senescence-related genes (p16 and p21) and cytoskeleton-related genes (PTK2, CD146, and CD49) already correlated to the decrease of potentials at this stage. To objectively compare the performance of morphology and gene expression for such early prediction, we tested a range of models using various combinations of features. Such comparison of predictive performances revealed that morphological features performed better overall than gene-expression profiles, balancing the predictive accuracy with the effort required for model construction. This benchmark list of various prediction models not only identifies the best morphological feature conversion

  8. Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays.

    PubMed

    Orgovan, Norbert; Ungai-Salánki, Rita; Lukácsi, Szilvia; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Szabó, Bálint; Horvath, Robert

    2016-01-01

    Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results

  9. Label-free Quantitative Proteomics Reveals a Role for the Mycobacterium tuberculosis SecA2 Pathway in Exporting Solute Binding Proteins and Mce Transporters to the Cell Wall*

    PubMed Central

    Feltcher, Meghan E.; Gunawardena, Harsha P.; Zulauf, Katelyn E.; Malik, Seidu; Griffin, Jennifer E.; Sassetti, Christopher M.; Chen, Xian; Braunstein, Miriam

    2015-01-01

    Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology. PMID:25813378

  10. On-Orbit, Immuno-Based, Label-Free White Blood Cell Counting System with Microelectromechanical Sensor Technology (OILWBCS-MEMS)

    NASA Technical Reports Server (NTRS)

    Edmonds, Jessica

    2015-01-01

    Aurora Flight Sciences, in partnership with Draper Laboratory, has developed a miniaturized system to count white blood cells in microgravity environments. The system uses MEMS technology to simultaneously count total white blood cells, the five white blood cell differential subgroups, and various lymphocyte subtypes. The OILWBCS-MEMS detection technology works by immobilizing an array of white blood cell-specific antibodies on small, gold-coated membranes. When blood flows across the membranes, specific cells' surface protein antigens bind to their corresponding antibodies. This binding can be measured and correlated to cell counts. In Phase I, the partners demonstrated surface chemistry sensitivity and specificity for total white blood cells and two lymphocyte subtypes. In Phase II, a functional prototype demonstrated end-to-end operation. This rugged, miniaturized device requires minimal blood sample preparation and will be useful for both space flight and terrestrial applications.

  11. Label Free Cell-Tracking and Division Detection Based on 2D Time-Lapse Images For Lineage Analysis of Early Embryo Development

    PubMed Central

    Cicconet, Marcelo; Gutwein, Michelle; Gunsalus, Kristin C; Geiger, Davi

    2014-01-01

    In this paper we report a database and a series of techniques related to the problem of tracking cells, and detecting their divisions, in time-lapse movies of mammalian embryos. Our contributions are: (1) a method for counting embryos in a well, and cropping each individual embryo across frames, to create individual movies for cell tracking; (2) a semi-automated method for cell tracking that works up to the 8-cell stage, along with a software implementation available to the public (this software was used to build the reported database); (3) an algorithm for automatic tracking up to the 4-cell stage, based on histograms of mirror symmetry coefficients captured using wavelets; (4) a cell-tracking database containing 100 annotated examples of mammalian embryos up to the 8-cell stage; (5) statistical analysis of various timing distributions obtained from those examples. PMID:24873887

  12. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    PubMed

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. PMID:27317400

  13. Label-free distinguishing between neurons and glial cells based on two-photon excited fluorescence signal of neuron perinuclear granules

    NASA Astrophysics Data System (ADS)

    Du, Huiping; Jiang, Liwei; Wang, Xingfu; Liu, Gaoqiang; Wang, Shu; Zheng, Liqin; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

    2016-08-01

    Neurons and glial cells are two critical cell types of brain tissue. Their accurate identification is important for the diagnosis of psychiatric disorders such as depression and schizophrenia. In this paper, distinguishing between neurons and glial cells by using the two-photon excited fluorescence (TPEF) signals of intracellular intrinsic sources was performed. TPEF microscopy combined with TUJ-1 and GFAP immunostaining and quantitative image analysis demonstrated that the perinuclear granules of neurons in the TPEF images of brain tissue and the primary cultured cortical cells were a unique characteristic of neurons compared to glial cells which can become a quantitative feature to distinguish neurons from glial cells. With the development of miniaturized TPEF microscope (‘two-photon fiberscopes’) imaging devices, TPEF microscopy can be developed into an effective diagnostic and monitoring tool for psychiatric disorders such as depression and schizophrenia.

  14. Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

    PubMed Central

    Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

    2015-01-01

    A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

  15. Multimodal label-free growth and morphology characterization of different cell types in a single culture with quantitative digital holographic phase microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Wibbeling, Jana; Kastl, Lena; Schnekenburger, Jürgen; Ketelhut, Steffi

    2015-03-01

    For the analysis of the impact of pharmaceuticals or pathogens on different cellular phenotypes under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of different cell types in a single culture is of particular interest. Digital holographic microscopy (DHM), a var-iant of quantitative phase microscopy (QPM), provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and has been demonstrated in particular to be suitable for long-term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth and motility. Thus, we studied the feasibility of QPM for the analysis of mixed cell cultures and explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with DHM was utilized. Mixed cell cultures with different cell types were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the area covered by the cells, the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability of the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different mor-phology features separately in a single culture.

  16. Label-free optical activation of astrocyte in vivo

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Yoon, Jonghee; Ku, Taeyun; Choi, Kyungsun; Choi, Chulhee

    2011-07-01

    As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.

  17. In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Yin, Linliang; Gonzalez-Malerva, Laura; Wang, Shaopeng; Yu, Xiaobo; Eaton, Seron; Zhang, Shengtao; Chen, Hong-Yuan; Labaer, Joshua; Tao, Nongjian

    2014-10-01

    Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

  18. In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance

    PubMed Central

    Wang, Wei; Yin, Linliang; Gonzalez-Malerva, Laura; Wang, Shaopeng; Yu, Xiaobo; Eaton, Seron; Zhang, Shengtao; Chen, Hong-Yuan; LaBaer, Joshua; Tao, Nongjian

    2014-01-01

    Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis. PMID:25312029

  19. Live-Cell, Label-Free Identification of GABAergic and Non-GABAergic Neurons in Primary Cortical Cultures Using Micropatterned Surface

    PubMed Central

    Kono, Sho; Kushida, Takatoshi; Hirano-Iwata, Ayumi; Niwano, Michio; Tanii, Takashi

    2016-01-01

    Excitatory and inhibitory neurons have distinct roles in cortical dynamics. Here we present a novel method for identifying inhibitory GABAergic neurons from non-GABAergic neurons, which are mostly excitatory glutamatergic neurons, in primary cortical cultures. This was achieved using an asymmetrically designed micropattern that directs an axonal process to the longest pathway. In the current work, we first modified the micropattern geometry to improve cell viability and then studied the axon length from 2 to 7 days in vitro (DIV). The cell types of neurons were evaluated retrospectively based on immunoreactivity against GAD67, a marker for inhibitory GABAergic neurons. We found that axons of non-GABAergic neurons grow significantly longer than those of GABAergic neurons in the early stages of development. The optimal threshold for identifying GABAergic and non-GABAergic neurons was evaluated to be 110 μm at 6 DIV. The method does not require any fluorescence labelling and can be carried out on live cells. The accuracy of identification was 98.2%. We confirmed that the high accuracy was due to the use of a micropattern, which standardized the development of cultured neurons. The method promises to be beneficial both for engineering neuronal networks in vitro and for basic cellular neuroscience research. PMID:27513933

  20. γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay.

    PubMed

    Seidel, Ursula Jördis Eva; Vogt, Fabian; Grosse-Hovest, Ludger; Jung, Gundram; Handgretinger, Rupert; Lang, Peter

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical

  1. Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells.

    PubMed

    Shi, Kai; Dou, Baoting; Yang, Jianmei; Yuan, Ruo; Xiang, Yun

    2016-04-15

    The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells. PMID:27016432

  2. Label-free and depth resolved optical sectioning of iron-complex deposits in sickle cell disease splenic tissue by multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Vigil, Genevieve D.; Adami, Alexander J.; Ahmed, Tahsin; Khan, Aamir; Chapman, Sarah; Andemariam, Biree; Thrall, Roger S.; Howard, Scott S.

    2015-06-01

    Multiphoton microscopy (MPM) imaging of intrinsic two-photon excited fluorescence (TPEF) is performed on humanized sickle cell disease (SCD) mouse model splenic tissue. Distinct morphological and spectral features associated with SCD are identified and discussed in terms of diagnostic relevance. Specifically, spectrally unique splenic iron-complex deposits are identified by MPM; this finding is supported by TPEF spectroscopy and object size to standard histopathological methods. Further, iron deposits are found at higher concentrations in diseased tissue than in healthy tissue by all imaging methods employed here including MPM, and therefore, may provide a useful biomarker related to the disease state. These newly characterized biomarkers allow for further investigations of SCD in live animals as a means to gain insight into the mechanisms impacting immune dysregulation and organ malfunction, which are currently not well understood.

  3. Maximizing throughput in label-free microspectroscopy with hybrid Raman imaging

    NASA Astrophysics Data System (ADS)

    Pavillon, Nicolas; Smith, Nicholas I.

    2015-01-01

    Raman spectroscopy is an optical method providing sample molecular composition, which can be analyzed (by point measurements) or spatially mapped by Raman imaging. These provide different information, signal-to-noise ratios, and require different acquisition times. Here, we quantitatively assess Raman spectral features and compare the two measurement methods by multivariate analysis. We also propose a hybrid method: scanning the beam through the sample but optically binning the signal at one location on the detector. This approach generates significantly more useful spectral signals in terms of peak visibility and statistical information. Additionally, by combination with a complementary imaging mode such as quantitative phase microscopy, hybrid imaging allows high throughput and robust spectral analysis while retaining sample spatial information. We demonstrate the improved ability to discriminate between cell lines when using hybrid scanning compared to typical point mode measurements, by quantitatively evaluating spectra taken from two macrophage-like cell lines. Hybrid scanning also provides better classification capability than the full Raman imaging mode, while providing higher signal-to-noise signals with shorter acquisition times. This hybrid imaging approach is suited for various applications including cytometry, cancer versus noncancer detection, and label-free discrimination of cell types or tissues.

  4. Label-free surface plasmon sensing towards cancer diagnostics

    NASA Astrophysics Data System (ADS)

    Sankaranarayanan, Goutham

    The main objective of this thesis is to develop a conventional, home-built SPR bio-sensor to demonstrate bio-sensing applications. This emphasizes the understanding of basic concepts of Surface Plasmon Resonance and various interrogation techniques. Intensity Modulation was opted to perform the label-free SPR bio-sensing experiments due to its cost-efficient and compact setup. Later, label-free surface plasmon sensing was carried out to study and understand the bio-molecular interactions between (1). BSA and Anti BSA molecules and (2). Exosome/Liposome on thin metal (Au) films. Exosomes are cell-derived vesicles present in bodily fluids like blood, saliva, urine, epididymal fluid containing miRNAs, RNA, proteins, etc., at stable quantities during normal health conditions. The exosomes comprise varied constituents based on their cell origin from where they are secreted and is specific to that particular origin. However an exacerbated release is observed during tumor or cancer conditions. This increased level of exosomes present in the sample, can be detected using the SPR bio-sensor demonstrated in this thesis and effective thickness of adsorption on Au surface can be estimated. Also, chemically synthesized liposome particles were studied to determine if they can generate an equivalent sensor response to that of exosomes to consider them as an alternate. Finally a 10ppb Mercury (Hg) sensing was performed as part of Environment Monitoring application and results have been tabulated and compared.

  5. DNA-fueled molecular machine for label-free and non-enzymatic ultrasensitive detection of telomerase activity.

    PubMed

    Sun, Panpan; Ran, Xiang; Liu, Chaoqun; Liu, Chaoying; Pu, Fang; Ren, Jinsong; Qu, Xiaogang

    2016-08-01

    Herein, a non-enzymatic and label-free strategy based on DNA-fueled molecular machine was developed for ultrasensitive detection of telomerase activity in cancer cell extracts even at the single-cell level. PMID:27405851

  6. Label-Free Quantitative Proteomics in Yeast.

    PubMed

    Léger, Thibaut; Garcia, Camille; Videlier, Mathieu; Camadro, Jean-Michel

    2016-01-01

    Label-free bottom-up shotgun MS-based proteomics is an extremely powerful and simple tool to provide high quality quantitative analyses of the yeast proteome with only microgram amounts of total protein. Although the experimental design of this approach is rather straightforward and does not require the modification of growth conditions, proteins or peptides, several factors must be taken into account to benefit fully from the power of this method. Key factors include the choice of an appropriate method for the preparation of protein extracts, careful evaluation of the instrument design and available analytical capabilities, the choice of the quantification method (intensity-based vs. spectral count), and the proper manipulation of the selected quantification algorithm. The elaboration of this robust workflow for data acquisition, processing, and analysis provides unprecedented insight into the dynamics of the yeast proteome. PMID:26483028

  7. The label free picomolar detection of insulin in blood serum.

    PubMed

    Xu, Mengyun; Luo, Xiliang; Davis, Jason J

    2013-01-15

    Insulin, a polypeptide hormone secreted by pancreatic cells, is a key regulator in glucose homeostasis. Its deficiency leads to insulin-dependent (type I) diabetes whereas resistance to insulin is common in type II diabetes, obesity and a range of endocrine disorders. Its determination is of considerable value, particularly in the clinical diagnosis of diabetes mellitus and the doping control of athletes. It has, additionally, been noted as a potential breast cancer marker (serum insulin levels being found to be raised in comparison to control patients). Electrochemical assays are potentially very cheap, highly sensitive, and very readily transposed to a point of care. Though there exist numerous examples of label free impedimetric or capacitative assaying of biomolecules, these are rarely demonstrated to be effective in complex biological mixtures or to be applicable to low molecular weight targets (since they operate through the interfacial displacement of water/ions and/or the steric blocking of a redox probe). We report herein an ultrasensitive electrochemical and label-free biosensor for insulin in blood serum with a clinically relevant linear range and detection limit of 1.2pM. The transducing surfaces, based on readily prepared, antibody modified, polyethylene glycol monolayer modified polycrystalline gold surfaces, respond in a highly specific and re-useable manner to the target in up to 50% blood serum. PMID:22840329

  8. Label-free integrative pharmacology on-target of drugs at the β2-adrenergic receptor

    NASA Astrophysics Data System (ADS)

    Ferrie, Ann M.; Sun, Haiyan; Fang, Ye

    2011-07-01

    We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the β2-adrenergic receptor. This method combines dynamic mass redistribution (DMR) assays using an array of probe molecule-hijacked cells with similarity analysis. The whole cell DMR assays track cell system-based, ligand-directed, and kinetics-dependent biased activities of the drugs, and translates their on-target pharmacology into numerical descriptors which are subject to similarity analysis. We demonstrate that the approach establishes an effective link between the label-free pharmacology and in vivo therapeutic indications of drugs.

  9. Label-free nonlinear optical imaging of mouse retina

    PubMed Central

    He, Sicong; Ye, Cong; Sun, Qiqi; Leung, Christopher K.S.; Qu, Jianan Y.

    2015-01-01

    A nonlinear optical (NLO) microscopy system integrating stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was developed to image fresh mouse retinas. The morphological and functional details of various retinal layers were revealed by the endogenous NLO signals. Particularly, high resolution label-free imaging of retinal neurons and nerve fibers in the ganglion cell and nerve fiber layers was achieved by capturing endogenous SRS and TPEF signals. In addition, the spectral and temporal analysis of TPEF images allowed visualization of different fluorescent components in the retinal pigment epithelium (RPE). Fluorophores with short TPEF lifetime, such as A2E, can be differentiated from other long-lifetime components in the RPE. The NLO imaging method would provide important information for investigation of retinal ganglion cell degeneration and holds the potential to study the biochemical processes of visual cycle in the RPE. PMID:25798325

  10. Label-free optical control of arterial contraction

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Yoon, Jonghee; Choi, Chulhee

    2010-01-01

    The diameters of blood vessels, especially in the brain, change dynamically over time to provide sufficient blood supply as needed. No existing technique allows noninvasive control of vascular diameter in vivo. We report that label-free irradiation with a femtosecond pulsed laser can trigger blood vessel contraction in vivo. In response to laser irradiation, cultured vascular smooth muscle cells showed a rapid increase in calcium concentration, followed by cell contraction. In a murine thinned skull window model, laser irradiation focused in the arterial vessel wall caused localized vascular contraction, followed by recovery. The nonlinear nature of the pulsed laser allowed highly specific targeting of subcortical vessels without affecting the surrounding region. We believe that femtosecond pulsed laser irradiation will become a useful experimental tool in the field of vascular biology.

  11. Label-free optical control of arterial contraction

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Yoon, Jonghee; Choi, Chulhee

    2010-02-01

    Blood vessels, especially in the brain, dynamically change the diameters over time to provide sufficient blood supply where needed. At present, there is no technique that allows noninvasive control of vascular diameter in vivo. Here we report that label-free irradiation of femtosecond pulsed laser can trigger blood vessel contraction in vivo. In response to laser irradiation, cultured vascular smooth muscle cell showed a rapid increase in calcium concentration followed by the cell contraction. In a murine thinned skull window model, laser irradiation focused in the arterial vessel wall caused localized vascular contraction followed by recovery. Nonlinear nature of the pulsed laser allowed highly specific targeting of subcortical vessels without affecting the surrounding region. We propose that femtosecond pulsed laser irradiation will be a useful experimental tool in the field of vascular biology.

  12. Label-Free Neurosurgical Pathology with Stimulated Raman Imaging.

    PubMed

    Lu, Fa-Ke; Calligaris, David; Olubiyi, Olutayo I; Norton, Isaiah; Yang, Wenlong; Santagata, Sandro; Xie, X Sunney; Golby, Alexandra J; Agar, Nathalie Y R

    2016-06-15

    The goal of brain tumor surgery is to maximize tumor removal without injuring critical brain structures. Achieving this goal is challenging as it can be difficult to distinguish tumor from nontumor tissue. While standard histopathology provides information that could assist tumor delineation, it cannot be performed iteratively during surgery as freezing, sectioning, and staining of the tissue require too much time. Stimulated Raman scattering (SRS) microscopy is a powerful label-free chemical imaging technology that enables rapid mapping of lipids and proteins within a fresh specimen. This information can be rendered into pathology-like images. Although this approach has been used to assess the density of glioma cells in murine orthotopic xenografts models and human brain tumors, tissue heterogeneity in clinical brain tumors has not yet been fully evaluated with SRS imaging. Here we profile 41 specimens resected from 12 patients with a range of brain tumors. By evaluating large-scale stimulated Raman imaging data and correlating this data with current clinical gold standard of histopathology for 4,422 fields of view, we capture many essential diagnostic hallmarks for glioma classification. Notably, in fresh tumor samples, we observe additional features, not seen by conventional methods, including extensive lipid droplets within glioma cells, collagen deposition in gliosarcoma, and irregularity and disruption of myelinated fibers in areas infiltrated by oligodendroglioma cells. The data are freely available in a public resource to foster diagnostic training and to permit additional interrogation. Our work establishes the methodology and provides a significant collection of reference images for label-free neurosurgical pathology. Cancer Res; 76(12); 3451-62. ©2016 AACR. PMID:27197198

  13. Label-Free Ultrasensitive Memristive Aptasensor.

    PubMed

    Tzouvadaki, Ioulia; Jolly, Pawan; Lu, Xiaoling; Ingebrandt, Sven; de Micheli, Giovanni; Estrela, Pedro; Carrara, Sandro

    2016-07-13

    We present the very first worldwide ever-reported electrochemical biosensor based on a memristive effect and DNA aptamers. This novel device is developed to propose a completely new approach in cancer diagnostics. In this study, an affinity-based technique is presented for the detection of the prostate specific antigen (PSA) using DNA aptamers. The hysteretic properties of memristive silicon nanowires functionalized with these DNA aptamers provide a label-free and ultrasensitive biodetection technique. The ultrasensitive detection is hereby demonstrated for PSA with a limit of detection down to 23 aM, best ever published value for electrochemical biosensors in PSA detection. The effect of polyelectrolytes on our memristive devices is also reported to further show how positive or negative charges affect the memristive hysteresis. With such an approach, combining memristive nanowires and aptamers, memristive aptamer-based biosensors can be proposed to detect a wide range of cancer markers with unprecedent ultrasensitivities to also address the issue of an early detection of cancer. PMID:27341189

  14. Label-free monitoring of individual DNA hybridization using SERS

    NASA Astrophysics Data System (ADS)

    Qi, Ji; Zeng, Jianbo; Zhao, Fusheng; Santos, Greggy M.; Lin, Steven Hsesheng; Raja, Balakrishnan; Strych, Ulrich; Willson, Richard C.; Shih, Wei-Chuan

    2015-03-01

    Sequence-specific detection of DNA hybridization at the single-molecule level has been instrumental and gradually become a ubiquitous tool in a wide variety of biological and biomedical applications such as clinical diagnostics, biosensors, and drug development. Label-free and amplification-free schemes are of particular interest because they could potentially provide in situ monitoring of individual hybridization events, which may lead to techniques for discriminating subtle variations due to single-base modification without stringency control or repetitive thermal cycling. Surface-enhanced Raman spectroscopy (SERS) has been widely used for molecular detection and identification by exploiting the localized surface plasmon resonance effect when the target molecules are near gold or silver nanostructures. However, effective and robust SERS assays have yet become a reality for trace detection. Recently, we have developed a SERS substrate by shaping nanoporous gold thin films into monolithic submicron disks, called nanoporous gold disks (NPGD). Here we demonstrate in situ monitoring of the same immobilized ssDNA molecules and their individual hybridization events.

  15. Simple and sensitive microbial pathogen detection using a label-free DNA amplification assay.

    PubMed

    Sun, Yuhuan; Zhao, Chuanqi; Yan, Zhengqing; Ren, Jinsong; Qu, Xiaogang

    2016-06-14

    By the combination of quaternized magnetic nanoparticles and a label-free exonuclease III-assisted DNA amplification assay, we report a simple and facile strategy for the convenient and highly sensitive detection of microbial pathogens, with a detection limit of down to 50 cells mL(-1). PMID:27210898

  16. Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

    PubMed Central

    Lu, Fa-Ke; Basu, Srinjan; Igras, Vivien; Hoang, Mai P.; Ji, Minbiao; Fu, Dan; Holtom, Gary R.; Neel, Victor A.; Freudiger, Christian W.; Fisher, David E.; Xie, X. Sunney

    2015-01-01

    Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time. PMID:26324899

  17. Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.

    PubMed

    Lu, Fa-Ke; Basu, Srinjan; Igras, Vivien; Hoang, Mai P; Ji, Minbiao; Fu, Dan; Holtom, Gary R; Neel, Victor A; Freudiger, Christian W; Fisher, David E; Xie, X Sunney

    2015-09-15

    Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time. PMID:26324899

  18. Label-Free Impedance Biosensors: Opportunities and Challenges

    PubMed Central

    Daniels, Jonathan S.; Pourmand, Nader

    2007-01-01

    Impedance biosensors are a class of electrical biosensors that show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in surface impedance when a target molecule binds to an immobilized probe. The affinity capture step leads to challenges shared by all label-free affinity biosensors; these challenges are discussed along with others unique to impedance readout. Various possible mechanisms for impedance change upon target binding are discussed. We critically summarize accomplishments of past label-free impedance biosensors and identify areas for future research. PMID:18176631

  19. Fast label-free detection of Legionella spp. in biofilms by applying immunomagnetic beads and Raman spectroscopy.

    PubMed

    Kusić, Dragana; Rösch, Petra; Popp, Jürgen

    2016-03-01

    Legionellae colonize biofilms, can form a biofilm by itself and multiply intracellularly within the protozoa commonly found in water distribution systems. Approximately half of the known species are pathogenic and have been connected to severe multisystem Legionnaires' disease. The detection methods for Legionella spp. in water samples are still based on cultivation, which is time consuming due to the slow growth of this bacterium. Here, we developed a cultivation-independent, label-free and fast detection method for legionellae in a biofilm matrix based on the Raman spectroscopic analysis of isolated single cells via immunomagnetic separation (IMS). A database comprising the Raman spectra of single bacterial cells captured and separated from the biofilms formed by each species was used to build the identification method based on a support vector machine (SVM) discriminative classifier. The complete method allows the detection of Legionella spp. in 100 min. Cross-reactivity of Legionella spp. specific immunomagnetic beads to the other studied genera was tested, where only small cell amounts of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli compared to the initial number of cells were isolated by the immunobeads. Nevertheless, the Raman spectra collected from isolated non-targeted bacteria were well-discriminated from the Raman spectra collected from isolated Legionella cells, whereby the Raman spectra of the independent dataset of Legionella strains were assigned with an accuracy of 98.6%. In addition, Raman spectroscopy was also used to differentiate between isolated Legionella species. PMID:26915495

  20. Microchannel DNA Sequencing by End-Labelled Free Solution Electrophoresis

    SciTech Connect

    Barron, A.

    2005-09-29

    The further development of End-Labeled Free-Solution Electrophoresis will greatly simplify DNA separation and sequencing on microfluidic devices. The development and optimization of drag-tags is critical to the success of this research.

  1. Label-Free Biosensor Imaging on Photonic Crystal Surfaces

    PubMed Central

    Zhuo, Yue; Cunningham, Brian T.

    2015-01-01

    We review the development and application of nanostructured photonic crystal surfaces and a hyperspectral reflectance imaging detection instrument which, when used together, represent a new form of optical microscopy that enables label-free, quantitative, and kinetic monitoring of biomaterial interaction with substrate surfaces. Photonic Crystal Enhanced Microscopy (PCEM) has been used to detect broad classes of materials which include dielectric nanoparticles, metal plasmonic nanoparticles, biomolecular layers, and live cells. Because PCEM does not require cytotoxic stains or photobleachable fluorescent dyes, it is especially useful for monitoring the long-term interactions of cells with extracellular matrix surfaces. PCEM is only sensitive to the attachment of cell components within ~200 nm of the photonic crystal surface, which may correspond to the region of most interest for adhesion processes that involve stem cell differentiation, chemotaxis, and metastasis. PCEM has also demonstrated sufficient sensitivity for sensing nanoparticle contrast agents that are roughly the same size as protein molecules, which may enable applications in “digital” diagnostics with single molecule sensing resolution. We will review PCEM’s development history, operating principles, nanostructure design, and imaging modalities that enable tracking of optical scatterers, emitters, absorbers, and centers of dielectric permittivity. PMID:26343684

  2. Label-Free Biosensor Imaging on Photonic Crystal Surfaces.

    PubMed

    Zhuo, Yue; Cunningham, Brian T

    2015-01-01

    We review the development and application of nanostructured photonic crystal surfaces and a hyperspectral reflectance imaging detection instrument which, when used together, represent a new form of optical microscopy that enables label-free, quantitative, and kinetic monitoring of biomaterial interaction with substrate surfaces. Photonic Crystal Enhanced Microscopy (PCEM) has been used to detect broad classes of materials which include dielectric nanoparticles, metal plasmonic nanoparticles, biomolecular layers, and live cells. Because PCEM does not require cytotoxic stains or photobleachable fluorescent dyes, it is especially useful for monitoring the long-term interactions of cells with extracellular matrix surfaces. PCEM is only sensitive to the attachment of cell components within ~200 nm of the photonic crystal surface, which may correspond to the region of most interest for adhesion processes that involve stem cell differentiation, chemotaxis, and metastasis. PCEM has also demonstrated sufficient sensitivity for sensing nanoparticle contrast agents that are roughly the same size as protein molecules, which may enable applications in "digital" diagnostics with single molecule sensing resolution. We will review PCEM's development history, operating principles, nanostructure design, and imaging modalities that enable tracking of optical scatterers, emitters, absorbers, and centers of dielectric permittivity. PMID:26343684

  3. Label-free detection of antigens using implantable SERS nanosensors

    NASA Astrophysics Data System (ADS)

    Li, Honggang; Baum, Caitlin E.; Cullum, Brian M.

    2005-11-01

    Monitoring the presence, production and transport of proteins inside individual living cells can provide vital information about cellular signaling pathways and the overall biological response of an organism. For example, cellular response to external stimuli, such as biological warfare (BW) agents, can be monitored by measuring interleukin-II (IL-2) expression inside T-cells as well as other chemical species associated with T-cell activation. By monitoring such species, pre-symptomatic detection of exposure to BW agents can be achieved, leading to significantly increased post-exposure survival rates. To accomplish such monitoring, we have developed and optimized implantable nanosphere-based nanosensors for the intracellular analysis of specific proteins in a label-free fashion. These sensors consist of 300-520 nm diameter silica spheres that have been coated with silver and antibodies to allow for trace protein detection via surface enhanced Raman spectroscopy (SERS). They have been optimized for SERS response by evaluating the size of the nanospheres best suited to 632.8 nm laser excitation, as well as the various nanosensor fabrication steps (i.e., silver deposition process, antibody binding, etc.). During usage, the presence of the specific protein of interest is monitored by either directly measuring SERS signals associated with the protein and/or changes in the SERS spectrum of the antibodies resulting from conformational changes after antigen binding. In this work, human insulin was used as a model compound for initial studies into the sensitivity of these optimized nanosensors.

  4. Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

    NASA Astrophysics Data System (ADS)

    Chirvi, Sajal

    Biomolecular interaction analysis (BIA) plays vital role in wide variety of fields, which include biomedical research, pharmaceutical industry, medical diagnostics, and biotechnology industry. Study and quantification of interactions between natural biomolecules (proteins, enzymes, DNA) and artificially synthesized molecules (drugs) is routinely done using various labeled and label-free BIA techniques. Labeled BIA (Chemiluminescence, Fluorescence, Radioactive) techniques suffer from steric hindrance of labels on interaction site, difficulty of attaching labels to molecules, higher cost and time of assay development. Label free techniques with real time detection capabilities have demonstrated advantages over traditional labeled techniques. The gold standard for label free BIA is surface Plasmon resonance (SPR) that detects and quantifies the changes in refractive index of the ligand-analyte complex molecule with high sensitivity. Although SPR is a highly sensitive BIA technique, it requires custom-made sensor chips and is not well suited for highly multiplexed BIA required in high throughput applications. Moreover implementation of SPR on various biosensing platforms is limited. In this research work spectral domain phase sensitive interferometry (SD-PSI) has been developed for label-free BIA and biosensing applications to address limitations of SPR and other label free techniques. One distinct advantage of SD-PSI compared to other label-free techniques is that it does not require use of custom fabricated biosensor substrates. Laboratory grade, off-the-shelf glass or plastic substrates of suitable thickness with proper surface functionalization are used as biosensor chips. SD-PSI is tested on four separate BIA and biosensing platforms, which include multi-well plate, flow cell, fiber probe with integrated optics and fiber tip biosensor. Sensitivity of 33 ng/ml for anti-IgG is achieved using multi-well platform. Principle of coherence multiplexing for multi

  5. Label-free electrochemical detection of human methyltransferase from tumors.

    PubMed

    Furst, Ariel L; Muren, Natalie B; Hill, Michael G; Barton, Jacqueline K

    2014-10-21

    The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications. PMID:25288757

  6. Applying label-free quantitation to top down proteomics.

    PubMed

    Ntai, Ioanna; Kim, Kyunggon; Fellers, Ryan T; Skinner, Owen S; Smith, Archer D; Early, Bryan P; Savaryn, John P; LeDuc, Richard D; Thomas, Paul M; Kelleher, Neil L

    2014-05-20

    With the prospect of resolving whole protein molecules into their myriad proteoforms on a proteomic scale, the question of their quantitative analysis in discovery mode comes to the fore. Here, we demonstrate a robust pipeline for the identification and stringent scoring of abundance changes of whole protein forms <30 kDa in a complex system. The input is ~100-400 μg of total protein for each biological replicate, and the outputs are graphical displays depicting statistical confidence metrics for each proteoform (i.e., a volcano plot and representations of the technical and biological variation). A key part of the pipeline is the hierarchical linear model that is tailored to the original design of the study. Here, we apply this new pipeline to measure the proteoform-level effects of deleting a histone deacetylase (rpd3) in S. cerevisiae. Over 100 proteoform changes were detected above a 5% false positive threshold in WT vs the Δrpd3 mutant, including the validating observation of hyperacetylation of histone H4 and both H2B isoforms. Ultimately, this approach to label-free top down proteomics in discovery mode is a critical technical advance for testing the hypothesis that whole proteoforms can link more tightly to complex phenotypes in cell and disease biology than do peptides created in shotgun proteomics. PMID:24807621

  7. Label-free microcavity biosensors: steps towards personalized medicine.

    PubMed

    Amarie, Dragos; Glazier, James A

    2012-01-01

    Personalized medicine has the potential to improve our ability to maintain health and treat disease, while ameliorating continuously rising healthcare costs. Translation of basic research findings to clinical applications within regulatory compliance is required for personalized medicine to become the new foundation for practice of medicine. Deploying even a few of the thousands of potential diagnostic biomarkers identified each year as part of personalized treatment workflows requires clinically efficient biosensor technologies to monitor multiple biomarkers in patients in real time. This paper discusses a critical component of a regulatory system, a microcavity optical biosensor for label-free monitoring of biomolecular interactions at physiologically-relevant concentrations. While most current biosensor research focuses on improving sensitivity, this paper emphasizes other characteristics a biosensor technology requires to be practical in a clinical setting, presenting robust microcavity biosensors which are easy to manufacture and integrate with microfluidics into flexible and redesignable platforms making the microcavity biosensors deployable for continuous monitoring of biomarkers in body fluids in the clinic,  in dense 2D random arrays for high-throughput applications like drug-library screening in interactomics, and of the secretory behavior of single cells in the laboratory. PMID:23443397

  8. Vertically coupled polymer microresonators for optofluidic label-free biosensors

    NASA Astrophysics Data System (ADS)

    Delezoide, Camille; Lautru, Joseph; Zyss, Joseph; Ledoux-Rak, Isabelle; Nguyen, Chi Thanh

    2012-01-01

    In this paper we report on the design and fabrication of polymeric microracetracks optical resonators for optofluidic label-free biosensing. In the domain of optical integrated devices, polymer materials offer the advantages of low cost, easy fabrication, low scattering loss on waveguide sidewalls, and high coupling efficiency to optical fibres and waveguides. Moreover, for biochemical sensing, polymer surfaces can be easily modified to immobilize a wide choice of target molecules. Polymers are also well compatible with microfluidic circuits, favoring the insertion of photonic circuits into optofluidic cells. The vertical coupling configuration, in which resonators are vertically coupled to the buried bus waveguide, presents several advantages in comparison with the lateral coupling configuration, particularly in the context of optofluidic biosensors. Polymeric microracetracks were fabricated using the SU-8 negative photoresist and the CYTOP fluorinated polymer, using a combination of a simple near UV lithography and reactive ion etching technology. Vertically coupled microracetracks immersed in deionized water display high Q-factors (> 35000) and finesse up to 25. Surface sensing experiments performed with these microresonators using TAMRA-cadaverine as a test molecule, which can be quantified through fluorescence analysis, demonstrated a very low detection limit of 0.22 attogram.

  9. Label-Free Microcavity Biosensors: Steps towards Personalized Medicine

    PubMed Central

    Amarie, Dragos; Glazier, James A.

    2012-01-01

    Personalized medicine has the potential to improve our ability to maintain health and treat disease, while ameliorating continuously rising healthcare costs. Translation of basic research findings to clinical applications within regulatory compliance is required for personalized medicine to become the new foundation for practice of medicine. Deploying even a few of the thousands of potential diagnostic biomarkers identified each year as part of personalized treatment workflows requires clinically efficient biosensor technologies to monitor multiple biomarkers in patients in real time. This paper discusses a critical component of a regulatory system, a microcavity optical biosensor for label-free monitoring of biomolecular interactions at physiologically-relevant concentrations. While most current biosensor research focuses on improving sensitivity, this paper emphasizes other characteristics a biosensor technology requires to be practical in a clinical setting, presenting robust microcavity biosensors which are easy to manufacture and integrate with microfluidics into flexible and redesignable platforms making the microcavity biosensors deployable for continuous monitoring of biomarkers in body fluids in the clinic, in dense 2D random arrays for high-throughput applications like drug-library screening in interactomics, and of the secretory behavior of single cells in the laboratory. PMID:23443397

  10. Label-free imaging of cellular malformation using high resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Zhongjiang; Li, Bingbing; Yang, Sihua

    2014-09-01

    A label-free high resolution photoacoustic microscopy (PAM) system for imaging cellular malformation is presented. The carbon fibers were used to testify the lateral resolution of the PAM. Currently, the lateral resolution is better than 2.7 μm. The human normal red blood cells (RBCs) were used to prove the imaging capability of the system, and a single red blood cell was mapped with high contrast. Moreover, the iron deficiency anemia RBCs were clearly distinguished from the cell morphology by using the PAM. The experimental results demonstrate that the photoacoustic microscopy system can accomplish label-free photoacoustic imaging and that it has clinical potential for use in the detection of erythrocytes and blood vessels malformation.

  11. Label-free photoelectrochemical strategy for hairpin DNA hybridization detection on titanium dioxide electrode

    SciTech Connect

    Lu Wu; Wang Geng; Jin Yan; Yao Xin; Hu Jianqiang; Li Jinghong

    2006-12-25

    A new photoelectrochemical strategy for hairpin DNA hybridization was devised, in which TiO{sub 2} served as the anchor and signal transducer, and no label or redox couples were required. Once the hybridization between hairpin DNA probe and target DNA occurred, the photocurrent would decrease, utilizing which the sequence of the target DNA could be identified. The sequence specificity experiment showed that one or more mismatches of DNA bases could be discriminated. This photoelectrochemical method would be a potential tool in DNA hybridization detection due to its great advantages: label-free, high sensitivity, specific recognition, low cost, and easy fabrication.

  12. Mass Spectrometry-Based Label-Free Quantitative Proteomics

    PubMed Central

    Zhu, Wenhong; Smith, Jeffrey W.; Huang, Chun-Ming

    2010-01-01

    In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies. PMID:19911078

  13. Label and Label-Free Detection Techniques for Protein Microarrays

    PubMed Central

    Syahir, Amir; Usui, Kenji; Tomizaki, Kin-ya; Kajikawa, Kotaro; Mihara, Hisakazu

    2015-01-01

    Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano-biological events.

  14. Label-free all-electronic biosensing in microfluidic systems

    NASA Astrophysics Data System (ADS)

    Stanton, Michael A.

    Label-free, all-electronic detection techniques offer great promise for advancements in medical and biological analysis. Electrical sensing can be used to measure both interfacial and bulk impedance changes in conducting solutions. Electronic sensors produced using standard microfabrication processes are easily integrated into microfluidic systems. Combined with the sensitivity of radiofrequency electrical measurements, this approach offers significant advantages over competing biological sensing methods. Scalable fabrication methods also provide a means of bypassing the prohibitive costs and infrastructure associated with current technologies. We describe the design, development and use of a radiofrequency reflectometer integrated into a microfluidic system towards the specific detection of biologically relevant materials. We developed a detection protocol based on impedimetric changes caused by the binding of antibody/antigen pairs to the sensing region. Here we report the surface chemistry that forms the necessary capture mechanism. Gold-thiol binding was utilized to create an ordered alkane monolayer on the sensor surface. Exposed functional groups target the N-terminus, affixing a protein to the monolayer. The general applicability of this method lends itself to a wide variety of proteins. To demonstrate specificity, commercially available mouse anti- Streptococcus Pneumoniae monoclonal antibody was used to target the full-length recombinant pneumococcal surface protein A, type 2 strain D39 expressed by Streptococcus Pneumoniae. We demonstrate the RF response of the sensor to both the presence of the surface decoration and bound SPn cells in a 1x phosphate buffered saline solution. The combined microfluidic sensor represents a powerful platform for the analysis and detection of cells and biomolecules.

  15. Label-free imaging of thick tissue at 1550 nm using a femtosecond optical parametric generator.

    PubMed

    Trägårdh, Johanna; Robb, Gillian; Gadalla, Kamal K E; Cobb, Stuart; Travis, Christopher; Oppo, Gian-Luca; McConnell, Gail

    2015-08-01

    We have developed a simple wavelength-tunable optical parametric generator (OPG), emitting broadband ultrashort pulses with peak wavelengths at 1530-1790 nm, for nonlinear label-free microscopy. The OPG consists of a periodically poled lithium niobate crystal, pumped at 1064 nm by a ultrafast Yb:fiber laser with high pulse energy. We demonstrate that this OPG can be used for label-free imaging, by third-harmonic generation, of nuclei of brain cells and blood vessels in a >150 μm thick brain tissue section, with very little decay of intensity with imaging depth and no visible damage to the tissue at an incident average power of 15 mW. PMID:26258338

  16. Label-free multi-photon imaging of dysplasia in Barrett’s esophagus

    PubMed Central

    Mehravar, Soroush; Banerjee, Bhaskar; Chatrath, Hemant; Amirsolaimani, Babak; Patel, Krunal; Patel, Charmi; Norwood, Robert A; Peyghambarian, Nasser; Kieu, Khanh

    2015-01-01

    Barrett’s esophagus (BE) is a metaplastic disorder where dysplastic and early cancerous changes are invisible to the naked eye and where the practice of blind biopsy is hampered by large sampling errors. Multi-photon microscopy (MPM) has emerged as an alternative solution for fast and label-free diagnostic capability for identifying the histological features with sub-micron accuracy. We developed a compact, inexpensive MPM system by using a handheld mode-locked fiber laser operating at 1560nm to study mucosal biopsies of BE. The combination of back-scattered THG, back-reflected forward THG and SHG signals generate images of cell nuclei and collagen, leading to label-free diagnosis in Barrett’s. PMID:26819824

  17. Nanostructured plasmonic interferometers for ultrasensitive label-free biosensing

    NASA Astrophysics Data System (ADS)

    Gao, Yongkang

    Optical biosensors that utilize surface plasmon resonance (SPR) technique to analyze the biomolecular interactions have been extensively explored in the last two decades and have become the gold standard for label-free biosensing. These powerful sensing tools allow fast, highly-sensitive monitoring of the interaction between biomolecules in real time, without the need for laborious fluorescent labeling, and have found widely ranging applications from biomedical diagnostics and drug discovery, to environmental sensing and food safety monitoring. However, the prism-coupling SPR geometry is complex and bulky, and has severely limited the integration of this technique into low-cost portable biomedical devices for point-of-care diagnostics and personal healthcare applications. Also, the complex prism-coupling scheme prevents the use of high numerical aperture (NA) optics to increase the spatial resolution for multi-channel, high-throughput detection in SPR imaging mode. This dissertation is focused on the design and fabrication of a promising new class of nanopatterned interferometric SPR sensors that integrate the strengths of miniaturized nanoplasmonic architectures with sensitive optical interferometry techniques to achieve bold advances in SPR biosensing. The nanosensor chips developed provide superior sensing performance comparable to conventional SPR systems, but employing a far simpler collinear optical transmission geometry, which largely facilitates system integration, miniaturization, and low-cost production. Moreover, the fabricated nanostructure-based SPR sensors feature a very small sensor footprint, allowing massive multiplexing on a chip for high-throughput detection. The successful transformation of SPR technique from bulky prism-coupling setup into this low-cost compact plasmonic platform would have a far-reaching impact on point-of-care diagnostic tools and also lead to advances in high-throughput sensing applications in proteomics, immunology, drug

  18. Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

    PubMed

    Oktem, Ozgur; Bildik, Gamze; Senbabaoglu, Filiz; Lack, Nathan A; Akin, Nazli; Yakar, Feridun; Urman, Defne; Guzel, Yilmaz; Balaban, Basak; Iwase, Akira; Urman, Bulent

    2016-04-01

    A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology. PMID:26746109

  19. Label-free optical resonant sensors for biochemical applications

    NASA Astrophysics Data System (ADS)

    Ciminelli, Caterina; Campanella, Clarissa Martina; Dell'Olio, Francesco; Campanella, Carlo Edoardo; Armenise, Mario Nicola

    2013-03-01

    For a number of years, the scientific community has been paying growing attention to the monitoring and enhancement of public health and the quality of life through the detection of all dangerous agents for the human body, including gases, proteins, virus, and bacterial agents. When these agents are detected through label-free biochemical sensors, the molecules are not modified structurally or functionally by adding fluorescent or radioactive dyes. This work focuses on label-free optical ring resonator-based configurations suited for bio-chemical sensing, highlighting their physical aspects and specific applications. Resonant wavelength shift and the modal splitting occurring when the analyte interacts with microresonant structures are the two major physical aspects analyzed in this paper. Competitive optical platforms proposed in the literature are also illustrated together with their properties and performance.

  20. Label-Free Technologies for Quantitative Multiparameter Biological Analysis

    PubMed Central

    Qavi, Abraham J.; Washburn, Adam L.; Byeon, Ji-Yeon; Bailey, Ryan C.

    2009-01-01

    In the post-genomic era, information is king and information-rich technologies are critically important drivers in both fundamental biology and medicine. It is now known that single-parameter measurements provide only limited detail and that quantitation of multiple biomolecular signatures can more fully illuminate complex biological function. Label-free technologies have recently attracted significant interest for sensitive and quantitative multiparameter analysis of biological systems. There are several different classes of label-free sensors that are currently being developed both in academia and in industry. In this critical review, we highlight, compare, and contrast some of the more promising approaches. We will describe the fundamental principles of these different methodologies and discuss advantages and disadvantages that might potentially help one in selecting the appropriate technology for a given bioanalytical application. PMID:19221722

  1. Low cost flatbed scanner label-free biosensor

    NASA Astrophysics Data System (ADS)

    Aygun, Ugur; Avci, Oguzhan; Seymour, Elif; Sevenler, Derin D.; Urey, Hakan; Ünlü, M. Selim; Ozkumur, Ayca Yalcin

    2016-03-01

    In this paper, we demonstrate utilization of a commercial flatbed document scanner as a label-free biosensor for highthroughput imaging of DNA and protein microarrays. We implemented an interferometric sensing technique through use of a silicon/oxide layered substrate, and easy to implement hardware modifications such as re-aligning moving parts and inserting a custom made sample plate. With a cost as low as 100USD, powered by a USB cable, and scan speed of 30 seconds for a 4mm x 4 mm area with ~10μm lateral resolution, the presented system offers a super low cost, easy to use alternative to commercially available label-free systems.

  2. Hybrid label-free multiphoton and optoacoustic microscopy (MPOM)

    NASA Astrophysics Data System (ADS)

    Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

    2015-07-01

    Many biological applications require a simultaneous observation of different anatomical features. However, unless potentially harmful staining of the specimens is employed, individual microscopy techniques do generally not provide multi-contrast capabilities. We present a hybrid microscope integrating optoacoustic microscopy and multiphoton microscopy, including second-harmonic generation, into a single device. This combined multiphoton and optoacoustic microscope (MPOM) offers visualization of a broad range of structures by employing different contrast mechanisms and at the same time enables pure label-free imaging of biological systems. We investigate the relative performance of the two microscopy modalities and demonstrate their multi-contrast abilities through the label-free imaging of a zebrafish larva ex vivo, simultaneously visualizing muscles and pigments. This hybrid microscopy application bears great potential for developmental biology studies, enabling more comprehensive information to be obtained from biological specimens without the necessity of staining.

  3. Label free detection of phospholipids by infrared absorption spectroscopy

    NASA Astrophysics Data System (ADS)

    Ahmed, Tahsin; Foster, Erick; Vigil, Genevieve; Khan, Aamir A.; Bohn, Paul; Howard, Scott S.

    2014-08-01

    We present our study on compact, label-free dissolved lipid sensing by combining capillary electrophoresis separation in a PDMS microfluidic chip online with mid-infrared (MIR) absorption spectroscopy for biomarker detection. On-chip capillary electrophoresis is used to separate the biomarkers without introducing any extrinsic contrast agent, which reduces both cost and complexity. The label free biomarker detection could be done by interrogating separated biomarkers in the channel by MIR absorption spectroscopy. Phospholipids biomarkers of degenerative neurological, kidney, and bone diseases are detectable using this label free technique. These phospholipids exhibit strong absorption resonances in the MIR and are present in biofluids including urine, blood plasma, and cerebrospinal fluid. MIR spectroscopy of a 12-carbon chain phosphatidic acid (PA) (1,2-dilauroyl-snglycero- 3-phosphate (sodium salt)) dissolved in N-methylformamide, exhibits a strong amide peak near wavenumber 1660 cm-1 (wavelength 6 μm), arising from the phosphate headgroup vibrations within a low-loss window of the solvent. PA has a similar structure to many important phospholipids molecules like phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylserine (PS), making it an ideal molecule for initial proof-of-concept studies. This newly proposed detection technique can lead us to minimal sample preparation and is capable of identifying several biomarkers from the same sample simultaneously.

  4. Novel optical approaches for label-free quantification of nano-cytotoxic effects

    NASA Astrophysics Data System (ADS)

    Mues, Sarah; Antunovic, Jan; Ketelhut, Steffi; Kemper, Björn; Schnekenburger, Jürgen

    2016-03-01

    Commonly used cytotoxicity assays to determine the formation of reactive oxygen species, cell viability or cell death are often affected by applied nanomaterials, which lead to false-positive or false-negative results. Thus, novel nanomaterial toxicity testing strategies that allow for high nanomaterial doses to determine Low Effect Levels (LOEL) even of low toxic materials are of high interest. We demonstrate novel approaches to quantify cytotoxic effects with new parameter sets such as cellular refractive index, volume, density and dry mass that are obtained by digital holographic microscopy (DHM). Furthermore, we correlate results obtained from spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with established cell viability and cell death assays. Moreover, in a label-free flow cytometry configuration, cell-nanoparticle-interaction-kinetics were determined by side scatter signal analysis. We demonstrate that silver spheres show a higher cytotoxicity than silver rods and found that this effect correlates with a decrease of the intracellular refractive index and a decreased temporal development of dry mass and cell covered surface area indicating reduced cell viability and increased cell death. Results from side scatter analysis suggest a dose-dependent uptake kinetics of both materials that correlates with cytotoxicity data of the established assays. Taken together, our results demonstrate DHM and flow cytometry as promising novel label-free tools for nanomaterial toxicity and cell particle interaction studies.

  5. Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy

    NASA Astrophysics Data System (ADS)

    Lu, Sijia; Min, Wei; Chong, Shasha; Holtom, Gary R.; Xie, X. Sunney

    2010-03-01

    Heme proteins, such as hemoglobins and cytochromes, play important roles in various biological processes. Here we employ the two-photon excited photothermal effect as a contrast mechanism to map heme proteins distribution. Particularly, both a thermal lens scheme and a high-frequency modulation are utilized to enhance the signal-to-noise ratio. We demonstrate label-free imaging of individual red blood cells, subcellular distribution of cytochromes in live mammalian cells, and the microvascular networks in mouse ear tissue and in a zebrafish gill.

  6. Detection of novel biomarkers for ovarian cancer with an optical nanotechnology detection system enabling label-free diagnostics

    NASA Astrophysics Data System (ADS)

    Kaja, Simon; Hilgenberg, Jill D.; Collins, Julie L.; Shah, Anna A.; Wawro, Debra; Zimmerman, Shelby; Magnusson, Robert; Koulen, Peter

    2012-08-01

    Ovarian carcinoma has the highest lethality rate of gynecologic tumors, largely attributed to the late-stage diagnosis of the disease. Reliable tools for both accurate diagnosis and early detection of disease onset are lacking, and presently less than 20% of ovarian cancers are detected at an early stage. Protein biomarkers that allow the discrimination of early and late stages of ovarian serous carcinomas are urgently needed as they would enable monitoring pre-symptomatic aspects of the disease, disease progression, and the efficacy of intervention therapies. We compare the absolute and relative protein levels of six protein biomarkers for ovarian cancer in five different established ovarian cancer cell lines, utilizing both quantitative immunoblot analysis and a guided-mode resonance (GMR) bioassay detection system that utilizes a label-free optical biosensor readout. The GMR sensor approach provided highly accurate, consistent, and reproducible quantification of protein biomarkers as validated by quantitative immunoblotting, as well as enhanced sensitivity, and is therefore suitable for quantification and detection of novel biomarkers for ovarian cancer. We identified fibronectin, apolipoprotein A1, and TIMP3 as potential protein biomarkers for the differential diagnosis of primary versus metastatic ovarian carcinoma. Future studies are needed to confirm the suitability of protein biomarkers tested herein in patient samples.

  7. Detection of novel biomarkers for ovarian cancer with an optical nanotechnology detection system enabling label-free diagnostics

    PubMed Central

    Kaja, Simon; Hilgenberg, Jill D.; Collins, Julie L.; Shah, Anna A.; Wawro, Debra; Zimmerman, Shelby; Magnusson, Robert

    2012-01-01

    Abstract. Ovarian carcinoma has the highest lethality rate of gynecologic tumors, largely attributed to the late-stage diagnosis of the disease. Reliable tools for both accurate diagnosis and early detection of disease onset are lacking, and presently less than 20% of ovarian cancers are detected at an early stage. Protein biomarkers that allow the discrimination of early and late stages of ovarian serous carcinomas are urgently needed as they would enable monitoring pre-symptomatic aspects of the disease, disease progression, and the efficacy of intervention therapies. We compare the absolute and relative protein levels of six protein biomarkers for ovarian cancer in five different established ovarian cancer cell lines, utilizing both quantitative immunoblot analysis and a guided-mode resonance (GMR) bioassay detection system that utilizes a label-free optical biosensor readout. The GMR sensor approach provided highly accurate, consistent, and reproducible quantification of protein biomarkers as validated by quantitative immunoblotting, as well as enhanced sensitivity, and is therefore suitable for quantification and detection of novel biomarkers for ovarian cancer. We identified fibronectin, apolipoprotein A1, and TIMP3 as potential protein biomarkers for the differential diagnosis of primary versus metastatic ovarian carcinoma. Future studies are needed to confirm the suitability of protein biomarkers tested herein in patient samples. PMID:23224173

  8. Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening.

    PubMed

    Martin, Julio

    2010-09-01

    Some drug targets are not amenable to screening because of the lack of a practical or validated biological assay. Likewise, some screening assays may not be predictive of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e., time, money or personnel) with limited knowledge of the actual tractability of the target. Label-free methodologies, implemented in microtiter plate format, may help address these issues and complement, simplify, or facilitate assays. Label-free biosensors, based on grating resonance or electrical impedance, are versatile platforms for detecting phenotypic changes in both engineered and native cells. Their non-invasive nature allows for the kinetic monitoring of multiple real-time cellular responses to external stimuli, as well as for the use of successive pharmacological challenges. The temporal signature recorded for a particular stimulus is characteristic of the cell type and the signaling pathway activated upon binding of a ligand to its receptor. Cellular label-free technology is an important technical advance in the study of functional pharmacological selectivity. Described in this overview are some of the hurdles encountered in modern drug discovery and the ways in which label-free technologies can be used to overcome these obstacles. PMID:22294376

  9. Label-free fluorescent molecular beacon based on a small fluorescent molecule non-covalently bound to the intentional gap site in the stem moiety.

    PubMed

    Gao, Qiang; Lin, Kai; Zhang, Hongge; Qi, Honglan; Zhang, Chengxiao

    2010-12-15

    A label-free fluorescent molecular beacon (MB) based on a fluorescent molecule, 5,6,7-trimethyl-1,8-naphthyridin-2-ylamine (ATMND) which is non-covalently bound to the intentional gap site in the stem moiety of the label-free MB, was developed. In the absence of a cDNA, ATMND fluorescence is significantly quenched because it binds to the unpaired cytosine at the gap site by hydrogen bonding. As a result, the label-free MB shows almost no fluorescence. Upon hybridization with cDNA, the label-free MB undergoes a conformational change to destroy the gap site. This results in an effective fluorescent enhancement because of the release of the ATMND from the gap site to the solution. Fluorescence titration shows that ATMND strongly binds to the cytosine at the gap site (K(11)>10(6)). Circular-dichroism spectroscopy indicates that the binding of ATMND at the gap site of the stem moiety does not induce a significant conformational change to the hairpin DNA. Under optimal conditions, the fluorescent intensity of the label-free MB increases with an increase in cDNA concentration from 50 nM to 1.5 μM. A detection limit of 20 nM cDNA was achieved. A single mismatched target ss-DNA can be effectively discriminated from cDNA. The advantage of the label-free MB is that both its ends can be left free to introduce other useful functionalities. In addition, the label-free MB synthesis introduced in this paper is relatively simple and inexpensive because no label is required. PMID:21111170

  10. Passivated aluminum nanohole arrays for label-free biosensing applications.

    PubMed

    Canalejas-Tejero, Víctor; Herranz, Sonia; Bellingham, Alyssa; Moreno-Bondi, María Cruz; Barrios, Carlos Angulo

    2014-01-22

    We report the fabrication and performance of a surface plasmon resonance aluminum nanohole array refractometric biosensor. An aluminum surface passivation treatment based on oxygen plasma is developed in order to circumvent the undesired effects of oxidation and corrosion usually found in aluminum-based biosensors. Immersion tests in deionized water and device simulations are used to evaluate the effectiveness of the passivation process. A label-free bioassay based on biotin analysis through biotin-functionalized dextran-lipase conjugates immobilized on the biosensor-passivated surface in aqueous media is performed as a proof of concept to demonstrate the suitability of these nanostructured aluminum films for biosensing. PMID:24354280

  11. Responsive Hydrogels for Label-Free Signal Transduction within Biosensors

    PubMed Central

    Gawel, Kamila; Barriet, David; Sletmoen, Marit; Stokke, Bjørn Torger

    2010-01-01

    Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications. PMID:22399885

  12. Optical birefringence of liquid crystals for label-free optical biosensing diagnosis

    PubMed Central

    Nguyen, Tan Tai; Han, Gyeo-Re; Jang, Chang-Hyun; Ju, Heongkyu

    2015-01-01

    Purpose We present a polarization-sensitive optical detection platform for label-free quantitative optical biosensing diagnosis using liquid crystals (LCs). This is capable of determining quantitatively the optical birefringence of optical cells containing LCs, whose orientation depends on the immobilized biomolecules. Patients and methods This technique uses a polarization-dependent double-port detection without any polarizer at a single wavelength and removes the need of aligning optical cells of LCs in the azimuthal direction, with respect to the light path through the optical cell. Thus, this technique enables a stand-alone detection in a relatively compact format without an additional optical instrument, such as a retardation compensator, a Michael–Levy chart, and a spectrophotometer, in order to determine the optical birefringence quantitatively. Results We demonstrate that bovine serum albumin immobilized on the gold surface of the cell hybrid interfaces that support both homeotropic and planar anchoring of LCs causes optical phase retardation change which can be determined quantitatively. We also provide estimation of the zenithal orientation of LCs near the gold surface of the hybrid interfaces, based on the phase retardation determined. The estimated limit of bovine serum albumin detection is approximately 2.1 μM. Conclusion This optical technique with LCs can serve an optical platform for label-free quantitative diagnosis of proteins in a real time manner. PMID:26347013

  13. A web-server of cell type discrimination system.

    PubMed

    Wang, Anyou; Zhong, Yan; Wang, Yanhua; He, Qianchuan

    2014-01-01

    Discriminating cell types is a daily request for stem cell biologists. However, there is not a user-friendly system available to date for public users to discriminate the common cell types, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and somatic cells (SCs). Here, we develop WCTDS, a web-server of cell type discrimination system, to discriminate the three cell types and their subtypes like fetal versus adult SCs. WCTDS is developed as a top layer application of our recent publication regarding cell type discriminations, which employs DNA-methylation as biomarkers and machine learning models to discriminate cell types. Implemented by Django, Python, R, and Linux shell programming, run under Linux-Apache web server, and communicated through MySQL, WCTDS provides a friendly framework to efficiently receive the user input and to run mathematical models for analyzing data and then to present results to users. This framework is flexible and easy to be expended for other applications. Therefore, WCTDS works as a user-friendly framework to discriminate cell types and subtypes and it can also be expended to detect other cell types like cancer cells. PMID:24578634

  14. A Web-Server of Cell Type Discrimination System

    PubMed Central

    Zhong, Yan

    2014-01-01

    Discriminating cell types is a daily request for stem cell biologists. However, there is not a user-friendly system available to date for public users to discriminate the common cell types, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and somatic cells (SCs). Here, we develop WCTDS, a web-server of cell type discrimination system, to discriminate the three cell types and their subtypes like fetal versus adult SCs. WCTDS is developed as a top layer application of our recent publication regarding cell type discriminations, which employs DNA-methylation as biomarkers and machine learning models to discriminate cell types. Implemented by Django, Python, R, and Linux shell programming, run under Linux-Apache web server, and communicated through MySQL, WCTDS provides a friendly framework to efficiently receive the user input and to run mathematical models for analyzing data and then to present results to users. This framework is flexible and easy to be expended for other applications. Therefore, WCTDS works as a user-friendly framework to discriminate cell types and subtypes and it can also be expended to detect other cell types like cancer cells. PMID:24578634

  15. Antimicrobial susceptibility assays based on the quantification of bacterial lipopolysaccharides via a label free lectin biosensor.

    PubMed

    Ma, Fen; Rehman, Abdul; Sims, Matthew; Zeng, Xiangqun

    2015-04-21

    A label free lectin biosensor developed in our laboratory that can quantitatively measure the binding between the lectin immobilized at the carbohydrate sensor surface and the lipopolysaccharide (LPS) on Gram-negative bacteria was demonstrated for an antibiotic susceptibility assay. The biosensor utilizes a polythiophene interface containing fused quinone moieties glycosylated to form a carbohydrate platform for the immobilization of Concanavalin A (Con A) and is capable of LPS binding measurements via orthogonal quartz crystal microbalance and electrochemical readouts (EQCM). Such orthogonal transduction provides cross-validation, better sensor sensitivity, and a large dynamic range of the measurements. We have applied this label free lectin biosensor for a new antibiotic susceptibility assay by characterizing the antimicrobial activities of various antibiotics (i.e., ciprofloxacin, ceftriaxone, and tetracycline) against Escherichia coli W1485 as a model system. The label free biosensor allows both end point and real time measurements of antibiotic effects on the bacterial cell surface LPS, which is shown to correlate to their antibiotic effects. At the end point, after 18 h incubation of bacterial cells with these three antibiotics respectively, the bacterial LPS binding signal was reduced to 23%, 27%, and 38%, respectively, for the three antibiotics, indicating that ciprofloxacin is the most effective against this E. coli strain. Real time measurements at the 1 h time point showed a similar trend with a reduction of binding to 91%, 93%, and 95%, respectively. From the binding kinetics of these measurements, the relaxation time (τ) was obtained, where higher τ value means slow binding interactions between the lectin and the bacterial LPS. The obtained order of τ, (i.e., τciprofloxacin > τceftriaxone > τtetracycline) again indicated that ciprofloxacin has more bactericidal activity than the other two antibiotics with the same concentrations. Thus, we are

  16. Semiconductor Electronic Label-Free Assay for Predictive Toxicology

    PubMed Central

    Mao, Yufei; Shin, Kyeong-Sik; Wang, Xiang; Ji, Zhaoxia; Meng, Huan; Chui, Chi On

    2016-01-01

    While animal experimentations have spearheaded numerous breakthroughs in biomedicine, they also have spawned many logistical concerns in providing toxicity screening for copious new materials. Their prioritization is premised on performing cellular-level screening in vitro. Among the screening assays, secretomic assay with high sensitivity, analytical throughput, and simplicity is of prime importance. Here, we build on the over 3-decade-long progress on transistor biosensing and develop the holistic assay platform and procedure called semiconductor electronic label-free assay (SELFA). We demonstrate that SELFA, which incorporates an amplifying nanowire field-effect transistor biosensor, is able to offer superior sensitivity, similar selectivity, and shorter turnaround time compared to standard enzyme-linked immunosorbent assay (ELISA). We deploy SELFA secretomics to predict the inflammatory potential of eleven engineered nanomaterials in vitro, and validate the results with confocal microscopy in vitro and confirmatory animal experiment in vivo. This work provides a foundation for high-sensitivity label-free assay utility in predictive toxicology. PMID:27117746

  17. Label-free detection and manipulation of single biological nanoparticles.

    PubMed

    DeSantis, Michael C; Cheng, Wei

    2016-09-01

    In the past several years, there have been significant advances in the field of nanoparticle detection for various biological applications. Of considerable interest are synthetic nanoparticles being designed as potential drug delivery systems as well as naturally occurring or biological nanoparticles, including viruses and extracellular vesicles. Many infectious diseases and several human cancers are attributed to individual virions. Because these particles likely display different degrees of heterogeneity under normal physiological conditions, characterization of these natural nanoparticles with single-particle sensitivity is necessary for elucidating information on their basic structure and function as well as revealing novel targets for therapeutic intervention. Additionally, biodefense and point-of-care clinical testing demand ultrasensitive detection of viral pathogens particularly with high specificity. Consequently, the ability to perform label-free virus sensing has motivated the development of multiple electrical-, mechanical-, and optical-based detection techniques, some of which may even have the potential for nanoparticle sorting and multi-parametric analysis. For each technique, the challenges associated with label-free detection and measurement sensitivity are discussed as are their potential contributions for future real-world applications. WIREs Nanomed Nanobiotechnol 2016, 8:717-729. doi: 10.1002/wnan.1392 For further resources related to this article, please visit the WIREs website. PMID:26846164

  18. Multiplexed label-free optical biosensor for medical diagnostics.

    PubMed

    Bottazzi, Barbara; Fornasari, Lucia; Frangolho, Ana; Giudicatti, Silvia; Mantovani, Alberto; Marabelli, Franco; Marchesini, Gerardo; Pellacani, Paola; Therisod, Rita; Valsesia, Andrea

    2014-01-01

    This paper describes a new multiplexed label-free biosensor. The detection technology is based on nanostructured gold-polymer surfaces. These surfaces support surface plasmon resonance modes that can be probed by a miniaturized optical setup. The optical characterization of the sensing chip shows the sensitivity and the limit-of-detection to refractive index changes. Moreover, by studying the progressive adhesion of molecular monolayers of polyelectrolytes, the decay of the plasmonic mode electric field above the surface has been reconstructed. A multiplexed label-free biosensing device is then described and characterized in terms of sensitivity, lateral resolution, and sensitivity to a model biological assay. The sensitivity in imaging mode of the device is of the order of 10-6 refractive index units, while the measured lateral resolution is 6.25 μm within a field of view of several tenths of mm2, making the instrument unique in terms of multiplexing capability. Finally, the proof-of-concept application of the technology as a point-of-care diagnostic tool for an inflammatory marker is demonstrated. PMID:24474511

  19. Label-free selection of RNA aptamers for metabolic engineering.

    PubMed

    Hwang, Chuhern; Carothers, James M

    2016-08-15

    RNA aptamers can be assembled into genetic regulatory devices that sense and respond to levels of specific cellular metabolites and thus serve an integral part of designing dynamic control into engineered metabolic pathways. Here, we describe a practical method for generating specific and high affinity aptamers to enable the wider use of in vitro selection and a broader application of aptamers for metabolic engineering. Conventional selection methods involving either radioactive labeling of RNA or the use of label-free methods such as SPR to track aptamer enrichment require resources that are not widely accessible to research groups. We present a label-free selection method that uses small volume spectrophotometers to track RNA enrichment paired with previously characterized affinity chromatography methods. Borrowing techniques used in solid phase peptide synthesis, we present an approach for immobilizing a wide range of metabolites to an amino PEGA matrix. As an illustration, we detail laboratory techniques employed to generate aptamers that bind p-aminophenylalanine, a metabolic precursor for bio-based production of plastics and the pristinamycin family of antibiotics. We focused on the development of methods for ligand immobilization, selection via affinity chromatography, and nucleic acid quantification that can be performed with common laboratory equipment. PMID:27339940

  20. An Overview of Label-free Electrochemical Protein Sensors

    PubMed Central

    Vestergaard, Mun'delanji; Kerman, Kagan; Tamiya, Eiichi

    2007-01-01

    Electrochemical-based protein sensors offer sensitivity, selectivity and reliability at a low cost, making them very attractive tools for protein detection. Although the sensors use a broad range of different chemistries, they all depend on the solid electrode surface, interactions with the target protein and the molecular recognition layer. Traditionally, redox enzymes have provided the molecular recognition elements from which target proteins have interacted with. This necessitates that the redox-active enzymes couple with electrode surfaces and usually requires the participation of added diffusional components, or assembly of the enzymes in functional chemical matrices. These complications, among many others, have seen a trend towards non-enzymatic-based electrochemical protein sensors. Several electrochemical detection approaches have been exploited. Basically, these have fallen into two categories: labeled and label-free detection systems. The former rely on a redox-active signal from a reporter molecule or a label, which changes upon the interaction of the target protein. In this review, we discuss the label-free electrochemical detection of proteins, paying particular emphasis to those that exploit intrinsic redox-active amino acids.

  1. Semiconductor Electronic Label-Free Assay for Predictive Toxicology.

    PubMed

    Mao, Yufei; Shin, Kyeong-Sik; Wang, Xiang; Ji, Zhaoxia; Meng, Huan; Chui, Chi On

    2016-01-01

    While animal experimentations have spearheaded numerous breakthroughs in biomedicine, they also have spawned many logistical concerns in providing toxicity screening for copious new materials. Their prioritization is premised on performing cellular-level screening in vitro. Among the screening assays, secretomic assay with high sensitivity, analytical throughput, and simplicity is of prime importance. Here, we build on the over 3-decade-long progress on transistor biosensing and develop the holistic assay platform and procedure called semiconductor electronic label-free assay (SELFA). We demonstrate that SELFA, which incorporates an amplifying nanowire field-effect transistor biosensor, is able to offer superior sensitivity, similar selectivity, and shorter turnaround time compared to standard enzyme-linked immunosorbent assay (ELISA). We deploy SELFA secretomics to predict the inflammatory potential of eleven engineered nanomaterials in vitro, and validate the results with confocal microscopy in vitro and confirmatory animal experiment in vivo. This work provides a foundation for high-sensitivity label-free assay utility in predictive toxicology. PMID:27117746

  2. Label-Free Electrical Detection of Enzymatic Reactions in Nanochannels.

    PubMed

    Duan, Chuanhua; Alibakhshi, Mohammad Amin; Kim, Dong-Kwon; Brown, Christopher M; Craik, Charles S; Majumdar, Arun

    2016-08-23

    We report label-free electrical detection of enzymatic reactions using 2-D nanofluidic channels and investigate reaction kinetics of enzymatic reactions on immobilized substrates in nanoscale-confined spaces. Trypsin proteolysis is chosen for demonstration of the detection scheme. When trypsin cleaves poly-l-lysine coated on the surface of silica nanochannels, the resulting change of surface charge density can be detected by monitoring the ionic conductance of the nanochannels. Our results show that detection of such surface enzymatic reactions is faster than detection of surface binding reactions in nanochannels for low-concentration analytes. Furthermore, the nanochannel sensor has a sensitivity down to 5 ng/mL, which statistically corresponds to a single enzyme per nanochannel. Our results also suggest that enzyme kinetics in nanochannels is fundamentally different from that in bulk solutions or plain surfaces. Such enzymatic reactions form two clear self-propagating reaction fronts inside the nanochannels, and the reaction fronts follow square-root time dependences at high enzyme concentrations due to significant nonspecific adsorption. However, at low enzyme concentrations when nonspecific adsorption is negligible, the reaction fronts propagate linearly with time, and the corresponding propagation speed is related to the channel geometry, enzyme concentration, catalytic reaction constant, diffusion coefficient, and substrate surface density. Optimization of this nanochannel sensor could lead to a quick-response, highly sensitive, and label-free sensor for enzyme assay and kinetic studies. PMID:27472431

  3. Hyperspectral backscatter imaging: a label-free approach to cytogenetics.

    PubMed

    Rebner, Karsten; Ostertag, Edwin; Kessler, Rudolf W

    2016-08-01

    Current techniques for chromosome analysis need to be improved for rapid, economical identification of complex chromosomal defects by sensitive and selective visualisation. In this paper, we present a straightforward method for characterising unstained human metaphase chromosomes. Backscatter imaging in a dark-field setup combined with visible and short near-infrared spectroscopy is used to monitor morphological differences in the distribution of the chromosomal fine structure in human metaphase chromosomes. The reasons for the scattering centres in the fine structure are explained. Changes in the scattering centres during preparation of the metaphases are discussed. FDTD simulations are presented to substantiate the experimental findings. We show that local scattering features consisting of underlying spectral modulations of higher frequencies associated with a high variety of densely packed chromatin can be represented by their scatter profiles even on a sub-microscopic level. The result is independent of the chromosome preparation and structure size. This analytical method constitutes a rapid, cost-effective and label-free cytogenetic technique which can be used in a standard light microscope. Graphical abstract Hyperspectral backscatter imaging for label-free characterization. PMID:27277813

  4. Label-free immunodetection with CMOS-compatible semiconducting nanowires.

    PubMed

    Stern, Eric; Klemic, James F; Routenberg, David A; Wyrembak, Pauline N; Turner-Evans, Daniel B; Hamilton, Andrew D; LaVan, David A; Fahmy, Tarek M; Reed, Mark A

    2007-02-01

    Semiconducting nanowires have the potential to function as highly sensitive and selective sensors for the label-free detection of low concentrations of pathogenic microorganisms. Successful solution-phase nanowire sensing has been demonstrated for ions, small molecules, proteins, DNA and viruses; however, 'bottom-up' nanowires (or similarly configured carbon nanotubes) used for these demonstrations require hybrid fabrication schemes, which result in severe integration issues that have hindered widespread application. Alternative 'top-down' fabrication methods of nanowire-like devices produce disappointing performance because of process-induced material and device degradation. Here we report an approach that uses complementary metal oxide semiconductor (CMOS) field effect transistor compatible technology and hence demonstrate the specific label-free detection of below 100 femtomolar concentrations of antibodies as well as real-time monitoring of the cellular immune response. This approach eliminates the need for hybrid methods and enables system-scale integration of these sensors with signal processing and information systems. Additionally, the ability to monitor antibody binding and sense the cellular immune response in real time with readily available technology should facilitate widespread diagnostic applications. PMID:17268465

  5. Label-free imaging of adipogenesis by coherent anti-stokes Raman scattering microscopy.

    PubMed

    Isomäki, Antti; Sillat, Tarvo; Ainola, Mari; Liljeström, Mikko; Konttinen, Yrjö T; Hukkanen, Mika

    2014-01-01

    Label-free imaging technologies to monitor the events associated with early, intermediate and late adipogenic differentiation in multipotent mesenchymal stromal cells (MSCs) offer an attractive and convenient alternative to conventional fixative based lipid dyes such as Oil Red O and Sudan Red, fluorescent labels such as LipidTOX, and more indirect methods such as qRT-PCR analyses of specific adipocyte differentiation markers such as peroxisome PPARγ and LPL. Coherent anti-Stokes Raman scattering (CARS) microscopy of live cells is a sensitive and fast imaging method enabling evaluation of the adipogenic differentiation with chemical specificity. CARS microscopy is based on imaging structures of interest by displaying the characteristic intrinsic vibrational contrast of chemical bonds. The method is nontoxic, non-destructive, and minimally invasive, thus presenting a promising method for longitudinal analyses of live cells and tissues. CARS provides a coherently emitted signal that is much stronger than the spontaneous Raman scattering. The anti-Stokes signal is blue shifted from the incident wavelength, thus reducing the non-vibrational background present in most biological materials. In this chapter, we aim to provide a detailed approach on how to induce adipogenic differentiation in MSC cultures, and present our methods related to label-free CARS imaging of the events associated with the adipogenesis. PMID:24706284

  6. Electrochemical lectin based biosensors as a label-free tool in glycomics

    PubMed Central

    Bertók, Tomáš; Katrlík, Jaroslav; Gemeiner, Peter; Tkac, Jan

    2016-01-01

    Glycans and other saccharide moieties attached to proteins and lipids, or present on the surface of a cell, are actively involved in numerous physiological or pathological processes. Their structural flexibility (that is based on the formation of various kinds of linkages between saccharides) is making glycans superb “identity cards”. In fact, glycans can form more “words” or “codes” (i.e., unique sequences) from the same number of “letters” (building blocks) than DNA or proteins. Glycans are physicochemically similar and it is not a trivial task to identify their sequence, or - even more challenging - to link a given glycan to a particular physiological or pathological process. Lectins can recognise differences in glycan compositions even in their bound state and therefore are most useful tools in the task to decipher the “glycocode”. Thus, lectin-based biosensors working in a label-free mode can effectively complement the current weaponry of analytical tools in glycomics. This review gives an introduction into the area of glycomics and then focuses on the design, analytical performance, and practical utility of lectin-based electrochemical label-free biosensors for the detection of isolated glycoproteins or intact cells. PMID:27239071

  7. Label-free oxygen-metabolic photoacoustic microscopy in vivo

    PubMed Central

    Yao, Junjie; Maslov, Konstantin I.; Zhang, Yu; Xia, Younan; Wang, Lihong V.

    2011-01-01

    Almost all diseases, especially cancer and diabetes, manifest abnormal oxygen metabolism. Accurately measuring the metabolic rate of oxygen (MRO2) can be helpful for fundamental pathophysiological studies, and even early diagnosis and treatment of disease. Current techniques either lack high resolution or rely on exogenous contrast. Here, we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution to noninvasively quantify MRO2in vivo in absolute units. mPAM is the unique modality for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed. Hyperthermia, cryotherapy, melanoma, and glioblastoma were longitudinally imaged in vivo. Counterintuitively, increased MRO2 does not necessarily cause hypoxia or increase oxygen extraction. In fact, early-stage cancer was found to be hyperoxic despite hypermetabolism. PMID:21806264

  8. CEST theranostics: label-free MR imaging of anticancer drugs

    PubMed Central

    Xu, Jiadi; Yadav, Nirbhay N.; Chan, Kannie W. Y.; Luo, Liangping; McMahon, Michael T.; Vogelstein, Bert; van Zijl, Peter C.M.; Zhou, Shibin; Liu, Guanshu

    2016-01-01

    Image-guided drug delivery is of great clinical interest. Here, we explored a direct way, namely CEST theranostics, to detect diamagnetic anticancer drugs simply through their inherent Chemical Exchange Saturation Transfer (CEST) MRI signal, and demonstrated its application in image-guided drug delivery of nanoparticulate chemotherapeutics. We first screened 22 chemotherapeutic agents and characterized the CEST properties of representative agents and natural analogs in three major categories, i.e., pyrimidine analogs, purine analogs, and antifolates, with respect to chemical structures. Utilizing the inherent CEST MRI signal of gemcitabine, a widely used anticancer drug, the tumor uptake of the i.v.-injected, drug-loaded liposomes was successfully detected in CT26 mouse tumors. Such label-free CEST MRI theranostics provides a new imaging means, potentially with an immediate clinical impact, to monitor the drug delivery in cancer. PMID:26837220

  9. Raman spectroscopy for label-free identification of calciphylaxis.

    PubMed

    Lloyd, William R; Agarwal, Shailesh; Nigwekar, Sagar U; Esmonde-White, Karen; Loder, Shawn; Fagan, Shawn; Goverman, Jeremy; Olsen, Bjorn R; Jumlongras, Dolrudee; Morris, Michael D; Levi, Benjamin

    2015-08-01

    Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, point-of-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment. PMID:26263412

  10. Plasmonic biosensor for label-free G-quadruplexes detection

    NASA Astrophysics Data System (ADS)

    Qiu, Suyan; Zhao, Fusheng; Santos, Greggy M.; Shih, Wei-Chuan

    2016-03-01

    G-quadruplex, readily formed by the G-rich sequence, potentially distributes in over 40 % of all human genes, such as the telomeric DNA with the G-rich sequence found at the end of the chromosome. The G-quadruplex structure is supposed to possess a diverse set of critical functions in the mammalian genome for transcriptional regulation, DNA replication and genome stability. However, most of the currently available methods for G-quadruplex identification are restricted to fluorescence techniques susceptible to poor sensitivity. It is essential to propose methods with higher sensitivity to specifically recognize the G-quadruplexes. In this study, we demonstrate a label-free plasmonic biosensor for G-quadruplex detection by relying on the advantages of nanoporous gold (NPG) disks that provide high-density plasmonic hot spots, suitable for molecular recognition capability without the requirement for labeling processes.

  11. Raman spectroscopy for label-free identification of calciphylaxis

    PubMed Central

    Lloyd, William R.; Agarwal, Shailesh; Nigwekar, Sagar U.; Esmonde-White, Karen; Loder, Shawn; Fagan, Shawn; Goverman, Jeremy; Olsen, Bjorn R.; Jumlongras, Dolrudee; Morris, Michael D.; Levi, Benjamin

    2015-01-01

    Abstract. Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, point-of-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment. PMID:26263412

  12. Recognition as a challenging label-free optical sensing system

    NASA Astrophysics Data System (ADS)

    Gauglitz, Günter

    2013-05-01

    Optical biosensors are increasingly used in application areas of environmental analysis, healthcare and food safety. The quality of the biosensor's results depends on the interaction layer, the detection principles, and evaluation strategies, not only on the biopolymer layer but also especially on recognition elements. Using label-free optical sensing, non-specific interaction between sample and transducer has to be reduced, and the selectivity of recognition elements has to be improved. For this reason, strategies to avoid non-specific interaction even in blood and milk are discussed, a variety of upcoming recognition is given. Based on the classification of direct optical detection methods, some examples for the above mentioned applications are reviewed. Trends as well as advantages of parallel multisport detection for kinetic evaluation are also part of the lecture.

  13. Hybrid Integrated Label-Free Chemical and Biological Sensors

    PubMed Central

    Mehrabani, Simin; Maker, Ashley J.; Armani, Andrea M.

    2014-01-01

    Label-free sensors based on electrical, mechanical and optical transduction methods have potential applications in numerous areas of society, ranging from healthcare to environmental monitoring. Initial research in the field focused on the development and optimization of various sensor platforms fabricated from a single material system, such as fiber-based optical sensors and silicon nanowire-based electrical sensors. However, more recent research efforts have explored designing sensors fabricated from multiple materials. For example, synthetic materials and/or biomaterials can also be added to the sensor to improve its response toward analytes of interest. By leveraging the properties of the different material systems, these hybrid sensing devices can have significantly improved performance over their single-material counterparts (better sensitivity, specificity, signal to noise, and/or detection limits). This review will briefly discuss some of the methods for creating these multi-material sensor platforms and the advances enabled by this design approach. PMID:24675757

  14. Label-free oxygen-metabolic photoacoustic microscopy in vivo

    NASA Astrophysics Data System (ADS)

    Yao, Junjie; Maslov, Konstantin I.; Zhang, Yu; Xia, Younan; Wang, Lihong V.

    2011-07-01

    Almost all diseases, especially cancer and diabetes, manifest abnormal oxygen metabolism. Accurately measuring the metabolic rate of oxygen (MRO2) can be helpful for fundamental pathophysiological studies, and even early diagnosis and treatment of disease. Current techniques either lack high resolution or rely on exogenous contrast. Here, we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution to noninvasively quantify MRO2 in vivo in absolute units. mPAM is the unique modality for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed. Hyperthermia, cryotherapy, melanoma, and glioblastoma were longitudinally imaged in vivo. Counterintuitively, increased MRO2 does not necessarily cause hypoxia or increase oxygen extraction. In fact, early-stage cancer was found to be hyperoxic despite hypermetabolism.

  15. Nanodroplet chemical microarrays and label-free assays.

    PubMed

    Gosalia, Dhaval; Diamond, Scott L

    2010-01-01

    The microarraying of chemicals or biomolecules on a glass surface allows for dense storage and miniaturized screening experiments and can be deployed in chemical-biology research or drug discovery. Microarraying allows the production of scores of replicate slides. Small molecule libraries are typically stored as 10 mM DMSO stock solutions, whereas libraries of biomolecules are typically stored in high percentages of glycerol. Thus, a method is required to print such libraries on microarrays, and then assay them against biological targets. By printing either small molecule libraries or biomolecule libraries in an aqueous solvent containing glycerol, each adherent nanodroplet remains fixed at a position on the microarray by surface tension without the use of wells, without evaporating, and without the need for chemically linking the compound to the surface. Importantly, glycerol is a high boiling point solvent that is fully miscible with DMSO and water and has the additional property of stabilizing various enzymes. The nanoliter volume of the droplet forms the reaction compartment once additional reagents are metered onto the microarray, either by aerosol spray deposition or by addressable acoustic dispensing. Incubation of the nanodroplet microarray in a high humidity environment controls the final water content of the reaction. This platform has been validated for fluorescent HTS assays of protease and kinases as well as for fluorogenic substrate profiling of proteases. Label-free HTS is also possible by running nanoliter HTS reactions on a MALDI target for mass spectrometry (MS) analysis without the need for desalting of the samples. A method is described for running nanoliter-scale multicomponent homogeneous reactions followed by label-free MALDI MS spectrometry analysis of the reactions. PMID:20857358

  16. Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis.

    PubMed

    Dai, Peng; Wang, Qin; Wang, Weihua; Jing, Ruirui; Wang, Wei; Wang, Fengqin; Azadzoi, Kazem M; Yang, Jing-Hua; Yan, Zhen

    2016-01-01

    Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients' tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis. PMID:26805816

  17. Label-Free Determination of Hemodynamic Parameters in the Microcirculaton with Third Harmonic Generation Microscopy

    PubMed Central

    Dietzel, Steffen; Pircher, Joachim; Nekolla, A. Katharina; Gull, Mazhar; Brändli, André W.; Pohl, Ulrich; Rehberg, Markus

    2014-01-01

    Determination of blood flow velocity and related hemodynamic parameters is an important aspect of physiological studies which in many settings requires fluorescent labeling. Here we show that Third Harmonic Generation (THG) microscopy is a suitable tool for label-free intravital investigations of the microcirculation in widely-used physiological model systems. THG microscopy is a non-fluorescent multi-photon scanning technique combining the advantages of label-free imaging with restriction of signal generation to a focal spot. Blood flow was visualized and its velocity was measured in adult mouse cremaster muscle vessels, non-invasively in mouse ear vessels and in Xenopus tadpoles. In arterioles, THG line scanning allowed determination of the flow pulse velocity curve and hence the heart rate. By relocating the scan line we obtained velocity profiles through vessel diameters, allowing shear rate calculations. The cell free layer containing the glycocalyx was also visualized. Comparison of the current microscopic resolution with theoretical, diffraction limited resolution let us conclude that an about sixty-fold THG signal intensity increase may be possible with future improved optics, optimized for 1200–1300 nm excitation. THG microscopy is compatible with simultaneous two-photon excited fluorescence detection. It thus also provides the opportunity to determine important hemodynamic parameters in parallel to common fluorescent observations without additional label. PMID:24933027

  18. Label-free in vivo imaging of human leukocytes using two-photon excited endogenous fluorescence

    NASA Astrophysics Data System (ADS)

    Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

    2013-04-01

    We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

  19. Automated, Reproducible, Titania-Based Phosphopeptide Enrichment Strategy for Label-Free Quantitative Phosphoproteomics

    PubMed Central

    Richardson, Brenna McJury; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur

    2013-01-01

    An automated phosphopeptide enrichment strategy is described using titanium dioxide (TiO2)-packed, fused silica capillaries for use with liquid chromatography (LC)-mass spectrometry (MS)/MS-based, label-free proteomics workflows. To correlate an optimum peptide:TiO2 loading ratio between different particle types, the ratio of phenyl phosphate-binding capacities was used. The optimum loading for the column was then verified through replicate enrichments of a range of quantities of digested rat brain tissue cell lysate. Fractions were taken during sample loading, multiple wash steps, and the elution steps and analyzed by LC-MS/MS to gauge the efficiency and reproducibility of the enrichment. Greater than 96% of the total phosphopeptides were detected in the elution fractions, indicating efficient trapping of the phosphopeptides on the first pass of enrichment. The quantitative reproducibility of the automated setup was also improved greatly with phosphopeptide intensities from replicate enrichments exhibiting a median coefficient of variation (CV) of 5.8%, and 80% of the identified phosphopeptides had CVs below 11.1%, while maintaining >85% specificity. By providing this high degree of analytical reproducibility, this method allows for label-free phosphoproteomics over large sample sets with complex experimental designs (multiple biological conditions, multiple biological replicates, multiple time-points, etc.), including large-scale clinical cohorts. PMID:23542237

  20. Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Kunstar, Aliz; Leijten, Jeroen; van Leuveren, Stefan; Hilderink, Janneke; Otto, Cees; van Blitterswijk, Clemens A.; Karperien, Marcel; van Apeldoorn, Aart A.

    2012-11-01

    Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

  1. Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance1[W

    PubMed Central

    Nikolovski, Nino; Shliaha, Pavel V.; Gatto, Laurent; Dupree, Paul; Lilley, Kathryn S.

    2014-01-01

    The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MSE [or HDMSE]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle. PMID:25122472

  2. Doppler Fourier Domain Optical Coherence Tomography for Label-Free Tissue Angiography

    NASA Astrophysics Data System (ADS)

    Leitgeb, Rainer A.; Szkulmowski, Maciej; Blatter, Cedric; Wojtkowski, Maciej

    Information about tissue perfusion and the vascular structure is certainly most important for assessment of tissue state or personal health and the diagnosis of any pathological conditions. It is therefore of key medical interest to have tools available for both quantitative blood flow assessment as well as qualitative vascular imaging. The strength of optical techniques is the unprecedented level of detail even for small capillary structures or microaneurysms and the possibility to combine different techniques for additional tissue spectroscopy giving insight into tissue metabolism. There is an immediate diagnostic and pharmacological demand for high-resolution, label-free, tissue angiography and flow assessment that in addition allow for precise depth gating of flow information. The most promising candidate is Doppler optical coherence tomography (DOCT) being noncontact, label free, and without employing hazardous radiation. DOCT provides fully quantitative volumetric information about blood flow together with the vascular and structural anatomy. Besides flow quantification, analysis of OCT signal fluctuations allows to contrast moving scatterers in tissue such as red blood cells from static tissue. This allows for non-invasive optical angiography and yields high resolution even for smallest capillaries. Because of the huge potential of DOCT and lable-free optical angiography for diagnosis, the last years saw a rapid increase of publications in this field with many different approaches. The present chapter gives an overview over existing Doppler OCT approaches and angiography techniques. It furthermore discusses limitations and noise issues, and gives examples for angiography in the eye and the skin.

  3. Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis

    PubMed Central

    Dai, Peng; Wang, Qin; Wang, Weihua; Jing, Ruirui; Wang, Wei; Wang, Fengqin; Azadzoi, Kazem M.; Yang, Jing-Hua; Yan, Zhen

    2016-01-01

    Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients’ tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis. PMID:26805816

  4. Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers

    PubMed Central

    Yamamoto, Kazuki; Chikaoka, Yoko; Hayashi, Gosuke; Sakamoto, Ryosuke; Yamamoto, Ryuji; Sugiyama, Akira; Kodama, Tatsuhiko; Okamoto, Akimitsu; Kawamura, Takeshi

    2015-01-01

    Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides. PMID:26819910

  5. Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis

    PubMed Central

    Latosinska, Agnieszka; Vougas, Konstantinos; Makridakis, Manousos; Klein, Julie; Mullen, William; Abbas, Mahmoud; Stravodimos, Konstantinos; Katafigiotis, Ioannis; Merseburger, Axel S.; Zoidakis, Jerome; Mischak, Harald; Vlahou, Antonia; Jankowski, Vera

    2015-01-01

    High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression

  6. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor

    PubMed Central

    Im, Hyungsoon; Shao, Huilin; Park, Yong Il; Peterson, Vanessa M.; Castro, Cesar M.; Weissleder, Ralph; Lee, Hakho

    2015-01-01

    Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analyses of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics. PMID:24752081

  7. Label-free biosensor based on long period grating

    NASA Astrophysics Data System (ADS)

    Baldini, Francesco; Chiavaioli, Francesco; Giannetti, Ambra; Brenci, Massimo; Trono, Cosimo

    2013-03-01

    Long period gratings have been recently proposed as label-free optical devices for biochemical sensing. A biochemical interaction along the grating region changes the biolayer refractive index and a change in the fiber transmission spectrum occurs. The fiber biofunctionalization was performed with a novel chemistry using Eudragit L100 copolymer as opposed to the commonly-used silanization procedure. An IgG/anti-IgG bioassay was carried out for studying the antigen/antibody interaction. The biosensor was fully characterized, monitoring the kinetics during the antibody immobilization and achieving the calibration curve of the assay. To compare the biosensor performance, two LPG-based biosensors with distinct grating periods were characterized following the same bioassay protocol. Experimental results demonstrated an enhancement of the biosensor performance when the fundamental core mode of a single-mode fiber couples with a higher order cladding mode. Considering an LPG manufactured on a bare optical fiber, in which the coupling occurs with the 7-th cladding mode, a dynamic signal range of 0.33 nm, a working range of 1.7 - 1450 mg L-1 and a LOD of 500 μg L-1 were achieved

  8. Part per trillion label-free electronic bioanalytical detection.

    PubMed

    Magliulo, Maria; Mallardi, Antonia; Gristina, Roberto; Ridi, Francesca; Sabbatini, Luigia; Cioffi, Nicola; Palazzo, Gerardo; Torsi, Luisa

    2013-04-16

    A Functional Bio-Interlayer Organic Field-Effect Transistor (FBI-OFET) sensor, embedding a streptavidin protein capturing layer, capable of performing label-free selective electronic detection of biotin at 3 part per trillion (mass fraction) or 15 pM, is proposed here. The response shows a logarithmic dependence spanning over 5 orders of magnitude of analyte concentration. The optimization of the FBI analytical performances is achieved by depositing the capturing layer through a controllable Layer-by-Layer (LbL) assembly, while an easy processable spin-coating deposition is proposed for potential low-cost production of equally highly performing sensors. Furthermore, a Langmuirian adsorption based model allows rationalizing the analyte binding process to the capturing layer. The FBI-OFET device is shown to operate also with an antibody interlayer as well as with an ad hoc designed microfluidic system. These occurrences, along with the proven extremely high sensitivity and selectivity, open to FBI-OFETs consideration as disposable electronic strip-tests for assays in biological fluids requiring very low detection limits. PMID:23323705

  9. Interpretation of interference signals in label free integrated interferometric biosensors

    NASA Astrophysics Data System (ADS)

    Heikkinen, Hanna; Wang, Meng; Okkonen, Matti; Hast, Jukka; Myllylä, Risto

    2006-02-01

    In the future fast, simple and reliable biosensors will be needed to detect various analytes from different biosamples. This is due to fact that the needs of traditional health care are changing. In the future homecare of patients and peoples' responsibility for their own health will increase. Also, different wellness applications need new parameters to be analysed, reducing costs of traditional health care, which are increasing rapidly. One fascinating and promising sensor type for these applications is an integrated optical interferometric immunosensor, which is manufactured using organic materials. The use of organic materials opens up enormous possibilities to develop different biochemical functions. In label free biosensors the measurement is based on detecting changes in refractive index, which typically are in the range of 10 -6-10 -8 [1]. In this research, theoretically generated interferograms are used to compare various signal processing methods. The goal is to develop an efficient method to analyse the interferogram. Different time domain signal processing methods are studied to determine the measuring resolution and efficiency of these methods. A low cost CCD -element is used in detecting the interferogram dynamics. It was found that in most of the signal processing methods the measuring resolution was mainly limited by pixel size. With calculation of Pearson's correlation coefficient, subpixel resolution was achieved which means that nanometer range optical path differences can be measured. This results in the refractive index resolution of the order of 10 -7.

  10. Label-free structural photoacoustic tomography of intact mouse brain

    NASA Astrophysics Data System (ADS)

    Li, Lei; Xia, Jun; Li, Guo; Garcia-Uribe, Alejandro; Wang, Lihong V.

    2015-03-01

    Capitalizing on endogenous hemoglobin contrast, photoacoustic computed tomography (PACT), a deep-tissue highresolution imaging modality, has drawn increasing interest in neuro-imaging. However, most existing studies are limited to functional imaging on the cortical surface, and the deep-brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep-brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can provide high-resolution label-free structural imaging through the entire mouse brain. With 100 μm in-plane resolution, we can clearly identify major structures of the brain, and the image quality is comparable to that of magnetic resonance microscopy. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome and lipid. The feasibility of imaging the structure of the brain in vivo has also been discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.

  11. Label-Free Pyrophosphate Recognition with Functionalized Asymmetric Nanopores.

    PubMed

    Ali, Mubarak; Ahmed, Ishtiaq; Ramirez, Patricio; Nasir, Saima; Niemeyer, Christof M; Mafe, Salvador; Ensinger, Wolfgang

    2016-04-01

    The label-free detection of pyrophosphate (PPi) anions with a nanofluidic sensing device based on asymmetric nanopores is demonstrated. The pore surface is functionalized with zinc complexes based on two di(2-picolyl)amine [bis(DPA)] moieties using carbodiimide coupling chemistry. The complexation of zinc (Zn(2+) ) ion is achieved by exposing the modified pore to a solution of zinc chloride to form bis(Zn(2+) -DPA) complexes. The chemical functionalization is demonstrated by recording the changes in the observed current-voltage (I-V) curves before and after pore modification. The bis(Zn(2+) -DPA) complexes on the pore walls serve as recognition sites for pyrophosphate anion. The experimental results show that the proposed nanofluidic sensor has the ability to sense picomolar concentrations of PPi anion in the surrounding environment. On the contrary, it does not respond to other phosphate anions, including monohydrogen phosphate, dihydrogen phosphate, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate. The experimental results are described theoretically by using a model based on the Poisson-Nernst-Planck equations. PMID:26939057

  12. Label free electrochemical aptasensor for ultrasensitive detection of ractopamine.

    PubMed

    Yang, Fei; Wang, Peilong; Wang, Ruiguo; Zhou, Ying; Su, Xiaoou; He, Yujian; Shi, Lei; Yao, Dongsheng

    2016-03-15

    A label free electrochemical (EC) aptasensor for ultrasensitive detection of ractopamine (RAC) was developed. A special immobilization media consisting of gold nanoparticles/poly dimethyl diallyl ammonium chloride-graphene composite (AuNPs/PDDA-GN) was utilized to improve conductivity and performance of the biosensor. The RAC aptamer was attached on AuNPs of the composite membrane via Au-S bond. The fabrication process of the EC aptasensor was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The peak currents obtained by differential pulse voltammetry decreased linearly with the increasing of RAC concentrations and the sensor responds approximately logarithmically over a wide dynamic range of RAC concentration from 1.0 × 10(-12)mol/L to 1.0 × 10(-8)mol/L. The linear correlation coefficient of the developed aptasensor was 0.998, the limit of detection was 5.0 × 10(-13)mol/L. The proposed EC aptasensor displayed good stability, reproducibility and robust operation in animal urine. Particularly, the generality of the fabrication approach of electrochemical aptasensor is highlighted with a further example for illegal drugs detection via the aptamer identification. PMID:26433067

  13. Bioplasmonic calligraphy for multiplexed label-free biodetection.

    PubMed

    Tian, Limei; Tadepalli, Sirimuvva; Park, Sang Hyun; Liu, Keng-Ku; Morrissey, Jeremiah J; Kharasch, Evan D; Naik, Rajesh R; Singamaneni, Srikanth

    2014-09-15

    Printable multi-marker biochips that enable simultaneous quantitative detection of multiple target biomarkers in point-of-care and resource-limited settings are a holy grail in the field of biodiagnostics. However, preserving the functionality of biomolecules, which are routinely employed as recognition elements, during conventional printing approaches remains challenging. In this article, we introduce a simple yet powerful approach, namely plasmonic calligraphy, for realizing multiplexed label-free bioassays. Plasmonic calligraphy involves a regular ballpoint pen filled with biofunctionalized gold nanorods as plasmonic ink for creating isolated test domains on paper substrates. Biofriendly plasmonic calligraphy approach serves as a facile method to miniaturize the test domain size to few mm(2), which significantly improves the sensitivity of the plasmonic biosensor compared to bioplasmonic paper fabricated using immersion approach. Furthermore, plasmonic calligraphy also serves as a simple and efficient means to isolate multiple test domains on a single test strip, which facilitates multiplexed biodetection and multi-marker biochips. Plasmonic calligraphy, which can be potentially automated by implementing with a robotic arm, serves as an alternate path forward to overcome the limitations of conventional ink-jet printing. PMID:24727607

  14. Bioplasmonic calligraphy for multiplexed label-free biodetection

    PubMed Central

    Tian, Limei; Tadepalli, Sirimuvva; Hyun Park, Sang; Liu, Keng-Ku; Morrissey, Jeremiah J.; Kharasch, Evan D.; Naik, Rajesh R.; Singamaneni, Srikanth

    2014-01-01

    Printable multi-marker biochips that enable simultaneous quantitative detection of multiple target biomarkers in point-of-care and resource-limited settings are a holy grail in the field of biodiagnostics. However, preserving the functionality of biomolecules, which are routinely employed as recognition elements, during conventional printing approaches remains challenging. In this article, we introduce a simple yet powerful approach, namely plasmonic calligraphy, for realizing multiplexed label-free bioassays. Plasmonic calligraphy involves a regular ballpoint pen filled with biofunctionalized gold nanorods as plasmonic ink for creating isolated test domains on paper substrates. Biofriendly plasmonic calligraphy approach serves as a facile method to miniaturize the test domain size to few mm2, which significantly improves the sensitivity of the plasmonic biosensor compared to bioplasmonic paper fabricated using immersion approach. Furthermore, plasmonic calligraphy also serves as a simple and efficient means to isolate multiple test domains on a single test strip, which facilitates multiplexed biodetection and multi-marker biochips. Plasmonic calligraphy, which can be potentially automated by implementing with a robotic arm, serves as an alternate path forward to overcome the limitations of conventional ink-jet printing. PMID:24727607

  15. Data from quantitative label free proteomics analysis of rat spleen.

    PubMed

    Dudekula, Khadar; Le Bihan, Thierry

    2016-09-01

    The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis. PMID:27358910

  16. Diatom-based label-free optical biosensor for biomolecules.

    PubMed

    Viji, S; Anbazhagi, M; Ponpandian, N; Mangalaraj, D; Jeyanthi, S; Santhanam, P; Devi, A Shenbaga; Viswanathan, C

    2014-10-01

    Diatoms are unicellular algae, which fabricates ornate biosilica shells called frustules that possess a surface rich in reactive silanol (Si-OH) groups. The intrinsic patterned porous structure of diatom frustules at nanoscale can be exploited in the effective detection of biomolecules. In this study, the frustules of a specific diatom Amphora sp. has been functionalized to detect bovine serum albumin (BSA). The functionalization of the diatom frustule substrate is achieved by using 3-aminopropyltriethoxysilane (APES). The field emission scanning electron microscopy (FESEM) results showed an ornately patterned surface of the frustule valve ordered at nanoscale. The Fourier transform infrared (FTIR) spectra confirmed the N-H bending and stretching of the amine group after amine functionalization. The emission peaks in the photoluminescence (PL) spectra of the amine-functionalized diatom biosilica selectively enhanced the intensity by a factor of ten when compared to that of a bare diatom biosilica. The result showed a significant quenching of PL intensity of BSA at around 445 nm due to the interaction of amine-functionalized diatom-BSA protein complex. The detection limit was found to be 3 × 10(-5) M of BSA protein. Hence, the study proves that the functionalized frustule of Amphora sp. is an effective quantitative analytical tool for optical label-free biosensing applications. PMID:24989453

  17. Label-free photoacoustic microscopy of myocardial sheet architecture.

    PubMed

    Zhang, Chi; Cheng, Ya-Jian; Chen, Junjie; Wickline, Samuel; Wang, Lihong V

    2012-06-01

    Cardiac myofibers are organized into sheet architectures, which contribute to up to 40% of the heart wall thickening for ejection of blood for circulation. It is important to delineate the sheet architecture for a better understanding of cardiac mechanisms. However, current sheet imaging technologies are limited by fixation-induced dehydration/deformation and low spatial resolution. Here we implemented high-resolution label-free photoacoustic microscopy (PAM) of the myocardial sheet architecture. With high endogenous optical-absorption contrast originating mainly from cytochrome, myoglobin, and melanin, PAM can image the unfixed, unstained and unsliced heart without introducing deformation artifacts. A fresh blood-free mouse heart was imaged by PAM ex vivo. The three-dimensional branching sheets were clearly identified within 150 [micro sign]m depth. Various morphological parameters were derived from the PAM image. The sheet thickness (80 ± 10 μm) and the cleavage height (11 ± 1 μm) were derived from an undehydrated heart for the first time. Therefore, PAM has the potential for the functional imaging of sheet architecture in ex vivo perfused and viable hearts. PMID:22734729

  18. Label-free DNA sequencing using Millikan detection.

    PubMed

    Dettloff, Roger; Leiske, Danielle; Chow, Andrea; Farinas, Javier

    2015-10-15

    A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications. PMID:26151683

  19. Multi-spot, label-free immunoassay on reflectionless glass.

    PubMed

    Salina, Matteo; Giavazzi, Fabio; Lanfranco, Roberta; Ceccarello, Erica; Sola, Laura; Chiari, Marcella; Chini, Bice; Cerbino, Roberto; Bellini, Tommaso; Buscaglia, Marco

    2015-12-15

    Biosensing platforms that combine high sensitivity, operational simplicity and affordable costs find wide application in many fields, including human diagnostics, food and environmental monitoring. In this work, we introduce a label-free biosensing chip made of glass with a single anti-reflective layer of SiO2. This common and economic material coated by a multi-functional copolymer based on dimethylacrylamide enables the detection even in turbid media. The copolymer coating provides covalent immobilization of antibodies onto the surface and prevents the non-specific adsorption of analytes and matrix constituents. The specific capture of target compounds yields a local increase of surface reflectivity measured by a simple imaging system. Chip design and quantitative interpretation of the data are based on a theoretical optical model. This approach enables the multiplex detection of biomolecular interactions with state-of-the-art sensitivity and minimal instrumental complexity. The detection performance is demonstrated by characterizing the interaction between human growth hormone in solution and the corresponding antibodies immobilized on the sensing surface, both in buffer and human serum, obtaining a clear signal for concentrations as small as 2.8 ng/ml. PMID:26188676

  20. Label-Free Optical Ring Resonator Bio/Chemical Sensors

    NASA Astrophysics Data System (ADS)

    Zhu, Hongying; Suter, Jonathan D.; Fan, Xudong

    Optical micro-ring resonator sensors are an emerging category of label-free optical sensors for bio/chemical sensing that have recently been under intensive investigation. Researchers of this technology have been motivated by a tremendous breadth of different applications, including medical diagnosis, environmental monitoring, homeland security, and food quality control, which require sensitive analytical tools. Ring resonator sensors use total internal reflection to support circulating optical resonances called whispering gallery modes (WGMs). The WGMs have an evanescent field of several hundred nanometers into the surrounding medium, and can therefore detect the refractive index change induced when the analyte binds to the resonator surface. Despite the small physical size of a resonator, the circulating nature of the WGM creates extremely long effective lengths, greatly increasing light-matter interaction and improving its sensing performance. Moreover, only small sample volume is needed for detection because the sensors can be fabricated in sizes well below 100 μm. The small footprint allows integration of those ring resonator sensors onto lab-on-a-chip types of devices for multiplexed detection.

  1. Optically Resonant Nanophotonic Devices for Label-Free Biomolecular Detection

    NASA Astrophysics Data System (ADS)

    Goddard, Julie; Mandal, Sudeep; Erickson, David

    Optical devices, such as surface plasmon resonance chips and waveguide-based Mach-Zehnder interferometers, have long been successfully used as label-free biomolecular sensors. Recently, however, there has been increased interest in developing new approaches to biomolecular detection that can improve on the limit of detection, specificity, and multiplexibility of these early devices and address emerging challenges in pathogen detection, disease diagnosis, and drug discovery. As we describe in this chapter, planar optically resonant nanophotonic devices (such as ring resonators, whispering gallery modes, and photonic crystal cavities) are one method that shows promise in significantly advancing the technology. Here we first provide a short review of these devices focusing on a handful of approaches illustrative of the state of the art. We then frame the major challenge to improving the technology as being the ability to provide simultaneously spatial localization of the electromagnetic energy and biomolecular binding events. We then introduce our “Nanoscale Optofluidic Sensor Arrays” which represents our approach to addressing this challenge. It is demonstrated how these devices serve to enable multiplexed detection while localizing the electromagnetic energy to a volume as small as a cubic wavelength. Challenges involved in the targeted immobilization of biomolecules over such a small area are discussed and our solutions presented. In general, we have tried to write this chapter with the novice in mind, providing details on the fabrication and immobilization methods that we have used and how one might adapt our approach to their designs.

  2. Label-free biosensing using silicon planar waveguide technology

    NASA Astrophysics Data System (ADS)

    Densmore, Adam; Xu, Dan-Xia; Waldron, Philip; Janz, Siegfried; Delâge, André; Lopinski, G.; Mischki, T.; Cheben, Pavel; Post, Edith; Lapointe, Jean; Schmid, Jens H.

    2007-06-01

    We exploit the unique properties of the silicon-on-insulator material platform to demonstrate a new series of planar waveguide evanescent field sensors for biological / chemical sensing. These sensors, combined with state-of-the-art surface functionalization chemistries, offer a sensitive, label-free means for the specific detection of biomolecules, without the need for fluorescent tags employed in conventional fluorescence-based biochips. The use of silicon photonic wire waveguide technology allows sensors with extremely small footprint and small radius of curvature to be fabricated, facilitating the development of densely packed sensor arrays for multi-parameter analysis, particularly attractive for drug discovery, pathogen detection, genomics and disease diagnostics. We show that high index contrast silicon photonic wire waveguides not only provide the above stated advantages but also offer increased sensitivity over that of evanescent field sensors constructed on other common waveguide material platforms. This results from the unique properties of the optical modes of silicon photonic wire waveguides, which exhibit very large surface electric field magnitude and strong localization near the waveguide surface. We discuss the design and fabrication of silicon-on-insulator-based Mach-Zehnder interferometer sensors and experimentally demonstrate their performance to detect bulk solution refractive index change and to monitor the specific adsorption of streptavidin to biotinylated waveguides.

  3. Recyclable optical microcavities for label-free sensing

    NASA Astrophysics Data System (ADS)

    Hunt, Heather K.; Armani, Andrea M.

    2011-10-01

    High-sensitivity, label-free biosensors, such as optical microcavities, have shown tremendous potential in medical diagnostics, environmental monitoring, and food safety evaluation, particularly when paired with a biochemical recognition element that grants high specificity towards a target of interest. Their primary limitation is that these systems are single-use, unless the recognition element can be regenerated. Therefore, the ability to selectively functionalize the optical microcavity for a specific target molecule and then recycle the system, without degrading device performance, is extremely important. Here, we present a bioconjugation strategy that not only imparts specificity to optical microcavities, but also allows for biosensor recycling. In this approach, we selectively functionalize the surface of silica microtoroids with a biotin recognition element. We then use a non-destructive O2 plasma treatment to remove the surface chemistry, refresh the recognition element, and recycle the device. The surface chemistry and optical performance of the functionalized and recycled devices are characterized by microcavity analysis, and typical spectroscopic techniques, respectively. The resulting devices can be recycled several times without performance degradation, and show high density surface coverage of biologically active recognition elements. This work represents one of the first examples of a recyclable, bioconjugation strategy for optical microtoroid resonators.

  4. Performance limitations of label-free sensors in molecular diagnosis using complex samples

    NASA Astrophysics Data System (ADS)

    Varma, Manoj

    2016-03-01

    Label-free biosensors promised a paradigm involving direct detection of biomarkers from complex samples such as serum without requiring multistep sample processing typical of labelled methods such as ELISA or immunofluorescence assays. Label-free sensors have witnessed decades of development with a veritable zoo of techniques available today exploiting a multitude of physical effects. It is appropriate now to critically assess whether label-free technologies have succeeded in delivering their promise with respect to diagnostic applications, particularly, ambitious goals such as early cancer detection using serum biomarkers, which require low limits of detection (LoD). Comparison of nearly 120 limits of detection (LoD) values reported by labelled and label-free sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Data from experiments where labelled and label-free assays were performed simultaneously using the same assay parameters also confirm that the LoD achieved by labelled techniques is 2 to 3 orders of magnitude better than that by label-free techniques. Furthermore, label-free techniques required significant signal amplification, for e.g. using nanoparticle conjugated secondary antibodies, to achieve LoDs comparable to labelled methods substantially deviating from the original "direct detection" paradigm. This finding has important implications on the practical limits of applying label-free detection methods for molecular diagnosis.

  5. Label-free detection repeatability of protein microarrays by oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Dai, Jun; Li, Lin; Wang, JingYi; He, LiPing; Lu, HuiBin; Ruan, KangCheng; Jin, KuiJuan; Yang, GuoZhen

    2012-12-01

    We examine the repeatabilities of oblique-incidence reflectivity difference (OIRD) method for label-free detecting biological molecular interaction using protein microarrays. The experimental results show that the repeatabilities are the same in a given microarray or microarray-microarray and are consistent, indicating that OIRD is a promising label-free detection technique for biological microarrays.

  6. Label-Free Biomarker Sensing in Undiluted Serum with Suspended Microchannel Resonators

    PubMed Central

    von Muhlen, Marcio G.; Brault, Norman D.; Knudsen, Scott M.; Jiang, Shaoyi; Manalis, Scott R.

    2010-01-01

    Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of cancer. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can directly quantify adsorbed mass via changes in resonant frequency. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from non-specific binding. In this report, we demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR SiO2 surfaces act as ultra-low fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL. PMID:20148583

  7. Carbon Nanostructure-Based Field-Effect Transistors for Label-Free Chemical/Biological Sensors

    PubMed Central

    Hu, PingAn; Zhang, Jia; Li, Le; Wang, Zhenlong; O’Neill, William; Estrela, Pedro

    2010-01-01

    Over the past decade, electrical detection of chemical and biological species using novel nanostructure-based devices has attracted significant attention for chemical, genomics, biomedical diagnostics, and drug discovery applications. The use of nanostructured devices in chemical/biological sensors in place of conventional sensing technologies has advantages of high sensitivity, low decreased energy consumption and potentially highly miniaturized integration. Owing to their particular structure, excellent electrical properties and high chemical stability, carbon nanotube and graphene based electrical devices have been widely developed for high performance label-free chemical/biological sensors. Here, we review the latest developments of carbon nanostructure-based transistor sensors in ultrasensitive detection of chemical/biological entities, such as poisonous gases, nucleic acids, proteins and cells. PMID:22399927

  8. Label-free identification of intestinal metaplasia in the stomach using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Wu, G.; Wei, J.; Zheng, Z.; Ye, J.; Zeng, S.

    2014-06-01

    The early diagnosis of intestinal metaplasia (IM) in the stomach together with effective therapeutic interventions is crucial to reducing the mortality-rates of the patients associated with gastric cancer. However, it is challenging during conventional white-light endoscopy, and histological analysis remains the ‘gold standard’ for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), for the identification of IM in the stomach. It was found that multiphoton imaging provides cellular and subcellular details to the identification of IM from normal gastric tissues. In particular, there is significant difference in the population density of goblet cells between normal and IM gastric tissues, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early gastric lesions. To our knowledge, this is the first demonstration of the potential of MPM for the identification of IM.

  9. Infrared Plasmonic Biosensor for Real-Time and Label-Free Monitoring of Lipid Membranes.

    PubMed

    Limaj, Odeta; Etezadi, Dordaneh; Wittenberg, Nathan J; Rodrigo, Daniel; Yoo, Daehan; Oh, Sang-Hyun; Altug, Hatice

    2016-02-10

    In this work, we present an infrared plasmonic biosensor for chemical-specific detection and monitoring of biomimetic lipid membranes in a label-free and real-time fashion. Lipid membranes constitute the primary biological interface mediating cell signaling and interaction with drugs and pathogens. By exploiting the plasmonic field enhancement in the vicinity of engineered and surface-modified nanoantennas, the proposed biosensor is able to capture the vibrational fingerprints of lipid molecules and monitor in real time the formation kinetics of planar biomimetic membranes in aqueous environments. Furthermore, we show that this plasmonic biosensor features high-field enhancement extending over tens of nanometers away from the surface, matching the size of typical bioassays while preserving high sensitivity. PMID:26761392

  10. Label-free functional nucleic acid sensors for detecting target agents

    DOEpatents

    Lu, Yi; Xiang, Yu

    2015-01-13

    A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or aptazyme) that have a tunable dynamic range through the introduction of an abasic site (e.g., dSpacer) or a vacant site into the functional nucleic acids. Also provided is a general method for designing label-free fluorescent aptamer sensors based on the regulation of malachite green (MG) fluorescence. A general method for designing label-free fluorescent catalytic and molecular beacons (CAMBs) is also provided. The methods demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. Sensors and methods of using the disclosed sensors are also provided.

  11. In situ label-free static cytometry by monitoring spatiotemporal fluctuations of image gray values

    NASA Astrophysics Data System (ADS)

    Wohl, Ishay; Zurgil, Naomi; Hakuk, Yaron; Sobolev, Maria; Galmidi, Moti; Deutsch, Mordechai

    2015-10-01

    Spatiotemporal fluctuation of homogeneity and randomness of gray values within an image was explored and utilized as a label-free means for cell examination. This was done by utilizing a user-friendly combination of simple bright field microscope and Cytocapture dish, wherein cells are individually held, each within a picoliter optical chamber, forming an array of cells to be repeatedly measured over time and biomanipulated in situ at single-cell resolution. First, the measured gray level information entropy (GLIE) was used and, based on the fact that living cells are not in a state of thermodynamic equilibrium but rather in a metastable state, two fluctuation-sensitive measures were proposed and examined: ASDE-the spatial average of temporal standard deviation (SD) of GLIE, and AA-the average time autocorrelation of GLIE. System performance was validated on cell-free solutions. This was followed by examining the performance of the measures AGLIE, ASDE, and AA to distinguish among individual live-still, dead and live cells from various cell lines, as well as between cells which were and were not induced to differentiate. Results, which were obtained on four types of cells, indicate advantages of the proposed measures which are believed to be significant additions to the microscope-based probe-free toolbox.

  12. Imaging label-free biosensor with microfluidic system

    NASA Astrophysics Data System (ADS)

    Jahns, S.; Glorius, P.; Hansen, M.; Nazirizadeh, Y.; Gerken, M.

    2015-06-01

    We present a microfluidic system suitable for parallel label-free detection of several biomarkers utilizing a compact imaging measurement system. The microfluidic system contains a filter unit to separate the plasma from human blood and a functionalized, photonic crystal slab sensor chip. The nanostructure of the photonic crystal slab sensor chip is fabricated by nanoimprint lithography of a period grating surface into a photoresist and subsequent deposition of a TiO2 layer. Photonic crystal slabs are slab waveguides supporting quasi-guided modes coupling to far-field radiation, which are sensitive to refractive index changes due to biomarker binding on the functionalized surface. In our imaging read-out system the resulting resonance shift of the quasi-guided mode in the transmission spectrum is converted into an intensity change detectable with a simple camera. By continuously taking photographs of the sensor surface local intensity changes are observed revealing the binding kinetics of the biomarker to its specific target. Data from two distinct measurement fields are used for evaluation. For testing the sensor chip, 1 μM biotin as well as 1 μM recombinant human CD40 ligand were immobilized in spotsvia amin coupling to the sensor surface. Each binding experiment was performed with 250 nM streptavidin and 90 nM CD40 ligand antibody dissolved in phosphate buffered saline. In the next test series, a functionalized sensor chip was bonded onto a 15 mm x 15 mm opening of the 75 mm x 25 mm x 2 mm microfluidic system. We demonstrate the functionality of the microfluidic system for filtering human blood such that only blood plasma was transported to the sensor chip. The results of first binding experiments in buffer with this test chip will be presented.

  13. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes.

    PubMed

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-05-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for 'lab-on-a-chip' type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  14. Photocatalytic electrosensor for label-free and ultrasensitive detection of BRCA1 gene.

    PubMed

    Qin, Xiaojiao; Xu, Shuxia; Deng, Li; Huang, Rongfu; Zhang, Xinfeng

    2016-11-15

    In this work, we have developed an electrochemical sensor for label-free and ultrasensitive detection of DNA (exemplified by breast cancer 1 gene) by using a photocatalytic reaction. Upon recognition of target DNA, the ethidium bromide molecules which were embedded in the hybridized double strand DNA (dsDNA, target DNA and capture DNA) could photo-catalytically generate singlet oxygen upon green light emitting diode irradiation, leading to an efficient cleave of the dsDNA. As a result, the voltammetry for the [Fe(CN)6](3-/4-) was improved remarkably because of less blocking of electrode and weaker charge repulsion. Such a simple strategy provided an ultrasensitive detection of breast cancer 1 gene down to the attomolar level with a broad linear range (10 aM-100 nM). The sensor is by far the most sensitive electrochemical method for detection of breast cancer 1 gene without an amplification procedure. Also the sensor can discriminate mismatched DNA from perfectly matched target DNA with high selectivity. Therefore, simplicity, high sensitivity and specificity provided by this photocatalytic eletrosensor will make it a promising tool for early diagnosis of gene-related diseases. PMID:27317999

  15. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  16. Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter

    PubMed Central

    Aftab, Obaid; Fryknäs, Mårten; Zhang, Xiaonan; De Milito, Angelo; Hammerling, Ulf; Linder, Stig; Larsson, Rolf; Gustafsson, Mats G

    2014-01-01

    Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced. PMID:24169509

  17. G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection.

    PubMed

    Nie, Ji; Zhang, De-Wen; Tie, Cai; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2014-06-15

    A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNA in the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in the second stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readout element. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidase-mimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Taking advantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermal cycling . As no complex synthesis or extra downstream operation is needed, the whole easy handling procedure can be finished in no more than 1h. This assay allows the sensing of the model DNA with the limit of detection to be 2.5pM. Moreover, it demonstrates good discrimination of mismatched sequences. The strategy has also been successfully implemented to sensitively detect Tay-Sachs genetic disorder mutant. PMID:24508547

  18. Marker-free cell discrimination by holographic optical tweezers

    NASA Astrophysics Data System (ADS)

    Schaal, F.; Warber, M.; Zwick, S.; van der Kuip, H.; Haist, T.; Osten, W.

    2009-06-01

    We introduce a method for marker-free cell discrimination based on optical tweezers. Cancerous, non-cancerous, and drug-treated cells could be distinguished by measuring the trapping forces using holographic optical tweezers. We present trapping force measurements on different cell lines: normal pre-B lymphocyte cells (BaF3; "normal cells"), their Bcr-Abl transformed counterparts (BaF3-p185; "cancer cells") as a model for chronic myeloid leukaemia (CML) and Imatinib treated BaF3-p185 cells. The results are compared with reference measurements obtained by a commercial flow cytometry system.

  19. In vivo, label-free, and noninvasive detection of melanoma metastasis by photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Liu, Rongrong; Wang, Cheng; Hu, Cheng; Wang, Xueding; Wei, Xunbin

    2014-02-01

    Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC's capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

  20. Kinetic discrimination in T-cell activation.

    PubMed Central

    Rabinowitz, J D; Beeson, C; Lyons, D S; Davis, M M; McConnell, H M

    1996-01-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model. PMID:8643643

  1. DNA-regulated silver nanoclusters for label-free ratiometric fluorescence detection of DNA.

    PubMed

    Liu, Lin; Yang, Qianhui; Lei, Jianping; Xu, Nan; Ju, Huangxian

    2014-11-18

    Two kinds of DNA-regulated Ag nanoclusters were one-pot synthesized on an oligonucleotide, and delicately utilized in the design of a label-free ratiometric fluorescence strategy for DNA detection with simplicity and high sensitivity. PMID:25247781

  2. Label-free probing of genes by time-domain terahertz sensing.

    PubMed

    Haring Bolivar, P; Brucherseifer, M; Nagel, M; Kurz, H; Bosserhoff, A; Büttner, R

    2002-11-01

    A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a freespace analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed. PMID:12452572

  3. Label-free integrative pharmacology on-target of opioid ligands at the opioid receptor family

    PubMed Central

    2013-01-01

    Background In vitro pharmacology of ligands is typically assessed using a variety of molecular assays based on predetermined molecular events in living cells. Many ligands including opioid ligands pose the ability to bind more than one receptor, and can also provide distinct operational bias to activate a specific receptor. Generating an integrative overview of the binding and functional selectivity of ligands for a receptor family is a critical but difficult step in drug discovery and development. Here we applied a newly developed label-free integrative pharmacology on-target (iPOT) approach to systematically survey the selectivity of a library of fifty-five opioid ligands against the opioid receptor family. All ligands were interrogated using dynamic mass redistribution (DMR) assays in both recombinant and native cell lines that express specific opioid receptor(s). The cells were modified with a set of probe molecules to manifest the binding and functional selectivity of ligands. DMR profiles were collected and translated to numerical coordinates that was subject to similarity analysis. A specific set of opioid ligands were then selected for quantitative pharmacology determination. Results Results showed that among fifty-five opioid ligands examined most ligands displayed agonist activity in at least one opioid receptor expressing cell line under different conditions. Further, many ligands exhibited pathway biased agonism. Conclusion We demonstrate that the iPOT effectively sorts the ligands into distinct clusters based on their binding and functional selectivity at the opioid receptor family. PMID:23497702

  4. Combined Labelled and Label-free SERS Probes for Triplex Three-dimensional Cellular Imaging

    NASA Astrophysics Data System (ADS)

    Chen, Yong; Bai, Xiangru; Su, Le; Du, Zhanwei; Shen, Aiguo; Materny, Arnulf; Hu, Jiming

    2016-01-01

    Cells are complex chemical systems, where the molecular composition at different cellular locations and specific intracellular chemical interactions determine the biological function. An in-situ nondestructive characterization of the complicated chemical processes (like e.g. apoptosis) is the goal of our study. Here, we present the results of simultaneous and three-dimensional imaging of double organelles (nucleus and membrane) in single HeLa cells by means of either labelled or label-free surface-enhanced Raman spectroscopy (SERS). This combination of imaging with and without labels is not possible when using fluorescence microscopy. The SERS technique is used for a stereoscopic description of the intrinsic chemical nature of nuclei and the precise localization of folate (FA) and luteinizing hormone-releasing hormone (LHRH) on the membrane under highly confocal conditions. We also report on the time-dependent changes of cell nuclei as well as membrane receptor proteins during apoptosis analyzed by statistical multivariate methods. The multiplex three-dimensional SERS imaging technique allows for both temporal (real time) and spatial (multiple organelles and molecules in three-dimensional space) live-cell imaging and therefore provides a new and attractive 2D/3D tracing method in biomedicine on subcellular level.

  5. Early apoptosis real-time detection by label-free SERS based on externalized phosphatidylserine.

    PubMed

    Zhou, Haibo; Wang, Qiqin; Yuan, Detian; Wang, Jinyong; Huang, Yang; Wu, Huihui; Jian, Jingyi; Yang, Danting; Huang, Ning; Haisch, Christoph; Jiang, Zhengjin; Chen, Shanze

    2016-07-21

    Apoptosis is a tightly regulated cellular process that plays an essential role in the development, aging, cancer biology, immune response, and pathogenesis of various diseases. Herein, we report a new SERS sensing strategy for in vitro sensitive detection of early apoptotic cells. The principle of this method is to in situ synthesize silver nanoparticles (AgNPs) on the phosphatidylserine (PS) of the apoptotic cell membrane during the early apoptosis, which enables distinguishing normal and apoptotic cells. The total assay time of the presented method is only 10 min, thus being faster, cheaper and simpler than current techniques for the detection of apoptosis. The intrinsic mechanism was verified by different approaches based on externalized phosphatidylserine. In addition, the detection process is real-time and label-free; i.e., the intrinsic SERS spectra from the cellular membrane are directly employed for apoptosis real-time detection, which avoids using additional chemical or biological reagents as external signal indicators. Therefore, our SERS approach may serve as a potentially practical tool for sensitive and real-time detection of early cell apoptosis, complementing the state-of-the-art strategies, e.g. flow cytometry. While further investigation is required to better understand the intrinsic mechanism of the in situ coating method, the current results may provide another choice for real-time detection of early apoptosis. PMID:27181439

  6. Combined Labelled and Label-free SERS Probes for Triplex Three-dimensional Cellular Imaging.

    PubMed

    Chen, Yong; Bai, Xiangru; Su, Le; Du, Zhanwei; Shen, Aiguo; Materny, Arnulf; Hu, Jiming

    2016-01-01

    Cells are complex chemical systems, where the molecular composition at different cellular locations and specific intracellular chemical interactions determine the biological function. An in-situ nondestructive characterization of the complicated chemical processes (like e.g. apoptosis) is the goal of our study. Here, we present the results of simultaneous and three-dimensional imaging of double organelles (nucleus and membrane) in single HeLa cells by means of either labelled or label-free surface-enhanced Raman spectroscopy (SERS). This combination of imaging with and without labels is not possible when using fluorescence microscopy. The SERS technique is used for a stereoscopic description of the intrinsic chemical nature of nuclei and the precise localization of folate (FA) and luteinizing hormone-releasing hormone (LHRH) on the membrane under highly confocal conditions. We also report on the time-dependent changes of cell nuclei as well as membrane receptor proteins during apoptosis analyzed by statistical multivariate methods. The multiplex three-dimensional SERS imaging technique allows for both temporal (real time) and spatial (multiple organelles and molecules in three-dimensional space) live-cell imaging and therefore provides a new and attractive 2D/3D tracing method in biomedicine on subcellular level. PMID:26781186

  7. Combined Labelled and Label-free SERS Probes for Triplex Three-dimensional Cellular Imaging

    PubMed Central

    Chen, Yong; Bai, Xiangru; Su, Le; Du, Zhanwei; Shen, Aiguo; Materny, Arnulf; Hu, Jiming

    2016-01-01

    Cells are complex chemical systems, where the molecular composition at different cellular locations and specific intracellular chemical interactions determine the biological function. An in-situ nondestructive characterization of the complicated chemical processes (like e.g. apoptosis) is the goal of our study. Here, we present the results of simultaneous and three-dimensional imaging of double organelles (nucleus and membrane) in single HeLa cells by means of either labelled or label-free surface-enhanced Raman spectroscopy (SERS). This combination of imaging with and without labels is not possible when using fluorescence microscopy. The SERS technique is used for a stereoscopic description of the intrinsic chemical nature of nuclei and the precise localization of folate (FA) and luteinizing hormone-releasing hormone (LHRH) on the membrane under highly confocal conditions. We also report on the time-dependent changes of cell nuclei as well as membrane receptor proteins during apoptosis analyzed by statistical multivariate methods. The multiplex three-dimensional SERS imaging technique allows for both temporal (real time) and spatial (multiple organelles and molecules in three-dimensional space) live-cell imaging and therefore provides a new and attractive 2D/3D tracing method in biomedicine on subcellular level. PMID:26781186

  8. Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique

    PubMed Central

    Laguna, Maríafe; Holgado, Miguel; Hernandez, Ana L.; Santamaría, Beatriz; Lavín, Alvaro; Soria, Javier; Suarez, Tatiana; Bardina, Carlota; Jara, Mónica; Sanza, Francisco J.; Casquel, Rafael

    2015-01-01

    The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. PMID:26287192

  9. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    PubMed Central

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18 kDa cationic antimicrobial protein (CAMP), and heat shock 70 kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  10. Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis.

    PubMed

    Poliakov, Anton; Russell, Calum W; Ponnala, Lalit; Hoops, Harold J; Sun, Qi; Douglas, Angela E; van Wijk, Klaas J

    2011-06-01

    Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont. PMID:21421797

  11. Identification of Phosphorylated Cyclin-Dependent Kinase 1 Associated with Colorectal Cancer Survival Using Label-Free Quantitative Analyses

    PubMed Central

    Tyan, Yu-Chang; Hsiao, Eric S. L.; Chu, Po-Chen; Lee, Chung-Ta; Lee, Jenq-Chang; Chen, Yi-Ming Arthur; Liao, Pao-Chi

    2016-01-01

    Colorectal cancer is the most common form of cancer in the world, and the five-year survival rate is estimated to be almost 90% in the early stages. Therefore, the identification of potential biomarkers to assess the prognosis of early stage colorectal cancer patients is critical for further clinical treatment. Dysregulated tyrosine phosphorylation has been found in several diseases that play a significant regulator of signaling in cellular pathways. In this study, this strategy was used to characterize the tyrosine phosphoproteome of colorectal cell lines with different progression abilities (SW480 and SW620). We identified a total of 280 phosphotyrosine (pTyr) peptides comprising 287 pTyr sites from 261 proteins. Label-free quantitative analysis revealed the differential level of a total of 103 pTyr peptides between SW480 and SW620 cells. We showed that cyclin-dependent kinase I (CDK1) pTyr15 level in SW480 cells was 3.3-fold greater than in SW620 cells, and these data corresponded with the label-free mass spectrometry-based proteomic quantification analysis. High level CDK1 pTyr15 was associated with prolonged disease-free survival for stage II colorectal cancer patients (n = 79). Taken together, our results suggest that the CDK1 pTyr15 protein is a potential indicator of the progression of colorectal cancer. PMID:27383761

  12. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    PubMed

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-01-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis. PMID:26908277

  13. Label-free chemical imaging of live Euglena gracilis by high-speed SRS spectral microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Wakisaka, Yoshifumi; Suzuki, Yuta; Tokunaga, Kyoya; Hirose, Misa; Domon, Ryota; Akaho, Rina; Kuroshima, Mai; Tsumura, Norimichi; Shimobaba, Tomoyoshi; Iwata, Osamu; Suzuki, Kengo; Nakashima, Ayaka; Goda, Keisuke; Ozeki, Yasuyuki

    2016-03-01

    Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

  14. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    PubMed Central

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-01-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 − 2.0 × 108 cells mL−1), a low limit of detection (79 cells mL−1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis. PMID:26908277

  15. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    NASA Astrophysics Data System (ADS)

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-02-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 - 2.0 × 108 cells mL-1), a low limit of detection (79 cells mL-1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  16. Glycoprofiling of cancer biomarkers: Label-free electrochemical lectin-based biosensors

    PubMed Central

    Pihíková, Dominika; Kasák, Peter

    2016-01-01

    Glycosylation of biomolecules is one of the most prevalent post- and co-translational modification in a human body, with more than half of all human proteins being glycosylated. Malignant transformation of cells influences glycosylation machinery resulting in subtle changes of the glycosylation pattern within the cell populations as a result of cancer. Thus, an altered terminal glycan motif on glycoproteins could provide a warning signal about disease development and progression and could be applied as a reliable biomarker in cancer diagnostics. Among all highly effective glycoprofiling tools, label-free electrochemical impedance spectroscopy (EIS)-based biosensors have emerged as especially suitable tool for point-of-care early-stage cancer detection. Herein, we highlight the current challenges in glycoprofiling of various cancer biomarkers by ultrasensitive impedimetric-based biosensors with low sample consumption, low cost fabrication and simple miniaturization. Additionally, this review provides a short introduction to the field of glycomics and lectinomics and gives a brief overview of glycan alterations in different types of cancer. PMID:27275016

  17. Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

    PubMed

    Scheerlinck, E; Dhaenens, M; Van Soom, A; Peelman, L; De Sutter, P; Van Steendam, K; Deforce, D

    2015-12-01

    Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest. PMID:26302362

  18. A light-sheet microscope compatible with mobile devices for label-free intracellular imaging and biosensing.

    PubMed

    Wu, Tsung-Feng; Yen, Tony Minghung; Han, Yuanyuan; Chiu, Yu-Jui; Lin, Eason Y-S; Lo, Yu-Hwa

    2014-09-01

    The inner structure, especially the nuclear structure, of cells carries valuable information about disease and health conditions of a person. Here we demonstrate a label-free technique to enable direct observations and measurements of the size, shape and morphology of the cell nucleus. With a microfabricated lens and a commercial CMOS imager, we form a scanning light-sheet microscope to produce a dark-field optical scattering image of the cell nucleus that overlays with the bright-field image produced in a separate regime of the same CMOS sensor. We have used the device to detect nuclear features that characterize the life cycle of cells and have used the nucleus volume as a new parameter for cell classification. The device can be developed into a portable, low-cost, point-of-care device leveraging the capabilities of the CMOS imagers to be pervasive in mobile electronics. PMID:24989638

  19. Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target

    PubMed Central

    Morse, Megan; Tran, Elizabeth; Sun, Haiyan; Levenson, Robert; Fang, Ye

    2011-01-01

    Development of new opioid drugs that provide analgesia without producing dependence is important for pain treatment. Opioid agonist drugs exert their analgesia effects primarily by acting at the mu opioid receptor (MOR) sites. High-resolution differentiation of opioid ligands is crucial for the development of new lead drug candidates with better tolerance profiles. Here, we use a label-free integrative pharmacology on-target (iPOT) approach to characterize the functional selectivity of a library of known opioid ligands for the MOR. This approach is based on the ability to detect dynamic mass redistribution (DMR) arising from the activation of the MOR in living cells. DMR assays were performed in HEK-MOR cells with and without preconditioning with probe molecules using label-free resonant waveguide grating biosensors, wherein the probe molecules were used to modify the activity of specific signaling proteins downstream the MOR. DMR signals obtained were then translated into high resolution heat maps using similarity analysis based on a numerical matrix of DMR parameters. Our data indicate that the iPOT approach clearly differentiates functional selectivity for distinct MOR signaling pathways among different opioid ligands, thus opening new avenues to discover and quantify the functional selectivity of currently used and novel opioid receptor drugs. PMID:22003401

  20. Versatile G-quadruplex-mediated strategies in label-free biosensors and logic systems.

    PubMed

    Ren, Jiangtao; Wang, Tianshu; Wang, Erkang; Wang, Jin

    2015-04-21

    G-quadruplex (G4), as one of the significant functional nucleic acids (FNAs), has attracted researchers' wide attention, and in particular has been employed for the construction of label-free molecular sensors and logic systems based on the peroxidase-like activity of the G4-hemin complex and G4-enhanced luminescence of G4-binding organic dyes. Its cation-dependent conformation and stability provide opportunities for the recognition of metal ion inputs and application of a split G4 strategy. Moreover, coupling the G4 sequence with other FNAs, e.g. metal ion-dependent DNAzymes and aptamers, has prominently broadened the range of possible targets from metal ions and DNA to diverse proteins and cells. Although there are limitations, such as a low ability of anti-interference and multiplex analysis, the excellent advantages (e.g. simplicity and low cost) endow the G4-mediated strategy with tremendous potential to be further exploited for practical bioanalysis and complicated DNA computing. PMID:25705973

  1. Comparative proteomic label-free analysis of Campylobacter jejuni NCTC 11168 cultured with porcine mucin.

    PubMed

    Hong, Sahyun; Cha, Injun; Kim, Nan-Ok; Seo, Jong-Bok; Kim, Soo-Young; Kim, Jong-Hyun; Chung, Gyung Tae; Jeon, Byeonghwa; Kang, Yeon-Ho

    2014-03-01

    Campylobacter jejuni is a major gastrointestinal pathogen in humans. Poultry is a primary reservoir for C. jejuni, and C. jejuni appears to be highly adapted to the gastrointestinal tracts of avian species. We determined the protein expression profiles of C. jejuni NCTC 11168 cultured in medium containing porcine mucin. Differentially expressed proteins in the presence and absence of porcine mucin were identified using the label-free method. We identified 52 proteins with expression that was either upregulated (32 proteins) or downregulated (20 proteins) by porcine mucin. These proteins are involved in diverse cellular functions, such as motility, cell wall synthesis, iron transport, energy production, and amino acid metabolism. In particular, the upregulated proteins were involved in chemotaxis (CheV and CetA), motility (FlaA), colonization and adherence (CadF, FrdA, CfrA, MapA, and HydA), and stress tolerance (TrxB and ClpB). These results suggest that C. jejuni changes its protein expression in response to porcine mucin and that this change in expression may contribute to host adaptation of C. jejuni NCTC 11168. PMID:24552179

  2. Subwavelength-resolution photoacoustic microscopy for label-free detection of optical absorption in vivo

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Maslov, Konstantin; Wang, Lihong V.

    2011-03-01

    Mainstream optical microscopy technologies normally detect fluorescence or scattering, which may require undesirable labeling, but cannot directly sense optical absorption, which provides essential biological functional information. Here we reported in vivo and label-free subwavelength-resolution photoacoustic microscopy (SW-PAM) by using a waterimmersion optical objective with a 1.23 NA. Capable of detecting nonfluorescent endogenous pigments, SW-PAM provides exquisitely high optical-absorption contrast. And, as a result of background-free detection, the sensitivity of SW-PAM to optical absorption reaches 100%. SW-PAM was demonstrated with wide-field optical microscopy by imaging gold nanospheres, ex vivo cells, and in vivo vasculature and melanoma. It was shown that SW-PAM has approached the ultimate diffraction-limited optical resolution-220 nm resolution at 532 nm wavelength. Subcellular organelles, such as melanosomes, can be resolved by SW-PAM. Vasculature and early-stage melanoma were imaged with 21:1 and 34:1 contrasts, respectively, without labeling. For all these applications, SW-PAM has contrasts orders of magnitude higher than wide-field optical microscopy. Therefore, SW-PAM is expected to join the mainstream microscopy technologies.

  3. 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

    2013-01-01

    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. PMID:23214408

  4. Label-free nucleic acids detection based on DNA templated silver nanoclusters fluorescent probe.

    PubMed

    Zhao, Haiyan; Wang, Lei; Zhu, Jing; Wei, Haiping; Jiang, Wei

    2015-06-01

    Based on DNA templated Ag NCs (DNA/Ag NCs) fluorescent probe, a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1 (HIV-1) DNA. Firstly, a hairpin probe, containing target DNA recognition sequence and guanine-rich sequence, was designed to hybridize with the target DNA and form a blunt 3'-terminus DNA duplex. Then, exonuclease III (Exo III) was employed to stepwise hydrolyze the mononucleotides from formed blunt 3'-terminus DNA duplex, releasing the target DNA and guanine-rich sequence. Finally, DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence, leading to an enhanced fluorescence signal for detection. The proposed method could detect as low as 2.9×10(-10) mol L(-1) HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA. Furthermore, the method possessed perfect recoveries in cells lysate and human serum, showing potential to be used in biological samples. PMID:25863386

  5. Label free targeted detection and quantification of celiac disease immunogenic epitopes by mass spectrometry.

    PubMed

    van den Broeck, Hetty C; Cordewener, Jan H G; Nessen, Merel A; America, Antoine H P; van der Meer, Ingrid M

    2015-04-24

    Celiac disease (CD) is a food-related disease caused by certain gluten peptides containing T-cell stimulating epitopes from wheat, rye, and barley. CD-patients have to maintain a gluten-free diet and are therefore dependent on reliable testing and labeling of gluten-free products. So far, the R5-ELISA is the approved method to detect if food products can be labeled gluten-free. Because the R5-ELISA detects gluten in general, there is a demand for an improved detection method that quantifies specifically CD-epitopes. Therefore, we developed a new method for detection and quantification of CD-epitopes, based on liquid chromatography (LC) coupled to mass spectrometry (MS) in multiple reaction monitoring (MRM) mode. This method enables targeted label free comparative analysis of the gluten proteins present in different wheat varieties and species, and in wheat-based food products. We have tested our method by analyzing several wheat varieties that vary in CD-epitope content, as was shown before using immunoblotting and specific monoclonal antibodies. The results showed that a modern bread wheat variety Toronto contained the highest amounts of CD immunogenic peptides compared with the older bread wheat variety Minaret and the tetraploid wheat variety Dibillik Sinde. Our developed method can detect quantitatively and simultaneously multiple specific CD-epitopes in a high throughput manner. PMID:25795397

  6. A universal label-free biosensing platform based on opto-fluidic ring resonators

    NASA Astrophysics Data System (ADS)

    Zhu, Hongying; White, Ian M.; Suter, Jonathan D.; Gohring, John; Fan, Xudong

    2009-02-01

    Rapid and accurate detection of biomolecules is important for medical diagnosis, pharmaceuticals, homeland security, food quality control, and environmental protection. A simple, low cost and highly sensitive label-free optical biosensor based on opto-fluidic ring resonator (OFRR) has been developed that naturally integrates microfluidics with ring resonators. The OFRR employs a piece of fused silica capillary with a diameter around 100 micrometers. The circular cross section of the capillary forms the ring resonator and light repeatedly travels along the resonator circumference in the form of whispering gallery modes (WGMs) through total internal reflection. When the capillary wall is as thin as a couple of micrometers (< 4 μm), an evanescent field of the WGMs exists at the OFRR inner surface and interacts with the sample when it flows through the OFRR. In order to detect the target molecules with high specificity, the OFRR inner surface is functionalized with receptors, such as antibodies, peptide-displayed bacteriophage or oligonucleotide DNA probes. The WGM spectral position shifts when biomolecules bind to the OFRR inner surface and change the local refractive index, which provides quantitative and kinetic information about the biomolecule interaction near the OFRR inner surface. The OFRR has been successfully demonstrated for detection of various types of biomoelcuels. Here, we will first introduce the basic operation principle of the OFRR as a sensor and then application examples of the OFRR in the detection of proteins, disease biomarkers, virus, DNA molecules, and cells with high sensitivities will be presented.

  7. Gallium plasmonic nanoparticles for label-free DNA and single nucleotide polymorphism sensing.

    PubMed

    Marín, Antonio García; García-Mendiola, Tania; Bernabeu, Cristina Navio; Hernández, María Jesús; Piqueras, Juan; Pau, Jose Luis; Pariente, Félix; Lorenzo, Encarnación

    2016-05-01

    A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori onto GaNP/Si substrates; complementary target sequences of Helicobacter pylori can be quantified over the range of 10 pM to 3.0 nM with a detection limit of 6.0 pM and a linear correlation coefficient of R(2) = 0.990. The selectivity of the device allows the detection of a single nucleotide polymorphism (SNP) in a specific sequence of Helicobacter pylori, without the need for a hybridization suppressor in solution such as formamide. Furthermore, it also allows the detection of this sequence in the presence of other pathogens, such as Escherichia coli in the sample. The broad applicability of the system was demonstrated by the detection of a specific gene mutation directly associated with cystic fibrosis in large genomic DNA isolated from blood cells. PMID:27120517

  8. Label-Free Optical Method for Quantifying Molecular Transport Across Cellular Membranes In Vitro.

    PubMed

    Sharifian Gh, Mohammad; Wilhelm, Michael J; Dai, Hai-Lung

    2016-09-01

    We demonstrate a nonlinear optical method for the label-free quantification of membrane transport rates of small/medium size molecules in living cells. Specifically, second-harmonic generation (SHG) laser scattering permits surface-specific characterization of transport across membranes. Unfortunately, most biologically relevant molecules are SHG-inactive. In the interest of extending this methodology for characterizing transport of any molecule, we monitor the SHG produced from an SHG-active reference molecule, in the presence of an SHG-inactive target molecule-of-interest as both molecules compete to cross a membrane. Of significance, the SHG-inactive target transport rate can be deduced as a perturbation in the measured transport rate of the reference. As proof-of-principle, we examine competitive transport of the strongly SHG-active cation, malachite green (MG), in the presence of a weakly SHG-active dication, propidium (Pro), across the outer-membrane protein channels in living bacteria. Comparison of the extracted and directly measured Pro transport rates validates the effectiveness of the method. PMID:27518496

  9. Miniaturized CARS microendoscope probe design for label-free intraoperative imaging

    NASA Astrophysics Data System (ADS)

    Chen, Xu; Wang, Xi; Xu, Xiaoyun; Cheng, Jie; Liu, Zhengfan; Weng, Sheng; Thrall, Michael J.; Goh, Alvin C.; McCormick, Daniel T.; Wong, Kelvin; Wong, Stephen T. C.

    2014-03-01

    A Coherent Anti-Stokes Raman Scattering (CARS) microendoscope probe for early stage label-free prostate cancer diagnosis at single cell resolution is presented. The handheld CARS microendoscope probe includes a customized micro-electromechanical systems (MEMS) scanning mirror as well as miniature optical and mechanical components. In our design, the excitation laser (pump and stokes beams) from the fiber is collimated, reflected by the reflecting mirror, and transmitted via a 2D MEMS scanning mirror and a micro-objective system onto the sample; emission in the epi-direction is returned through the micro-objective lens, MEMS and reflecting mirror, and collimation system, and finally the emission signal is collected by a photomultiplier tube (PMT). The exit pupil diameter of the collimator system is designed to match the diameter of the MEMS mirror and the entrance pupil diameter of the micro-objective system. The back aperture diameter of the micro-objective system is designed according to the largest MEMS scanning angle and the distance between the MEMS mirror and the back aperture. To increase the numerical aperture (NA) of the micro-objective system in order to enhance the signal collection efficiency, the back aperture diameter of the micro-objective system is enlarged with an upfront achromatic wide angle Keplerian telescope beam expander. The integration of a miniaturized micro-optics probe with optical fiber CARS microscopy opens up the possibility of in vivo molecular imaging for cancer diagnosis and surgical intervention.

  10. Label-free three dimensional reconstruction of biological samples (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Aknoun, Sherazade; Bon, Pierre; Savatier, Julien; Monneret, Serge; Wattellier, Benoit F.

    2016-03-01

    We describe the use of spatially incoherent illumination combined with quantitative phase imaging (QPI) [1] to make tridimensional reconstruction of semi-transparent biological samples. Quantitative phase imaging is commonly used with coherent illumination for the relatively simple interpretation of the phase measurement. We propose to use spatially incoherent illumination which is known to increase lateral and axial resolution compared to classical coherent illumination. The goal is to image thick samples with intracellular resolution [2]. The 3D volume is imaged by axially scanning the sample with a quadri-wave lateral shearing interferometer used as a conventional camera while using spatially incoherent white-light illumination (native microscope halogen source) or NIR light. We use a non-modified inverted microscope equipped with a Z-axis piezo stage. A z-stack is recorded by objective translation along the optical axis. The main advantages of this approach are its easy implementation, compared to the other state-of-the-art diffraction tomographic setups, and its speed which makes even label-free 3D living sample imaging possible. A deconvolution algorithm is used to compensate for the loss in contrast due to spatially incoherent illumination. This makes the tomographic volume phase values quantitative. Hence refractive index could be recovered from the optical slices. We will present tomographic reconstruction of cells, thick fixed tissue of few tens of micrometers using white light, and the use of NIR light to reach deeper planes in the tissue.

  11. Label-free three-dimensional reconstruction of biological samples (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Aknoun, Sherazade; Bon, Pierre; Savatier, Julien; Monneret, Serge; Wattellier, Benoit F.

    2016-03-01

    We describe the use of spatially incoherent illumination combined with quantitative phase imaging (QPI) [1] to make tridimensional reconstruction of semi-transparent biological samples. Quantitative phase imaging is commonly used with coherent illumination for the relatively simple interpretation of the phase measurement. We propose to use spatially incoherent illumination which is known to increase lateral and axial resolution compared to classical coherent illumination. The goal is to image thick samples with intracellular resolution [2]. The 3D volume is imaged by axially scanning the sample with a quadri-wave lateral shearing interferometer used as a conventional camera while using spatially incoherent white-light illumination (native microscope halogen source) or NIR light. We use a non-modified inverted microscope equipped with a Z-axis piezo stage. A z-stack is recorded by objective translation along the optical axis. The main advantages of this approach are its easy implementation, compared to the other state-of-the-art diffraction tomographic setups, and its speed which makes even label-free 3D living sample imaging possible. A deconvolution algorithm is used to compensate for the loss in contrast due to spatially incoherent illumination. This makes the tomographic volume phase values quantitative. Hence refractive index could be recovered from the optical slices. We will present tomographic reconstruction of cells, thick fixed tissue of few tens of micrometers using white light, and the use of NIR light to reach deeper planes in the tissue.

  12. Diamagnetic repulsion--a versatile tool for label-free particle handling in microfluidic devices.

    PubMed

    Peyman, Sally A; Kwan, Er Yee; Margarson, Oliver; Iles, Alexander; Pamme, Nicole

    2009-12-25

    We report the exploration of diamagnetic repulsion forces for the selective manipulation of microparticles inside microfluidic devices. Diamagnetic materials such as polymers are repelled from magnetic fields, an effect greatly enhanced by suspending a diamagnetic object in a paramagnetic Mn(2+) solution. The versatility of diamagnetic repulsion is demonstrated for the trapping, focussing and deflection of polystyrene particles for three example applications. Firstly, magnet pairs with unlike poles facing each other were arranged along a microcapillary to trap plugs of differently functionalised particles for a simultaneous surface-based assay in which biotin was selectively bound to a plug of streptavidin coated particles utilising only 22nL of reagent. Secondly, by slightly modifying the magnetic field design, the rapid focussing of particles into a narrow central stream at a flow rate of 650microms(-1) was accomplished for particle pre-concentration. In a third application, 5 and 10microm polystyrene particles were separated from each other in continuous flow by passing the particle mixture through a microfluidic chamber with a perpendicular magnetic field, a method termed diamagnetophoresis. The separation was investigated between flow rates of 20-100microL h(-1), with full resolution of the particle populations being achieved at 20microL h(-1). These experiments show the potential of diamagnetic repulsion for simple, label-free manipulation of particles and other diamagnetic objects such as cells for a range of bioanalytical techniques. PMID:19592004

  13. Progress of new label-free techniques for biosensors: a review.

    PubMed

    Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

    2016-06-01

    The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future. PMID:25608959

  14. Label-Free Imaging of Membrane Potential Using Membrane Electromotility

    PubMed Central

    Oh, Seungeun; Fang-Yen, Christopher; Choi, Wonshik; Yaqoob, Zahid; Fu, Dan; Park, YongKeun; Dassari, Ramachandra R.; Feld, Michael S.

    2012-01-01

    Electrical activity may cause observable changes in a cell's structure in the absence of exogenous reporter molecules. In this work, we report a low-coherence interferometric microscopy technique that can detect an optical signal correlated with the membrane potential changes in individual mammalian cells without exogenous labels. By measuring milliradian-scale phase shifts in the transmitted light, we can detect changes in the cells' membrane potential. We find that the observed optical signals are due to membrane electromotility, which causes the cells to deform in response to the membrane potential changes. We demonstrate wide-field imaging of the propagation of electrical stimuli in gap-junction-coupled cell networks. Membrane electromotility-induced cell deformation may be useful as a reporter of electrical activity. PMID:22828327

  15. Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis.

    PubMed

    Noothalapati, Hemanth; Sasaki, Takahiro; Kaino, Tomohiro; Kawamukai, Makoto; Ando, Masahiro; Hamaguchi, Hiro-O; Yamamoto, Tatsuyuki

    2016-01-01

    Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, β-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and β-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future. PMID:27278218

  16. Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis

    PubMed Central

    Noothalapati, Hemanth; Sasaki, Takahiro; Kaino, Tomohiro; Kawamukai, Makoto; Ando, Masahiro; Hamaguchi, Hiro-o; Yamamoto, Tatsuyuki

    2016-01-01

    Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, β-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and β-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future. PMID:27278218

  17. Biosensing platform combining label-free and labelled analysis using Bloch surface waves

    NASA Astrophysics Data System (ADS)

    Danz, Norbert; Sinibaldi, Alberto; Munzert, Peter; Anopchenko, Aleksei; Förster, Erik; Schmieder, Stefan; Chandrawati, Rona; Rizzo, Riccardo; Heller, Rene; Sonntag, Frank; Mascioletti, Alessandro; Rana, Subinoy; Schubert, Thomas; Stevens, Molly M.; Michelotti, Francesco

    2015-05-01

    Bloch surface waves (BSW) propagating at the boundary of truncated photonic crystals (1D-PC) have emerged as an attractive approach for label-free sensing in plasmon-like sensor configurations. Due to the very low losses in such dielectric thin film stacks, BSW feature very low angular resonance widths compared to the surface plasmon resonance (SPR) case. Besides label-free operation, the large field enhancement and the absence of quenching allow utilizing BSW coupled fluorescence detection to additionally sense the presence of fluorescent labels. This approach can be adapted to the case of angularly resolved resonance detection, thus giving rise to a combined label-free / labelled biosensor platform. It features a parallel analysis of multiple spots arranged as a one-dimensional array inside a microfluidic channel of a disposable chip. Application of such a combined biosensing approach to the detection of the Angiopoietin-2 cancer biomarker in buffer solutions is reported.

  18. An Adaptive Alignment Algorithm for Quality-controlled Label-free LC-MS*

    PubMed Central

    Sandin, Marianne; Ali, Ashfaq; Hansson, Karin; Månsson, Olle; Andreasson, Erik; Resjö, Svante; Levander, Fredrik

    2013-01-01

    Label-free quantification using precursor-based intensities is a versatile workflow for large-scale proteomics studies. The method however requires extensive computational analysis and is therefore in need of robust quality control during the data mining stage. We present a new label-free data analysis workflow integrated into a multiuser software platform. A novel adaptive alignment algorithm has been developed to minimize the possible systematic bias introduced into the analysis. Parameters are estimated on the fly from the data at hand, producing a user-friendly analysis suite. Quality metrics are output in every step of the analysis as well as actively incorporated into the parameter estimation. We furthermore show the improvement of this system by comprehensive comparison to classical label-free analysis methodology as well as current state-of-the-art software. PMID:23306530

  19. Nanochannel array device operating through Prussian blue nanoparticles for sensitive label-free immunodetection of a cancer biomarker.

    PubMed

    Espinoza-Castañeda, Marisol; de la Escosura-Muñiz, Alfredo; Chamorro, Alejandro; de Torres, Carmen; Merkoçi, Arben

    2015-05-15

    A novel nanochannel array (NC) device that operates through Prussian blue nanoparticles (PBNPs) as redox indicator for sensitive label free immunodetection of a cancer biomarker is presented. Stable and narrow-sized (around 4 nm) PBNPs, protected by polyvinylpyrrolidone, exhibited a well-defined and reproducible redox behavior and were successfully applied for the voltammetric evaluation of the nanochannels (20 nm pore sized) blockage due to the immunocomplex formation. The bigger size of the PBNPs compared with ionic indicators such as the [Fe(CN)6](4-/3-) system leads to an increase in the steric effects hindering their diffusion toward the signaling electrode which in turn is transduced to an improvement of the detection limit from 200 µg mL(-1) to 34 pg human IgG mL(-1). This novel and effective PBNPs-NC technology for the detection of small proteins captured inside the nanochannels is successfully applied for the quantification of a cancer biomarker (parathyroid hormone-related protein, PTHrP) in a real clinical scenario such as cell culture medium. The achieved label-free detection of PTHrP at levels of 50 ng mL(-1) is with great interest to study relevant functions that this protein exerts in normal tissues and cancer. PMID:25103338

  20. Label-free multimodal protease detection based on protein/perylene dye coassembly and enzyme-triggered disassembly.

    PubMed

    Lin, Yiyang; Chapman, Robert; Stevens, Molly M

    2014-07-01

    The development of novel assays for protease sensing plays an important role in clinical diagnostics and therapeutics. Herein, we report a supramolecular platform for label-free protease detection, based on protein/dye self-assembly and enzyme-triggered disassembly. In a typical case, coassembly of protamine sulfate and perylene dye via electrostatic attractions and π-π interactions caused significant colorimetric and fluorescent responses. Subsequent addition of trypsin was found to cleave the amide bonds of protein, triggering the dissociation of protein/dye aggregates and the release of perylene dyes. The enzyme-triggered disassembly was transduced into multiple readouts including absorption, fluorescence, and polarization, which were exploited for trypsin detection and inhibitor testing. This assay was also used for turn-on fluorescence detection of cathepsin B, an enzyme known to be overexpressed in mammalian cancer cells. The integration of supramolecular self-assembly into enzyme detection in this work has provided a novel label-free biosensing platform which is highly sensitive with multimodal readouts. The relative simplicity of the approach avoids the need for time-consuming substrate synthesis, and is also amenable to naked eye detection. PMID:24914622

  1. Label-free nonenzymatic glycation monitoring of collagen scaffolds in type 2 diabetic mice by confocal Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Shi, Panpan; Liu, Hanping; Deng, Xiaoyuan; Jin, Ying; Wang, Qiannan; Liu, Hao; Chen, Maosheng; Han, Xue

    2015-02-01

    Collagen is the key target of nonenzymatic glycation during physiopathological processes such as diabetes. The induced changes in the biochemical property of collagen by nonenzymatic glycation remain a major challenge to probe. This study investigated the use of confocal Raman microspectroscopy to label-free monitor the nonenzymatic glycation of collagen scaffolds from type 2 diabetic (T2D) mice at different timepoints (0, 4, 8, and 12 weeks). The glycated collagen scaffolds were obtained through the decellularized dermal matrix method to remove the epidermis layer, subcutaneous tissue, and cells in the dermis and to retain the collagen fibrils. Raman spectra showed no changes in Raman peak positions, which indicated that nonenzymatic glycation could produce no significant changes in the triple-helix structure of collagen in T2D mice. However, the relative intensity of the Raman bands at 921, 1033, 1244, 1274, 1346, 1635, and 1672 cm-1 increased as diabetic time progressed. Correlation analysis suggested that the spectra of these bands had a high positive correlation with the expression of anti-advanced glycation end products obtained by immunofluorescence imaging of the same collagen scaffolds. Confocal Raman microspectroscopy proves a potential tool to label-free monitor the collagen changes caused by nonenzymatic glycation in T2D mice.

  2. Differential Plasma Glycoproteome of p19ARF Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform

    PubMed Central

    Letarte, Simon; Brusniak, Mi-Youn; Campbell, David; Eddes, James; Kemp, Christopher J.; Lau, Hollis; Mueller, Lukas; Schmidt, Alexander; Shannon, Paul; Kelly-Spratt, Karen S.; Vitek, Olga; Zhang, Hui; Aebersold, Ruedi; Watts, Julian D.

    2010-01-01

    A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow

  3. The potential of label-free nonlinear optical molecular microscopy to non-invasively characterize the viability of engineered human tissue constructs.

    PubMed

    Chen, Leng-Chun; Lloyd, William R; Kuo, Shiuhyang; Kim, Hyungjin Myra; Marcelo, Cynthia L; Feinberg, Stephen E; Mycek, Mary-Ann

    2014-08-01

    Nonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine. PMID:24854093

  4. Dip-and-read method for label-free renewable sensing enhanced using complex DNA structures.

    PubMed

    Zhang, Min; Jiang, Xiao-Qin; Le, Huynh-Nhu; Wang, Ping; Ye, Bang-Ce

    2013-02-01

    A label-free assay is reported in this work for the detection of DNA with enhanced sensitivity using complex DNA structures (DNA tetrahedrons) based on the biolayer interferometry. The DNA tetrahedrons help to amplify the optical signals of the biolayer interferometry, thus improving the detection limit of DNA by about 100-fold. We further demonstrated that this method could be expanded to ATP detection by taking advantage of the target-dependent adaptability of aptamers. It appears to us that this new label-free assay promises new opportunities for developing novel biolayer interferometry assays. PMID:23298262

  5. Rapid and label-free amplification and detection assay for genotyping of cancer biomarker.

    PubMed

    Shin, Yong; Soo, Ross A; Yoon, Jaeyun; Perera, Agampodi Promoda; Yoon, Yong-Jin; Park, Mi Kyoung

    2015-06-15

    As understanding of the molecular pathways that drive malignancy in human cancer improves, personalized genotype-based therapy in combination with the predictive biomarker for the efficacy of targeted therapy is becoming more popular in cancer management. Sanger sequencing, that has been the gold standard for mutation analysis in cancer since the 1970s, suffers from low sensitivity, complexity, and time-consuming and labor-intensive procedure. Although several PCR based molecular testing methods are being emerged, there is no universal assay available for genotyping of cancer biomarkers. Here we present a rapid, simple and sensitive assay for the detection of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancers (NSCLCs). The assay employs a novel double mis-matched primer (DMP) set to improve the detection ability of isothermal solid-phase amplification/detection (ISAD) based on silicon microring biosensor. We show that the EGFR-DMP can detect EGFR gene mutations within 20 min in a label-free and real-time manner. The EGFR-DMP was able to detect a mutation in a sample containing only 1% of the mutant cells in a mixture of wild-type cells. Furthermore, to validate the proposed assay for potential applications in clinical diagnostics, we examined paraffin-embedded tissue samples from 10 NSCLC patients for the presence of EGFR mutations by performing EGFR-DMP and direct sequencing. The EGFR-DMP assay was able to rapidly detect the mutation, with high sensitivity and specificity. The EGFR-DMP assay offers a robust and sensitive approach for the rapid identification of the EGFR mutation. The high sensitivity and specificity and rapidity of this approach may make it useful for predicting the clinical response to targeted EGFR TKIs as a companion diagnostic. PMID:25569872

  6. Label-free probe of HIV-1 TAT peptide binding to mimetic membranes.

    PubMed

    Rao, Yi; Kwok, Sheldon J J; Lombardi, Julien; Turro, Nicholas J; Eisenthal, Kenneth B

    2014-09-01

    The transacting activator of transduction (TAT) protein plays a key role in the progression of AIDS. Studies have shown that a +8 charged sequence of amino acids in the protein, called the TAT peptide, enables the TAT protein to penetrate cell membranes. To probe mechanisms of binding and translocation of the TAT peptide into the cell, investigators have used phospholipid liposomes as cell membrane mimics. We have used the method of surface potential sensitive second harmonic generation (SHG), which is a label-free and interface-selective method, to study the binding of TAT to anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1'-rac-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. It is the SHG sensitivity to the electrostatic field generated by a charged interface that enabled us to obtain the interfacial electrostatic potential. SHG together with the Poisson-Boltzmann equation yielded the dependence of the surface potential on the density of adsorbed TAT. We obtained the dissociation constants Kd for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC Kd was found to be 7.5 ± 2 μM, and for POPG Kd was 29.0 ± 4.0 μM. As TAT was added to the liposome solution the POPC surface potential changed from 0 mV to +37 mV, and for POPG it changed from -57 mV to -37 mV. A numerical calculation of Kd, which included all terms obtained from application of the Poisson-Boltzmann equation to the TAT liposome SHG data, was shown to be in good agreement with an approximated solution. PMID:25136100

  7. Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

    PubMed Central

    Rao, Yi; Kwok, Sheldon J. J.; Lombardi, Julien; Turro, Nicholas J.; Eisenthal, Kenneth B.

    2014-01-01

    The transacting activator of transduction (TAT) protein plays a key role in the progression of AIDS. Studies have shown that a +8 charged sequence of amino acids in the protein, called the TAT peptide, enables the TAT protein to penetrate cell membranes. To probe mechanisms of binding and translocation of the TAT peptide into the cell, investigators have used phospholipid liposomes as cell membrane mimics. We have used the method of surface potential sensitive second harmonic generation (SHG), which is a label-free and interface-selective method, to study the binding of TAT to anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-rac-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. It is the SHG sensitivity to the electrostatic field generated by a charged interface that enabled us to obtain the interfacial electrostatic potential. SHG together with the Poisson–Boltzmann equation yielded the dependence of the surface potential on the density of adsorbed TAT. We obtained the dissociation constants Kd for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC Kd was found to be 7.5 ± 2 μM, and for POPG Kd was 29.0 ± 4.0 μM. As TAT was added to the liposome solution the POPC surface potential changed from 0 mV to +37 mV, and for POPG it changed from −57 mV to −37 mV. A numerical calculation of Kd, which included all terms obtained from application of the Poisson–Boltzmann equation to the TAT liposome SHG data, was shown to be in good agreement with an approximated solution. PMID:25136100

  8. High-throughput label-free image cytometry and image-based classification of live Euglena gracilis

    PubMed Central

    Lei, Cheng; Ito, Takuro; Ugawa, Masashi; Nozawa, Taisuke; Iwata, Osamu; Maki, Masanori; Okada, Genki; Kobayashi, Hirofumi; Sun, Xinlei; Tiamsak, Pimsiri; Tsumura, Norimichi; Suzuki, Kengo; Di Carlo, Dino; Ozeki, Yasuyuki; Goda, Keisuke

    2016-01-01

    We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner. PMID:27446699

  9. High-throughput label-free image cytometry and image-based classification of live Euglena gracilis.

    PubMed

    Lei, Cheng; Ito, Takuro; Ugawa, Masashi; Nozawa, Taisuke; Iwata, Osamu; Maki, Masanori; Okada, Genki; Kobayashi, Hirofumi; Sun, Xinlei; Tiamsak, Pimsiri; Tsumura, Norimichi; Suzuki, Kengo; Di Carlo, Dino; Ozeki, Yasuyuki; Goda, Keisuke

    2016-07-01

    We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner. PMID:27446699

  10. Label-Free Characterization of Emerging Human Neuronal Networks

    NASA Astrophysics Data System (ADS)

    Mir, Mustafa; Kim, Taewoo; Majumder, Anirban; Xiang, Mike; Wang, Ru; Liu, S. Chris; Gillette, Martha U.; Stice, Steven; Popescu, Gabriel

    2014-03-01

    The emergent self-organization of a neuronal network in a developing nervous system is the result of a remarkably orchestrated process involving a multitude of chemical, mechanical and electrical signals. Little is known about the dynamic behavior of a developing network (especially in a human model) primarily due to a lack of practical and non-invasive methods to measure and quantify the process. Here we demonstrate that by using a novel optical interferometric technique, we can non-invasively measure several fundamental properties of neural networks from the sub-cellular to the cell population level. We applied this method to quantify network formation in human stem cell derived neurons and show for the first time, correlations between trends in the growth, transport, and spatial organization of such a system. Quantifying the fundamental behavior of such cell lines without compromising their viability may provide an important new tool in future longitudinal studies.

  11. Discriminating dengue-infected hepatic cells (WRL-68) using dielectrophoresis.

    PubMed

    Yafouz, Bashar; Kadri, Nahrizul Adib; Rothan, Hussin A; Yusof, Rohana; Ibrahim, Fatimah

    2016-02-01

    Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a nonuniform electric field, has been used as a potential technique for manipulation and separation of many biological samples without destructive consequences to the cell. Cells of the same genotype in different physiological and pathological states have unique morphological and structural features, therefore, it is possible to differentiate between them using their DEP responses. This paper reports the experimental discrimination of normal and dengue-infected human hepatic fetal epithelial cells (WRL-68 cells) based on their DEP crossover frequency, at which no resultant movement occurs in the cells in response to the DEP force. A microarray dot electrode was used to conduct the DEP experiments. The DEP forces applied to the cells were quantified by analyzing the light intensity shift within the electrode's dot region based on the Cumulative Modal Intensity Shift image analysis technique. The differences in dielectric properties between infected and uninfected cells were exploited by plotting a unique DEP spectrum for each set of cells. We observed that the crossover frequency decreased from 220 kHz for the normal WRL-68 cells to 140 kHz after infection with the dengue virus in a medium conductivity of 100 μS/cm. We conclude that the change in the DEP crossover frequency between dengue-infected cells and their healthy counterparts should allow direct characterization of these cell types by exploiting their electrophysiological properties. PMID:26530354

  12. KDAC8 substrate specificity quantified by a biologically relevant, label-free deacetylation assay.

    PubMed

    Toro, Tasha B; Watt, Terry J

    2015-12-01

    Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme-substrate pairs of label-free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine-based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady-state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry-based experiments and cell-based experiments will allow the identification of specific KDAC-substrate pairs and lead to a better understanding of the biological consequences of these interactions. PMID:26402585

  13. New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

    PubMed Central

    Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay L.; Barker, N. Scott; Haverstick, Doris; Utz, Marcel; Landers, James P.

    2012-01-01

    Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, and enables label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques. PMID:22423674

  14. Asynchronous magnetic bead rotation (AMBR) micro-viscometer for rapid, sensitive and label-free studies of bacterial growth and drug sensitivity

    PubMed Central

    Sinn, Irene; Albertson, Theodore; Kinnunen, Paivo; Breslauer, David N.; McNaughton, Brandon H.; Burns, Mark A.; Kopelman, Raoul

    2012-01-01

    The long turnaround time in antimicrobial susceptibility testing (AST) endangers patients and encourages the administration of wide spectrum antibiotics, thus resulting in alarming increases of multi-drug resistant pathogens. A method for faster detection of bacterial proliferation presents one avenue towards addressing this global concern. We report on a label-free asynchronous magnetic bead rotation (AMBR) based viscometry method that rapidly detects bacterial growth and determines drug sensitivity by measuring changes in the suspension’s viscosity. With this platform, we observed the growth of a uropathogenic Escherichia coli isolate, with an initial concentration of 50 cells per drop, within 20 minutes; in addition, we determined the gentamicin minimum inhibitory concentration (MIC) of the E. coli isolate within 100 minutes. We thus demonstrated a label-free, micro-viscometer platform that can measure bacterial growth and drug susceptibility more rapidly, with lower initial bacterial counts than existing commercial systems, and potentially with any microbial strains. PMID:22507307

  15. Ag nanocluster-based label-free catalytic and molecular beacons for amplified biosensing.

    PubMed

    Gong, Liang; Kuai, Hailan; Ren, Songlei; Zhao, Xu-Hua; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong

    2015-08-01

    By employing DNAzyme as a recognition group and amplifier, and DNA-stabilized silver nanoclusters (DNA/AgNCs) as signal reporters, we reported for the first time a label-free catalytic and molecular beacon as an amplified biosensing platform for highly selective detection of cofactors such as Pb(2+) and L-histidine. PMID:26120805

  16. Functionalized Polymer Microgel Particles Enable Customizable Production of Label-Free Sensor Arrays.

    PubMed

    Lifson, Mark A; Carter, Jared A; Miller, Benjamin L

    2015-08-01

    Probe molecule immobilization onto surfaces is a critical step in the production of many analytical devices, including labeled and label-free microarrays. New methods to increase the density and uniformity of probe deposition have the potential to significantly enhance the ultimate limits of detection and reproducibility. Hydrogel-based materials have been employed in the past to provide a 3D protein-friendly surface for deposition of antibodies and nucleic acids. However, these methods are susceptible to variation during polymerization of the hydrogel scaffold and provide limited opportunities for tuning deposition parameters on an antibody-by-antibody basis. In this work, a versatile hydrogel nanoparticle deposition method was developed for the production of label-free microarrays and tested in the context of antibody-antigen binding. Poly(N-isopropylacrylamide) nanoparticles (PNIPAM) were conjugated to antibodies using an avidin/biotin system and deposited onto surfaces using a noncontact printing system. After drying, these gel spots formed uniform and thin layers <10 nm in height. The conjugates were characterized with dynamic light scattering, scanning electron microscopy, and atomic force microscopy. We tested this format in the context of tumor necrosis factor-alpha (TNF-α) detection via arrayed imaging reflectometry (AIR), a label-free protein microarray method. This method of probe molecule deposition should be generally useful in the production of microarrays for label-free detection. PMID:26140413

  17. Surface Plasmon Resonance Label-Free Monitoring of Antibody Antigen Interactions in Real Time

    ERIC Educational Resources Information Center

    Kausaite, Asta; van Dijk, Martijn; Castrop, Jan; Ramanaviciene, Almira; Baltrus, John P.; Acaite, Juzefa; Ramanavicius, Arunas

    2007-01-01

    Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without…

  18. A scanning acoustic microscope discriminates cancer cells in fluid

    NASA Astrophysics Data System (ADS)

    Miura, Katsutoshi; Yamamoto, Seiji

    2015-10-01

    Scanning acoustic microscopy (SAM) discriminates lesions in sections by assessing the speed of sound (SOS) or attenuation of sound (AOS) through tissues within a few minutes without staining; however, its clinical use in cytological diagnosis is unknown. We applied a thin layer preparation method to observe benign and malignant effusions using SAM. Although SAM is inferior in detecting nuclear features than light microscopy, it can differentiate malignant from benign cells using the higher SOS and AOS values and large irregular cell clusters that are typical features of carcinomas. Moreover, each single malignant cell exhibits characteristic cytoplasmic features such as a large size, irregular borders and secretory or cytoskeletal content. By adjusting the observation range, malignant cells are differentiated from benign cells easily using SAM. Subtle changes in the functional and structural heterogeneity of tumour cells were pursuable with a different digital data of SAM. SAM can be a useful tool for screening malignant cells in effusions before light microscopic observation. Higher AOS values in malignant cells compared with those of benign cells support the feasibility of a novel sonodynamic therapy for malignant effusions.

  19. Gallium plasmonic nanoparticles for label-free DNA and single nucleotide polymorphism sensing

    NASA Astrophysics Data System (ADS)

    Marín, Antonio García; García-Mendiola, Tania; Bernabeu, Cristina Navio; Hernández, María Jesús; Piqueras, Juan; Pau, Jose Luis; Pariente, Félix; Lorenzo, Encarnación

    2016-05-01

    A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori onto GaNP/Si substrates; complementary target sequences of Helicobacter pylori can be quantified over the range of 10 pM to 3.0 nM with a detection limit of 6.0 pM and a linear correlation coefficient of R2 = 0.990. The selectivity of the device allows the detection of a single nucleotide polymorphism (SNP) in a specific sequence of Helicobacter pylori, without the need for a hybridization suppressor in solution such as formamide. Furthermore, it also allows the detection of this sequence in the presence of other pathogens, such as Escherichia coli in the sample. The broad applicability of the system was demonstrated by the detection of a specific gene mutation directly associated with cystic fibrosis in large genomic DNA isolated from blood cells.A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori

  20. Label-free in vivo imaging of Drosophila melanogaster by multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Chiao-Ying; Hovhannisyan, Vladimir; Wu, June-Tai; Lin, Sung-Jan; Lin, Chii-Wann; Chen, Jyh-Horng; Dong, Chen-Yuan

    2008-02-01

    The fruit fly Drosophila melanogaster is one of the most valuable organisms in genetic and developmental biology studies. Drosophila is a small organism with a short life cycle, and is inexpensive and easy to maintain. The entire genome of Drosophila has recently been sequenced (cite the reference). These advantages make fruit fly an attractive model organism for biomedical researches. Unlike humans, Drosophila can be subjected to genetic manipulation with relative ease. Originally, Drosophila was mostly used in classical genetics studies. In the model era of molecular biology, the fruit fly has become a model organ for developmental biology researches. In the past, numerous molecularly modified mutants with well defined genetic defects affecting different aspects of the developmental processes have been identified and studied. However, traditionally, the developmental defects of the mutant flies are mostly examined in isolated fixed tissues which preclude the observation of the dynamic interaction of the different cell types and the extracellular matrix. Therefore, the ability to image different organelles of the fruit fly without extrinsic labeling is invaluable for Drosophila biology. In this work, we successfully acquire in vivo images of both developing muscles and axons of motor neurons in the three larval stages by using the minimially invasive imaging modality of multiphoton (SHG) microscopy. We found that while SHG imaging is useful in revealing the muscular architecture of the developing larva, it is the autofluorescence signal that allows label-free imaging of various organelles to be achieved. Our results demonstrate that multiphoton imaging is a powerful technique for investigation the development of Drosophila.

  1. Label-Free Density Measurements of Radial Peripapillary Capillaries in the Human Retina

    PubMed Central

    Yu, Paula K.; Balaratnasingam, Chandrakumar; Xu, Jing; Morgan, William H.; Mammo, Zaid; Han, Sherry; Mackenzie, Paul; Merkur, Andrew; Kirker, Andrew; Albiani, David; Sarunic, Marinko V.; Yu, Dao-Yi

    2015-01-01

    Radial peripapillary capillaries (RPCs) comprise a unique network of capillary beds within the retinal nerve fibre layer (RNFL) and play a critical role in satisfying the nutritional requirements of retinal ganglion cell (RGC) axons. Understanding the topographical and morphological characteristics of these networks through in vivo techniques may improve our understanding about the role of RPCs in RGC axonal health and disease. This study utilizes a novel, non-invasive and label-free optical imaging technique, speckle variance optical coherence tomography (svOCT), for quantitatively studying RPC networks in the human retina. Six different retinal eccentricities from 16 healthy eyes were imaged using svOCT. The same eccentricities were histologically imaged in 9 healthy donor eyes with a confocal scanning laser microscope. Donor eyes were subject to perfusion-based labeling techniques prior to retinal dissection, flat mounting and visualization with the microscope. Capillary density and diameter measurements from each eccentricity in svOCT and histological images were compared. Data from svOCT images were also analysed to determine if there was a correlation between RNFL thickness and RPC density. The results are as follows: (1) The morphological characteristics of RPC networks on svOCT images are comparable to histological images; (2) With the exception of the nasal peripapillary region, there were no significant differences in RPC density measurements between svOCT and histological images; (3) Capillary diameter measurements were significantly greater in svOCT images compared to histology; (4) There is a positive correlation between RPC density and RNFL thickness. The findings in this study suggest that svOCT is a reliable modality for analyzing RPC networks in the human retina. It may therefore be a valuable tool for aiding our understanding about vasculogenic mechanisms that are involved in RGC axonopathies. Further work is required to explore the reason for

  2. Label-free CEST MRI Detection of Citicoline-Liposome Drug Delivery in Ischemic Stroke

    PubMed Central

    Liu, Huanling; Jablonska, Anna; Li, Yuguo; Cao, Suyi; Liu, Dexiang; Chen, Hanwei; Van Zijl, Peter CM; Bulte, Jeff W.M.; Janowski, Miroslaw; Walczak, Piotr; Liu, Guanshu

    2016-01-01

    ABSTRACT Citicoline (CDPC) is a natural supplement with well-documented neuroprotective effects in the treatment of neurodegenerative diseases. In the present study, we sought to exploit citicoline as a theranostic agent with its inherent chemical exchange saturation transfer (CEST) MRI signal, which can be directly used as an MRI guidance in the citicoline drug delivery. Our in vitro CEST MRI results showed citicoline has two inherent CEST signals at +1 and +2 ppm, attributed to exchangeable hydroxyl and amine protons, respectively. To facilitate the targeted drug delivery of citicoline to ischemic regions, we prepared liposomes encapsulating citicoline (CDPC-lipo) and characterized the particle properties and CEST MRI properties. The in vivo CEST MRI detection of liposomal citicoline was then examined in a rat brain model of unilateral transient ischemia induced by a two-hour middle cerebral artery occlusion. The results showed that the delivery of CPDC-lipo to the brain ischemic areas could be monitored and quantified by CEST MRI. When administered intra-arterially, CDPC-lipo clearly demonstrated a detectable CEST MRI contrast at 2 ppm. CEST MRI revealed that liposomes preferentially accumulated in the areas of ischemia with a disrupted blood-brain-barrier. We furthermore used CEST MRI to detect the improvement in drug delivery using CDPC-lipo targeted against vascular cell adhesion molecule (VCAM)-1 in the same animal model. The MRI findings were validated using fluorescence microscopy. Hence, liposomal citicoline represents a prototype theranostic system, where the therapeutic agent can be detected directly by CEST MRI in a label-free fashion. PMID:27446492

  3. Label-free CEST MRI Detection of Citicoline-Liposome Drug Delivery in Ischemic Stroke.

    PubMed

    Liu, Huanling; Jablonska, Anna; Li, Yuguo; Cao, Suyi; Liu, Dexiang; Chen, Hanwei; Van Zijl, Peter Cm; Bulte, Jeff W M; Janowski, Miroslaw; Walczak, Piotr; Liu, Guanshu

    2016-01-01

    ABSTRACT Citicoline (CDPC) is a natural supplement with well-documented neuroprotective effects in the treatment of neurodegenerative diseases. In the present study, we sought to exploit citicoline as a theranostic agent with its inherent chemical exchange saturation transfer (CEST) MRI signal, which can be directly used as an MRI guidance in the citicoline drug delivery. Our in vitro CEST MRI results showed citicoline has two inherent CEST signals at +1 and +2 ppm, attributed to exchangeable hydroxyl and amine protons, respectively. To facilitate the targeted drug delivery of citicoline to ischemic regions, we prepared liposomes encapsulating citicoline (CDPC-lipo) and characterized the particle properties and CEST MRI properties. The in vivo CEST MRI detection of liposomal citicoline was then examined in a rat brain model of unilateral transient ischemia induced by a two-hour middle cerebral artery occlusion. The results showed that the delivery of CPDC-lipo to the brain ischemic areas could be monitored and quantified by CEST MRI. When administered intra-arterially, CDPC-lipo clearly demonstrated a detectable CEST MRI contrast at 2 ppm. CEST MRI revealed that liposomes preferentially accumulated in the areas of ischemia with a disrupted blood-brain-barrier. We furthermore used CEST MRI to detect the improvement in drug delivery using CDPC-lipo targeted against vascular cell adhesion molecule (VCAM)-1 in the same animal model. The MRI findings were validated using fluorescence microscopy. Hence, liposomal citicoline represents a prototype theranostic system, where the therapeutic agent can be detected directly by CEST MRI in a label-free fashion. PMID:27446492

  4. Direct label-free electrochemical detection of proteins using the polarized oil/water interface.

    PubMed

    Osakai, Toshiyuki; Yuguchi, Yukiko; Gohara, Emi; Katano, Hajime

    2010-07-01

    Voltammetric behaviors of various globular proteins, including cytochrome c, ribonuclease A, lysozyme, albumin, myoglobin, and alpha-lactalbumin, were studied at the polarized 1,2-dichloroethane/water (DCE/W) interface in the presence of four different anionic surfactants, that is, dinonylnaphthalenesulfonate (DNNS), bis(2-ethylhexyl)sulfosuccinate (Aerosol-OT; AOT), bis(2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl)sulfosuccinate (BDFHS), and bis(2-ethylhexyl)phosphate (BEHP). When the W phase was acidic (pH = approximately 3.4), the surfactants (except for BEHP) added to DCE facilitated the adsorption of the above proteins to the DCE/W interface and gave a well-developed voltammetric wave due to the adsorption/desorption of the proteins. This voltammetric wave, which we here call "protein wave", is promising for direct label-free electrochemical detection of proteins. The current for the adsorption of a protein to the interface showed a linear dependence on the protein concentration in the presence of excess surfactant. The foot potential at which the protein wave appeared in cyclic voltammetry showed different values depending on the natures of the protein and surfactant. Multivariate analysis for the foot potentials determined for different proteins with different surfactants revealed that the protein selectivity should depend on the charged, polar, and nonpolar surface areas of a protein molecule. On the basis of these voltammetric studies, it was shown in principle that online electrochemical separation/determination of proteins could be performed using a two-step oil/water-type flow-cell system. PMID:20462245

  5. Label-Free Density Measurements of Radial Peripapillary Capillaries in the Human Retina.

    PubMed

    Yu, Paula K; Balaratnasingam, Chandrakumar; Xu, Jing; Morgan, William H; Mammo, Zaid; Han, Sherry; Mackenzie, Paul; Merkur, Andrew; Kirker, Andrew; Albiani, David; Sarunic, Marinko V; Yu, Dao-Yi

    2015-01-01

    Radial peripapillary capillaries (RPCs) comprise a unique network of capillary beds within the retinal nerve fibre layer (RNFL) and play a critical role in satisfying the nutritional requirements of retinal ganglion cell (RGC) axons. Understanding the topographical and morphological characteristics of these networks through in vivo techniques may improve our understanding about the role of RPCs in RGC axonal health and disease. This study utilizes a novel, non-invasive and label-free optical imaging technique, speckle variance optical coherence tomography (svOCT), for quantitatively studying RPC networks in the human retina. Six different retinal eccentricities from 16 healthy eyes were imaged using svOCT. The same eccentricities were histologically imaged in 9 healthy donor eyes with a confocal scanning laser microscope. Donor eyes were subject to perfusion-based labeling techniques prior to retinal dissection, flat mounting and visualization with the microscope. Capillary density and diameter measurements from each eccentricity in svOCT and histological images were compared. Data from svOCT images were also analysed to determine if there was a correlation between RNFL thickness and RPC density. The results are as follows: (1) The morphological characteristics of RPC networks on svOCT images are comparable to histological images; (2) With the exception of the nasal peripapillary region, there were no significant differences in RPC density measurements between svOCT and histological images; (3) Capillary diameter measurements were significantly greater in svOCT images compared to histology; (4) There is a positive correlation between RPC density and RNFL thickness. The findings in this study suggest that svOCT is a reliable modality for analyzing RPC networks in the human retina. It may therefore be a valuable tool for aiding our understanding about vasculogenic mechanisms that are involved in RGC axonopathies. Further work is required to explore the reason for

  6. Real time and label free profiling of clinically relevant exosomes.

    PubMed

    Sina, Abu Ali Ibn; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G; Shiddiky, Muhammad J A; Trau, Matt

    2016-01-01

    Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(-) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14-35% of their bulk population express HER2. PMID:27464736

  7. Sub-micrometric reflectometry for localized label-free biosensing.

    PubMed

    Casquel, R; Soler, J A; Holgado, M; López, A; Lavín, A; de Vicente, J; Sanza, F J; Laguna, M F; Bañuls, M J; Puchades, R

    2015-05-18

    In this work we present an optical technique for characterizing sub-micrometric areas based on reflectivity of the light as a function of angle of incidence for the two pure polarizations s and p, covering a range of angles of incidence from -71.80° to 71.80° with a resolution of 0.1°. Circular areas with a diameter in the order of 600 nm can be characterized, and the spectra for the two polarizations can be obtained with a single measurement. For biosensing purposes, we have fabricated several Bio Photonic Sensing Cells (BICELLs) consisting of interferometers of 1240 nm of SU-8 polymer over silicon. An indirect immunoassay is performed over these BICELLs and compared experimentally with FT-VIS-NIR spectrometry and theoretical calculations. The Limit of Detection (LoD) achieved is comparable with standard high resolution spectrometry, but with the capability of analyzing sub-micrometric domains for immunoassays reactions onto a sensing surface. PMID:26074509

  8. Real time and label free profiling of clinically relevant exosomes

    PubMed Central

    Sina, Abu Ali Ibn; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G.; Shiddiky, Muhammad J. A.; Trau, Matt

    2016-01-01

    Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(−) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14–35% of their bulk population express HER2. PMID:27464736

  9. Label-Free Analysis of Cellular Lipid Droplet Formation by Non-Linear Microscopy

    NASA Astrophysics Data System (ADS)

    Schie, Iwan W.

    Cellular lipid droplets (LD) are cellular organelles that can be found in every cell type. Recent research indicates that cellular LD are involved in a large number of cellular metabolic functions, such as lipid metabolism, protection from lipotoxicity, protein storage and degradation, and many more. LD formation is frequently associated with adverse health effects, i.e. alcoholic and non-alcoholic fatty liver disease, diabetes type-2, as well as many cardiovascular disorders. Despite their wide presence, LDs are the least studied and most poorly understood cellular organelles. Typically, LDs are investigated using fluorescence-based techniques that require staining with exogenous fluorophores. Other techniques, e.g. biochemical assays, require the destruction of cells that prohibit the analysis of living cells. Therefore, in my thesis research I developed a novel compound fast-scanning nonlinear optical microscope equipped with the ability to also acquire Raman spectra at specific image locations. This system allows us to image label-free cellular LD formation in living cells and analyze the composition of single cellular LDs. Images can be acquired at near video-rate (˜16 frames/s). Furthermore, the system has the ability to acquire very large images of tissue of up to 7.5x15 cm2 total area by stitching together scans with dimensions of 1x1 mm2 in less than 1 minute. The system also enables the user to acquire Raman spectra from points of interest in the multiphoton images and provides chemically-specific data from sample volumes as small as 1 femtoliter. In my thesis I used this setup to determine the effects of VLDL lipolysis products on primary rat hepatocytes. By analyzing the Raman spectra and comparing the peak ratios for saturated and unsaturated fatty acid it was determined that the small cellular LD are highly saturated, while large cellular LDs contain mostly unsaturated lipids. Furthermore, I established a method to determine the specific contribution

  10. Tethering of spherical DOTAP liposome gold nanoparticles on cysteamine monolayer for sensitive label free electrochemical detection of DNA and transfection.

    PubMed

    Bhuvana, Mohanlal; Dharuman, Venkataraman

    2014-05-21

    Construction of spherical liposomes is critical for developing tools for targeted gene and drug delivery applications in biotechnology and medicine, however, it has been demonstrated only in solution phase until now. Spherical liposome tethering on pristine thiol monolayer on gold transducer and its application to label free DNA sensing and transfection has rarely been reported. Here, we report tethering of spherical 1,2-dioleoyltrimethylammoniumpropane liposome-gold nanoparticle (DOTAP-AuNP) on amine terminated monolayer by simple electrostatic interaction on gold transducer for the first time. Cuddling of cationic liposome by AuNP prevents spherical vesicle fusion in both liquid and solid phases, an essential criterion required for gene and drug delivery applications. The spherical nature of DOTAP-AuNPs on a gold surface is confirmed electrochemically using both [Fe(CN)6](3-/4-) and [Ru(NH3)6](3+) redox probes. Atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS) and ultraviolet-visible (UV) spectroscopic techniques confirm the robust nature of spherical liposome-AuNPs on solid and in liquid phases. The surface is applied for label free DNA hybridization and single nucleotide polymorphism detections sensitively and selectively without signal amplification. The lowest target DNA concentration detected is 100 attomole. DNA transfection is made simply by dropping E. coli cells on DOTAP-AuNP-DNA immobilized transducer surface. The difference between the fluorescent image of transfected E. coli and the differential interference contrast image of E. coli cells by confocal laser scanning microscopy (CLSM) confirms the efficiency and simplicity of the transfection method developed in terms of reduced cost and reagents. PMID:24652193

  11. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    NASA Astrophysics Data System (ADS)

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  12. Diazonium-based impedimetric aptasensor for the rapid label-free detection of Salmonella typhimurium in food sample.

    PubMed

    Bagheryan, Zahra; Raoof, Jahan-Bakhsh; Golabi, Mohsen; Turner, Anthony P F; Beni, Valerio

    2016-06-15

    Fast and accurate detection of microorganisms is of key importance in clinical analysis and in food and water quality monitoring. Salmonella typhimurium is responsible for about a third of all cases of foodborne diseases and consequently, its fast detection is of great importance for ensuring the safety of foodstuffs. We report the development of a label-free impedimetric aptamer-based biosensor for S. typhimurium detection. The aptamer biosensor was fabricated by grafting a diazonium-supporting layer onto screen-printed carbon electrodes (SPEs), via electrochemical or chemical approaches, followed by chemical immobilisation of aminated-aptamer. FTIR-ATR, contact angle and electrochemical measurements were used to monitor the fabrication process. Results showed that electrochemical immobilisation of the diazonium-grafting layer allowed the formation of a denser aptamer layer, which resulted in higher sensitivity. The developed aptamer-biosensor responded linearly, on a logarithm scale, over the concentration range 1 × 10(1) to 1 × 10(8)CFU mL(-1), with a limit of quantification (LOQ) of 1 × 10(1) CFU mL(-1) and a limit of detection (LOD) of 6 CFU mL(-1). Selectivity studies showed that the aptamer biosensor could discriminate S. typhimurium from 6 other model bacteria strains. Finally, recovery studies demonstrated its suitability for the detection of S. typhimurium in spiked (1 × 10(2), 1 × 10(4) and 1 × 10(6) CFU mL(-1)) apple juice samples. PMID:26894987

  13. Label-free DNA-based detection of Mycobacterium tuberculosis and rifampicin resistance through hydration induced stress in microcantilevers.

    PubMed

    Domínguez, Carmen M; Kosaka, Priscila M; Sotillo, Alma; Mingorance, Jesús; Tamayo, Javier; Calleja, Montserrat

    2015-02-01

    We have developed a label-free assay for the genomic detection of Mycobacterium tuberculosis and rifampicin resistance. The method relies on the quantification of the hydration induced stress on microcantilever biosensors functionalized with oligonucleotide probes, before and after hybridization with specific targets. We have found a limit of detection of 10 fg/mL for PCR amplified products of 122 bp. Furthermore, the technique can successfully target genomic DNA (gDNA) fragments of length >500 bp, and it can successfully discriminate single mismatches. We have used both loci IS6110 and rpoB as targets to detect the mycobacteria and the rifampicin resistance from gDNA directly extracted from bacterial culture and without PCR amplification. We have been able to detect 2 pg/mL target concentration in samples with an excess of interfering DNA and in a total analysis time of 1 h and 30 min. The detection limit found demonstrates the capability to develop direct assays without the need for long culture steps or PCR amplification. The methodology can be easily translated to different microbial targets, and it is suitable for further development of miniaturized devices and multiplexed detection. PMID:25599922

  14. Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor

    NASA Astrophysics Data System (ADS)

    Zaffino, R. L.; Mir, M.; Samitier, J.

    2014-03-01

    We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications.

  15. Label-free detection of Phytophthora ramorum using surface-enhanced Raman spectroscopy.

    PubMed

    Yüksel, Sezin; Schwenkbier, Lydia; Pollok, Sibyll; Weber, Karina; Cialla-May, Dana; Popp, Jürgen

    2015-11-01

    In this study, we report on a novel approach for the label-free and species-specific detection of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman scattering (SERS). In this context, we consider the entire analysis chain including sample preparation, DNA isolation, amplification and hybridization on SERS substrate-immobilized adenine-free capture probes. Thus, the SERS-based detection of target DNA is verified by the strong spectral feature of adenine which indicates the presence of hybridized target DNA. This property was realized by replacing adenine moieties in the species-specific capture probes with 2-aminopurine. In the case of the matching capture and target sequence, the characteristic adenine peak serves as an indicator for specific DNA hybridization. Altogether, this is the first assay demonstrating the detection of a plant pathogen from an infected plant material by label-free SERS employing DNA hybridization on planar SERS substrates consisting of silver nanoparticles. PMID:26393411

  16. Combined label-free optical and optoacoustic imaging of model organisms at mesoscopy and microscopy resolutions

    NASA Astrophysics Data System (ADS)

    Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

    2016-03-01

    We present a multi-scale imaging system that integrates five optoacoustic and multi-photon modalities into the same device. The hybrid microscope offers a unique zoom-in ability by allowing for optoacoustic microscopy and mesoscopy scans of the sample within the same imaging framework. Furthermore, by combining several label-free modalities, we are able to visualize a broad range of anatomical features, taking advantage of their complementary contrast mechanisms. We characterize the spatial resolution and relative orientation of the different sub-modalities and demonstrate the system's performance by the imaging of several model organisms ex vivo. The presented ability to dynamically vary scanning volume and resolution together with its multi-contrast and label-free imaging capabilities make the hybrid microscope a promising tool for comprehensive biological imaging.

  17. Enzymatic Polymerization on DNA Modified Gold Nanowire for Label-Free Detection of Pathogen DNA

    PubMed Central

    Jeong, Jaepil; Kim, Hyejin; Lee, Jong Bum

    2015-01-01

    This paper presents a label-free biosensor for the detection of single-stranded pathogen DNA through the target-enhanced gelation between gold nanowires (AuNW) and the primer DNAs branched on AuNW. The target DNA enables circularization of the linear DNA template, and the primer DNA is elongated continuously via rolling circle amplification. As a result, in the presence of the target DNA, a macroscopic hydrogel was fabricated by the entanglement of the elongated DNA with AuNWs as a scaffold fiber for effective gelation. In contrast, very small separate particles were generated in the absence of the target DNA. This label-free biosensor might be a promising tool for the detection of pathogen DNAs without any devices for further analysis. Moreover, the biosensor based on the weaving of AuNW and DNAs suggests a novel direction for the applications of AuNWs in biological engineering. PMID:26084045

  18. Fabrication of a label-free plasmon immunosensor based on triangular silver nanoplates

    NASA Astrophysics Data System (ADS)

    Dong, Peipei; Lin, Yuanyuan; Di, Junwei

    2013-08-01

    In this work, we have firstly electrodeposited small gold seeds (average diameter of ~40 nm) onto transparent indium tin oxide (ITO) thin film coated glass. Then silver triangular nanoplates with edge lengths of ~200 nm were fabricated using seed-mediated growth method. The localized surface plasmon resonance (LSPR) peak was located at ~700 nm. Finally, a label-free plasmon immunosensor was prepared by directly immobilizing goat anti-mouse IgG onto silver surface. The performance of the LSPR immunosensor was investigated. The red-shift of the biosensor was linearly proportional to mouse IgG concentration ranged from 5 ng/mL to 500 ng/mL, with a detection limit of 2 ng/mL. The label-free immunosensor was simple, sensitive and selective.

  19. Plasma Biomarker Discovery Using 3D Protein Profiling Coupled with Label-Free Quantitation

    PubMed Central

    Beer, Lynn A.; Tang, Hsin-Yao; Barnhart, Kurt T.; Speicher, David W.

    2011-01-01

    In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC–MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC–MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as “apparent” biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC–MS/MS with label-free quantitation of candidate biomarkers. PMID:21468938

  20. Microgel photonics and lab on fiber technology for advanced label-free fiber optic nanoprobes

    NASA Astrophysics Data System (ADS)

    Giaquinto, M.; Micco, A.; Aliberti, A.; Ricciardi, A.; Ruvo, M.; Cutolo, A.; Cusano, A.

    2016-05-01

    We experimentally demonstrate a novel optical fiber label free optrode platform resulting from the integration between two rapidly emerging technologies such as Lab-on-Fiber Technology (LOFT) and Microgel Photonics (MPs). The device consists of a microgel (MG) layer painted on a metallic slabs supporting plasmonic resonances, directly integrated on the optical fiber tip. A molecular binding event induces significant changes in the MG layer thickness (and consequently in its 'equivalent' refractive index) resulting in an evident wavelength shift of the resonant feature. As a case of study, glucose-responsive MGs have been synthesized by incorporating into the gel matrix boronic acid moieties, whose interaction with glucose rules the driving forces for gel swelling. Our results pave the way for new technological routes aimed to develop advanced label free fiber optic nanoprobes.

  1. Combining microscopy with mesoscopy using optical and optoacoustic label-free modes

    NASA Astrophysics Data System (ADS)

    Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

    2015-08-01

    Biology requires observations at multiple geometrical scales, a feature that is not typically offered by a single imaging modality. We developed a hybrid optical system that not only provides different contrast modes but also offers imaging at different geometrical scales, achieving uniquely broad resolution and a 1000-fold volume sampling increase compared to volumes scanned by optical microscopy. The system combines optoacoustic mesoscopy, optoacoustic microscopy and two-photon microscopy, the latter integrating second and third harmonic generation modes. Label-free imaging of a mouse ear and zebrafish larva ex-vivo demonstrates the contrast and scale complementarity provided by the hybrid system. We showcase the superior anatomical orientation offered by the label-free capacity and hybrid operation, over fluorescence microscopy, and the dynamic selection between field of view and resolution achieved, leading to new possibilities in biological visualization.

  2. Label-free proteomics uncovers energy metabolism and focal adhesion regulations responsive for endometrium receptivity.

    PubMed

    Chen, Qian; Zhang, Aijun; Yu, Feng; Gao, Jing; Liu, Yue; Yu, Chengli; Zhou, Hu; Xu, Chen

    2015-04-01

    The menstrual cycle of the female uterus leads to periodic changes of the endometrium. These changes are important for developing the endometrial receptivity and for achieving competency of embryo implantation. However, the molecular events underlying the endometrial receptivity process remain poorly understood. Here we applied an LC-MS-based label-free quantitative proteomic approach to compare the endometrial tissues in the midsecretory (receptive) phase with the endometrial tissues in the proliferative phase from age-matched woman (n = 6/group). The proteomes of endometrial tissues were extracted using an SDS-based detergent, digested by the filter-aided sample preparation procedures, and subsequently analyzed by nano-LC-MS/MS (Orbitrap XL) with a 4 h gradient. Reliable protein expression profiles were reproducibly obtained from the endometrial tissues in the receptive and proliferative phases. A total of 2138 protein groups were quantified under highly stringent criteria with a false discovery rate of <1% for peptide and protein groups. Among these proteins, 317 proteins had differences in expression that were statistically significant between the receptive and proliferative phases. Direct protein-protein interaction network analyses of these significantly changed proteins showed that the up-regulation of creatine kinase B-type (CKB) in the receptive phase may be related to endometrium receptivity. The interaction network also showed that proteins related to cell-cell adhesion were down-regulated. Moreover, the results from KEGG pathway analyses are consistent with the protein-protein interaction results. The proteins, including alpha-actinin (ACTN), extracellular matrix proteins, integrin alpha-V, and so on, that are involved in the focal adhesion pathway were down-regulated in the receptive phase compared with the proliferative phase, which may facilitate the implantation of the fertilized ovum. Selected proteins were validated by Western blot analysis and

  3. Monolithically-integrated Young interferometers for label-free and multiplexed detection of biomolecules

    NASA Astrophysics Data System (ADS)

    Savra, Eleftheria; Malainou, Antonia; Salapatas, Alexandros; Botsialas, Athanasios; Petrou, Panagiota; Raptis, Ioannis; Makarona, Eleni; Kakabakos, Sotirios E.; Misiakos, Konstantinos

    2016-03-01

    In this work, interferometric silicon chips with monolithically-integrated light-emitting devices coupled to co-integrated monomodal waveguides shaped as Young interferometers through mainstream silicon technology, are presented. Although the light sources are broad-band emitters, Young interferometry is possible through filtering. Chips with arrays of ten multiplexed interferometers have been employed for the label-free determination of pesticides in drinking water currently achieving detection limits in the ng/ml range.

  4. Novel image processing method study for a label-free optical biosensor

    NASA Astrophysics Data System (ADS)

    Yang, Chenhao; Wei, Li'an; Yang, Rusong; Feng, Ying

    2015-10-01

    Optical biosensor is generally divided into labeled type and label-free type, the former mainly contains fluorescence labeled method and radioactive-labeled method, while fluorescence-labeled method is more mature in the application. The mainly image processing methods of fluorescent-labeled biosensor includes smooth filtering, artificial gridding and constant thresholding. Since some fluorescent molecules may influence the biological reaction, label-free methods have been the main developing direction of optical biosensors nowadays. The using of wider field of view and larger angle of incidence light path which could effectively improve the sensitivity of the label-free biosensor also brought more difficulties in image processing, comparing with the fluorescent-labeled biosensor. Otsu's method is widely applied in machine vision, etc, which choose the threshold to minimize the intraclass variance of the thresholded black and white pixels. It's capacity-constrained with the asymmetrical distribution of images as a global threshold segmentation. In order to solve the irregularity of light intensity on the transducer, we improved the algorithm. In this paper, we present a new image processing algorithm based on a reflectance modulation biosensor platform, which mainly comprises the design of sliding normalization algorithm for image rectification and utilizing the improved otsu's method for image segmentation, in order to implement automatic recognition of target areas. Finally we used adaptive gridding method extracting the target parameters for analysis. Those methods could improve the efficiency of image processing, reduce human intervention, enhance the reliability of experiments and laid the foundation for the realization of high throughput of label-free optical biosensors.

  5. Study and development of label-free optical biosensors for biomedical applications

    NASA Astrophysics Data System (ADS)

    Choi, Charles J.

    For the majority of assays currently performed, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so that they may be readily visualized. The methods of using labels to track biomolecular binding events are very sensitive and effective, and are employed as standardized assay protocol across research labs worldwide. However, using labels induces experimental uncertainties due to the effect of the label on molecular conformation, active binding sites, or inability to find an appropriate label that functions equivalently for all molecules in an experiment. Therefore, the ability to perform highly sensitive biochemical detection without the use of fluorescent labels would further simplify assay protocols and would provide quantitative kinetic data, while removing experimental artifacts from fluorescent quenching, shelf-life, and background fluorescence phenomena. In view of the advantages mentioned above, the study and development of optical label-free sensor technologies have been undertaken here. In general, label-free photonic crystal (PC) biosensors and metal nanodome array surface-enhanced Raman scattering (SERS) substrates, both of which are fabricated by nanoreplica molding process, have been used as the method to attack the problem. Chapter 1 shows the work on PC label-free biosensor incorporated microfluidic network for bioassay performance enhancement and kinetic reaction rate constant determination. Chapter 2 describes the work on theoretical and experimental comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays. Chapter 3 shows the work on integration of PC biosensor with actuate-to-open valve microfluidic chip for pL-volume combinatorial mixing and screening application. In Chapter 4, the development and characterization of SERS nanodome array is shown. Lastly, Chapter 5 describes SERS nanodome sensor incorporated tubing for point-of-care monitoring of

  6. Label-free DNA Biosensor Based on SERS Molecular Sentinel on Nanowave Chip

    PubMed Central

    Ngo, Hoan Thanh; Wang, Hsin-Neng; Fales, Andrew M.; Vo-Dinh, Tuan

    2013-01-01

    Development of a rapid, cost-effective, label-free biosensor for DNA detection is important for many applications in clinical diagnosis, homeland defense, and environment monitoring. A unique label-free DNA biosensor based on Molecular Sentinel (MS) immobilized on a plasmonic ‘Nanowave’ chip, which is also referred to as a metal film over nanosphere (MFON), is presented. Its sensing mechanism is based upon the decrease of the surface-enhanced Raman scattering (SERS) intensity when Raman label tagged at one end of MS is physically separated from the MFON's surface upon DNA hybridization. This method is label-free as the target does not have to be labeled. The MFON fabrication is relatively simple and low-cost with high reproducibility based on depositing a thin shell of gold over close-packed arrays of nanospheres. The sensing process involves a single hybridization step between the DNA target sequences and the complementary MS probes on the Nanowave chip without requiring secondary hybridization or post-hybridization washing, thus resulting in rapid assay time and low reagent usage. The usefulness and potential application of the biosensor for medical diagnostics is demonstrated by detecting the human radical S-adenosyl methionine domain containing 2 (RSAD2) gene, a common inflammation biomarker. PMID:23718777

  7. freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis

    PubMed Central

    Li, Zhenye; Pan, Chao; Duan, Huilong

    2015-01-01

    Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range. PMID:26665161

  8. Accurate label-free reaction kinetics determination using initial rate heat measurements

    PubMed Central

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Jacobs, Denise; Hagen, Wilfred R.

    2015-01-01

    Accurate label-free methods or assays to obtain the initial reaction rates have significant importance in fundamental studies of enzymes and in application-oriented high throughput screening of enzyme activity. Here we introduce a label-free approach for obtaining initial rates of enzyme activity from heat measurements, which we name initial rate calorimetry (IrCal). This approach is based on our new finding that the data recorded by isothermal titration calorimetry for the early stages of a reaction, which have been widely ignored, are correlated to the initial rates. Application of the IrCal approach to various enzymes led to accurate enzyme kinetics parameters as compared to spectroscopic methods and enabled enzyme kinetic studies with natural substrate, e.g. proteases with protein substrates. Because heat is a label-free property of almost all reactions, the IrCal approach holds promise in fundamental studies of various enzymes and in use of calorimetry for high throughput screening of enzyme activity. PMID:26574737

  9. Arrayed imaging reflectometry for inexpensive and label-free protein arrays

    NASA Astrophysics Data System (ADS)

    Striemer, Christopher C.; Mace, Charles R.; Carter, Jared A.; Mehta, Sourabh D.; Miller, Benjamin L.

    2009-02-01

    Highly sensitive optical techniques, capable of detecting very small quantities of specific proteins in a label-free format, offer great promise for pathogen detection because they avoid the complexity, expense, and process time associated with the use of secondary reporter elements. Arrayed Imaging Reflectometry (AIR) is one of the simplest label-free methodologies, combining laser reflectance imaging of a thermally oxidized silicon chip with standard microarray printing technology to create a platform with the potential to identify and quantify 100's of target proteins in a matter of minutes. This technique exploits a reflectance zero condition that is formed when s-polarized light strikes the surface of a silicon wafer with a single-layer oxide coating. In the vicinity of this deep reflectance minimum, picometer-scale variations in film thickness (surface relief) can be imaged directly in a reflected laser signal imaged with a CCD camera. By directly arraying probe molecules onto this substrate, minute changes in the optical thickness of each spot, corresponding to binding of the target of interest, can be measured. Array size is limited only by the resolution of the imaging system and the array printer, enabling complex protein signatures, indicative of specific pathogens or disease states to be measured in a biosample. The cost-effectiveness of a low-complexity substrate and reader, combined with the short assay times associated with label-free detection make AIR a promising new technology for pathogen and toxic exposure assessment.

  10. Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone.

    PubMed

    Lee, Kuang-Li; You, Meng-Lin; Tsai, Chia-Hsin; Lin, En-Hung; Hsieh, Shu-Yi; Ho, Ming-Hsun; Hsu, Ju-Chun; Wei, Pei-Kuen

    2016-01-15

    The widespread and intensive use of neonicotinoid insecticides induces negative cascading effects on ecosystems. It is desirable to develop a portable sensitive sensing platform for on-site screening of high-risk pesticides. We combined an indirect competitive immunoassay, highly sensitive surface plasmon resonance (SPR) biochip and a simple portable imaging setup for label-free detection of imidacloprid pesticides. The SPR biochip consists of several capped nanoslit arrays with different periods which form a spectral image on the chip. The qualitative and semiquantitative analyses of pesticides can be directly observed from the spot shift on the chip. The precise semiquantitative analyses can be further completed by using image processing in a smartphone. We demonstrate simultaneous detection of four different concentrations of imidacloprid pesticides. The visual detection limit is about 1ppb, which is well below the maximum residue concentration permitted by law (20ppb). Compared to the one-step strip assay, the proposed chip is capable of performing semiquantitative analyses and multiple detection. Compared to the enzyme-linked immunosorbent assay, our method is label-free and requires simple washing steps and short reaction time. In addition, the label-free chip has a comparable sensitivity but wider working range than those labeling techniques. PMID:26298639

  11. Experimental and theoretical bases for mechanisms of antigen discrimination by T cells

    PubMed Central

    Kajita, Masashi K.; Yokota, Ryo; Aihara, Kazuyuki; Kobayashi, Tetsuya J.

    2015-01-01

    Interaction only within specific molecules is a requisite for accurate operations of a biochemical reaction in a cell where bulk of background molecules exist. While structural specificity is a well-established mechanism for specific interaction, biophysical and biochemical experiments indicate that the mechanism is not sufficient for accounting for the antigen discrimination by T cells. In addition, the antigen discrimination by T cells also accompanies three intriguing properties other than the specificity: sensitivity, speed, and concentration compensation. In this work, we review experimental and theoretical works on the antigen discrimination by focusing on these four properties and show future directions towards understanding of the fundamental principle for molecular discrimination. PMID:27493520

  12. Smart CuS Nanoparticles as Peroxidase Mimetics for the Design of Novel Label-Free Chemiluminescent Immunoassay.

    PubMed

    Yang, Zhanjun; Cao, Yue; Li, Juan; Lu, Mimi; Jiang, Zhikang; Hu, Xiaoya

    2016-05-18

    In the present work, a novel label-free chemiluminescent (CL) immunoassay method was designed by employing smart CuS nanoparticles (CuSNPs) as peroxidase mimetics. The CuSNPs were synthesized through a simple coprecipitation method, and showed high catalytic activity and stability. This efficient label-free CL immunoassay could be easily achieved through a simple strategy. First, CuSNPs dispersed in chitosan were modified on the epoxy-functionalized glass slide to form a solid CL signal interface. Streptavidin was then used to functionalize CuSNPs to capture the biotinylated antibody, further producing a sensing interface. After online incubation with antigen molecules, the formed antibody-antigen complex on the biosensing substrate could prevent the diffusion channel of CL substrate toward the signal interface, and restrained the mimic enzyme-catalyzed CL reaction, finally resulting in the decrease of CL signals of the assay system. Compared to the label-based CL immunoassay, the proposed label-free assay mode is more simple, cheap and fast. Using a model analyte alpha-fetoprotein, the label-free CL immunoassay method had a linear range of 0.1-60 ng/mL and a low detection limit of 0.07 ng/mL. Moreover, the peroxidase mimetic-based label-free CL immunoassay system showed good specificity, acceptable repeatability, and good accuracy. The study provided a promising strategy for the development of highly efficient label-free CL immunoassay system. PMID:27137349

  13. Label-free real-time imaging of myelination in the Xenopus laevis tadpole by in vivo stimulated Raman scattering microscopy

    PubMed Central

    Hu, Chun-Rui; Zhang, Delong; Slipchenko, Mikhail N.; Cheng, Ji-Xin; Hu, Bing

    2014-01-01

    Abstract. The myelin sheath plays an important role as the axon in the functioning of the neural system, and myelin degradation is a hallmark pathology of multiple sclerosis and spinal cord injury. Electron microscopy, fluorescent microscopy, and magnetic resonance imaging are three major techniques used for myelin visualization. However, microscopic observation of myelin in living organisms remains a challenge. Using a newly developed stimulated Raman scattering microscopy approach, we report noninvasive, label-free, real-time in vivo imaging of myelination by a single-Schwann cell, maturation of a single node of Ranvier, and myelin degradation in the transparent body of the Xenopus laevis tadpole. PMID:25104411

  14. Label-free real-time imaging of myelination in the Xenopus laevis tadpole by in vivo stimulated Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Hu, Chun-Rui; Zhang, Delong; Slipchenko, Mikhail N.; Cheng, Ji-Xin; Hu, Bing

    2014-08-01

    The myelin sheath plays an important role as the axon in the functioning of the neural system, and myelin degradation is a hallmark pathology of multiple sclerosis and spinal cord injury. Electron microscopy, fluorescent microscopy, and magnetic resonance imaging are three major techniques used for myelin visualization. However, microscopic observation of myelin in living organisms remains a challenge. Using a newly developed stimulated Raman scattering microscopy approach, we report noninvasive, label-free, real-time in vivo imaging of myelination by a single-Schwann cell, maturation of a single node of Ranvier, and myelin degradation in the transparent body of the Xenopus laevis tadpole.

  15. Label-free fluorescent biosensor based on the target recycling and Thioflavin T-induced quadruplex formation for short DNA species of c-erbB-2 detection.

    PubMed

    Chen, Jinghua; Lin, Jia; Zhang, Xi; Cai, Shuxian; Wu, Dongzhi; Li, Chunyan; Yang, Sheng; Zhang, Jing

    2014-03-19

    Non-invasive early diagnosis of breast cancer is the most effective way to improve the survival rate and increase more chances of breast-conserving. In this paper, we developed a label-free fluorescent biosensor based on nuclease assisted target recycling and Thioflavin T-induced quadruplex formation for short DNA species of c-erbB-2 detection in saliva. By employing the strategy, the sensor can detect as low as 20fM target DNA with high discrimination ability even against single-base mismatch sequence. To the best of our knowledge, the proposed sensor is the first attempt to apply Thioflavin T that possesses outstanding structural selectivity for G-quadruplex in DNA amplification techniques, which may represent a promising path toward direct breast cancer detection in saliva at the point of care. PMID:24594816

  16. Origin and prediction of free-solution interaction studies performed label-free

    PubMed Central

    Bornhop, Darryl J.; Kammer, Michael N.; Kussrow, Amanda; Flowers, Robert A.; Meiler, Jens

    2016-01-01

    Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for free-solution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution. PMID:26960999

  17. Label-free detection of insulin and glucagon within human islets of Langerhans using Raman spectroscopy.

    PubMed

    Hilderink, Janneke; Otto, Cees; Slump, Cees; Lenferink, Aufried; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

    2013-01-01

    Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm(-1) band assigned to disulfide bridges in insulin, and the 1552 cm(-1) band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans. PMID:24167603

  18. Origin and prediction of free-solution interaction studies performed label-free.

    PubMed

    Bornhop, Darryl J; Kammer, Michael N; Kussrow, Amanda; Flowers, Robert A; Meiler, Jens

    2016-03-22

    Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for free-solution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R(2)> 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution. PMID:26960999

  19. Design optimization of structural parameters for highly sensitive photonic crystal label-free biosensors.

    PubMed

    Ju, Jonghyun; Han, Yun-ah; Kim, Seok-min

    2013-01-01

    The effects of structural design parameters on the performance of nano-replicated photonic crystal (PC) label-free biosensors were examined by the analysis of simulated reflection spectra of PC structures. The grating pitch, duty, scaled grating height and scaled TiO2 layer thickness were selected as the design factors to optimize the PC structure. The peak wavelength value (PWV), full width at half maximum of the peak, figure of merit for the bulk and surface sensitivities, and surface/bulk sensitivity ratio were also selected as the responses to optimize the PC label-free biosensor performance. A parametric study showed that the grating pitch was the dominant factor for PWV, and that it had low interaction effects with other scaled design factors. Therefore, we can isolate the effect of grating pitch using scaled design factors. For the design of PC-label free biosensor, one should consider that: (1) the PWV can be measured by the reflection peak measurement instruments, (2) the grating pitch and duty can be manufactured using conventional lithography systems, and (3) the optimum design is less sensitive to the grating height and TiO2 layer thickness variations in the fabrication process. In this paper, we suggested a design guide for highly sensitive PC biosensor in which one select the grating pitch and duty based on the limitations of the lithography and measurement system, and conduct a multi objective optimization of the grating height and TiO2 layer thickness for maximizing performance and minimizing the influence of parameter variation. Through multi-objective optimization of a PC structure with a fixed grating height of 550 nm and a duty of 50%, we obtained a surface FOM of 66.18 RIU-1 and an S/B ratio of 34.8%, with a grating height of 117 nm and TiO2 height of 210 nm. PMID:23470487

  20. Development of a label-free immunosensor system for detecting plasma cortisol levels in fish.

    PubMed

    Wu, Haiyun; Ohnuki, Hitoshi; Hibi, Kyoko; Ren, Huifeng; Endo, Hideaki

    2016-02-01

    Fishes display a wide variation in their physiological responses to stress, which is clearly evident in the plasma corticosteroid changes, chiefly cortisol levels in fish. In the present study, we describe a novel label-free immunosensor for detecting plasma cortisol levels. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry. For the immobilization of the antibody on the surface of sensing electrode, we used a self-assembled monolayer of thiol-containing compounds. Using this electrode, we detect the CV signal change caused by the generation of antigen-antibody complex. The immunosensor showed a response to cortisol levels, and the anodic peak value linearly decreased with a correlation coefficient of 0.990 in diluted plasma. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as 17α, 20β-dihydroxy-4-pregnen-3-one, progesterone, estriol, estradiol, and testosterone. The specific detection of cortisol was suggested by a minimal change from -0.32 to 0.51 μA in the anodic peak value of the other steroid hormones. The sensor system was used to determine the plasma cortisol levels in Nile tilapia (Oreochromis niloticus), and the results were compared with those of the same samples determined using the conventional method (ELISA). A good correlation was obtained between values determined using both methods (correlation coefficient 0.993). These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish plasma samples. PMID:26254257

  1. A novel label-free and reusable electrochemical cytosensor for highly sensitive detection and specific collection of CTCs.

    PubMed

    Shen, Huawei; Yang, Juan; Chen, Zhongping; Chen, Xueping; Wang, Li; Hu, Jing; Ji, Feihu; Xie, Guoming; Feng, Wenli

    2016-07-15

    Circulating tumor cells (CTCs) contain a great deal of information of tumor phenotype. Therefore, highly sensitive detection and specific enrichment of CTCs are of intense interest. Herein, a label-free electrochemical impedance spectroscopy cytosensor with effective surface recognition between specific epithelial cell adhesion molecules (EpCAM) over-expressed on the cell membrane and EpCAM aptamer was developed for the detection of CTCs. After immobilization of 6-mercapto-1-hexanol (MCH) onto the gold electrode, the capture probe can be directionally inserted in MCH interspaces, which can improve the sensitivity of the cytosensor. A wide detection range from 30 to 1×10(6)cellsmL(-1) with a detection limit as low as 10cellsmL(-1) is reached on the condition of acceptable stability and reproducibility. The cytosensor can easily distinguish CTCs from the real blood sample due to the specific combination of EpCAM and EpCAM aptamer. Furthermore, the cytosensor can be reused 8 times and enrich CTCs by Uracil DNA Excision Mix specific cleaving the deoxyuridines (dUs) of the aptamer. The collected CTCs can contribute to further study. Thus, we reported that this cytosensor is a promising technique for the early monitoring and therapy of cancer. PMID:27016910

  2. Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics.

    PubMed

    Nanjo, Yohei; Nakamura, Takuji; Komatsu, Setsuko

    2013-11-01

    Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean. PMID:23659366

  3. Label-free imaging of intracellular motility by low-coherent quantitative phase microscope in reflection geometry

    NASA Astrophysics Data System (ADS)

    Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

    2011-11-01

    We demonstrate tomographic imaging of intracellular activity of living cells by a low-coherent quantitative phase microscope. The intracellular organelles, such as the nucleus, nucleolus, and mitochondria, are moving around inside living cells, driven by the cellular physiological activity. In order to visualize the intracellular motility in a label-free manner we have developed a reflection-type quantitative phase microscope which employs the phase shifting interferometric technique with a low-coherent light source. The phase shifting interferometry enables us to quantitatively measure the intensity and phase of the optical field, and the low-coherence interferometry makes it possible to selectively probe a specific sectioning plane in the cell volume. The results quantitatively revealed the depth-resolved fluctuations of intracellular surfaces so that the plasma membrane and the membranes of intracellular organelles were independently measured. The transversal and the vertical spatial resolutions were 0.56 μm and 0.93 μm, respectively, and the mechanical sensitivity of the phase measurement was 1.2 nanometers. The mean-squared displacement was applied as a statistical tool to analyze the temporal fluctuation of the intracellular organelles. To the best of our knowledge, our system visualized depth-resolved intracellular organelles motion for the first time in sub-micrometer resolution without contrast agents.

  4. Intensity interrogation near cutoff resonance for label-free cellular profiling.

    PubMed

    Nazirizadeh, Yousef; Behrends, Volker; Prósz, Aurél; Orgovan, Norbert; Horvath, Robert; Ferrie, Ann M; Fang, Ye; Selhuber-Unkel, Christine; Gerken, Martina

    2016-01-01

    We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems. PMID:27086879

  5. Label-Free Imaging of Eosinophilic Esophagitis Mouse Models Using Optical Coherence Tomography.

    PubMed

    Alex, Aneesh; Tait Wojno, Elia D; Artis, David; Zhou, Chao

    2016-01-01

    Eosinophilic esophagitis (EoE) is an immune-mediated disorder characterized by esophageal inflammation and related structural changes causing symptoms such as feeding difficulties and food impaction. The pathophysiological mechanisms underlying EoE remain poorly understood. Preclinical studies using mouse models have been critical in comprehending human disease mechanisms and associated pathways. In this chapter, we describe an experimental method using a noninvasive label-free optical imaging technique, optical coherence tomography, to characterize the pathophysiological changes in the esophagus of mice with EoE-like disease ex vivo. PMID:27246028

  6. Highly Sensitive Colorimetric Detection of Ochratoxin A by a Label-Free Aptamer and Gold Nanoparticles

    PubMed Central

    Luan, Yunxia; Chen, Jiayi; Li, Cheng; Xie, Gang; Fu, Hailong; Ma, Zhihong; Lu, Anxiang

    2015-01-01

    A label-free aptamer-based assay for the highly sensitive and specific detection of Ochratoxin A (OTA) was developed using a cationic polymer and gold nanoparticles (AuNPs). The OTA aptamer was used as a recognition element for the colorimetric detection of OTA based on the aggregation of AuNPs by the cationic polymer. By spectroscopic quantitative analysis, the colorimetric assay could detect OTA down to 0.009 ng/mL with high selectivity in the presence of other interfering toxins. This study offers a new alternative in visual detection methods that is rapid and sensitive for OTA detection. PMID:26690477

  7. Label-free electrochemical aptasensing of the human prostate-specific antigen using gold nanospears.

    PubMed

    Rahi, A; Sattarahmady, N; Heli, H

    2016-08-15

    Gold nanospears were electrodeposited with the assistance of arginine as a soft template and precise selection of experimental parameters. The nanospears were then employed as a transducer to immobilize an aptamer of prostate-specific antigen (PSA) and fabrication of a label-free electrochemical aptasensor. The aptasensor was employed for the detection of PSA with a linear concentration range of 0.125-200ngmL(-1) and a limit of detection of 50pgmL(-1). The aptasensor was successfully applied to detect PSA in blood serum samples of healthy and patient persons. PMID:27260456

  8. Label-free optical quantification of structural alterations in Alzheimer's disease.

    PubMed

    Lee, Moosung; Lee, Eeksung; Jung, JaeHwang; Yu, Hyeonseung; Kim, Kyoohyun; Yoon, Jonghee; Lee, Shinhwa; Jeong, Yong; Park, YongKeun

    2016-01-01

    We present a wide-field quantitative label-free imaging of mouse brain tissue slices with sub-micrometre resolution, employing holographic microscopy and an automated scanning platform. From the measured light field images, scattering coefficients and anisotropies are quantitatively retrieved by using the modified the scattering-phase theorem, which enables access to structural information about brain tissues. As a proof of principle, we demonstrate that these scattering parameters enable us to quantitatively address structural alteration in the brain tissues of mice with Alzheimer's disease. PMID:27485313

  9. Gold nanoparticles as a label-free probe for the detection of amyloidogenic protein.

    PubMed

    Zhang, Hai-Jie; Zheng, Hu-Zhi; Long, Yi-Juan; Xiao, Geng-Fu; Zhang, Ling-Yan; Wang, Qin-Long; Gao, Mei; Bai, Wen-Jun

    2012-01-30

    Because amyloidogenic proteins, such as prion protein, β-amyloid peptide and α-synuclein, are associated with a variety of diseases, methods for their detection are important. Recombinant prion protein (rPrP) can selectively induce aggregation of dihydrolipoic acid capped gold nanoparticles (DHLA-AuNPs), which reduces the absorbance of the DHLA-AuNPs and changes their color from red to blue. These changes were used for label-free qualitative and quantitative detection of amyloidogenic protein. The addition of NaCl improved the detection sensitivity considerably, and the detection limit was as low as 33 pmol/L. PMID:22284509

  10. Third harmonic generation imaging for fast, label-free pathology of human brain tumors

    PubMed Central

    Kuzmin, N. V.; Wesseling, P.; Hamer, P. C. de Witt; Noske, D. P.; Galgano, G. D.; Mansvelder, H. D.; Baayen, J. C.; Groot, M. L.

    2016-01-01

    In brain tumor surgery, recognition of tumor boundaries is key. However, intraoperative assessment of tumor boundaries by the neurosurgeon is difficult. Therefore, there is an urgent need for tools that provide the neurosurgeon with pathological information during the operation. We show that third harmonic generation (THG) microscopy provides label-free, real-time images of histopathological quality; increased cellularity, nuclear pleomorphism, and rarefaction of neuropil in fresh, unstained human brain tissue could be clearly recognized. We further demonstrate THG images taken with a GRIN objective, as a step toward in situ THG microendoscopy of tumor boundaries. THG imaging is thus a promising tool for optical biopsies. PMID:27231629

  11. A label free aptamer-based LPG sensor for detection of mercury in aquatic solutions

    NASA Astrophysics Data System (ADS)

    Nikbakht, Hamed; Latifi, Hamid; Ziaee, Farzaneh

    2015-09-01

    We demonstrate a label free fiber optic sensor for detection of mercury ions in aquatic solutions. This sensor utilizes aptamers as bio-recognition element which traps mercury ions and cause a refractive index change in the vicinity of the sensor. Refractive index variations lead to a change in the transmission spectrum that can be used to calculate the concentration of mercury ions in that solution. The concentration of 1 nM mercury ions was detected which is below the specific amount determined by the US environmental protection agency as the maximum authorized contaminant level of Hg2+ ions in drinking water.

  12. Datasets from label-free quantitative proteomic analysis of human glomeruli with sclerotic lesions

    PubMed Central

    Zhang, Ying; Xu, Bo; Kinoshita, Naohiko; Yoshida, Yutaka; Tasaki, Masayuki; Fujinaka, Hidehiko; Magdeldin, Sameh; Yaoita, Eishin; Yamamoto, Tadashi

    2015-01-01

    Human glomeruli with intermediate (i-GS) and advanced (GS) sclerotic lesions as well as the normal control (Nor) were captured from laser microdissection, digested by trypsin and subjected to shotgun LC-MS/MS analysis (LTQ-Orbitrap XL). The label-free quantification was performed using the Normalized Spectral Index (SIN) to assess the relative molar concentration of each protein identified in a sample. All the experimental data are shown in this article. The data is associated to the research article submitted to Journal of Proteomics [1]. PMID:26217785

  13. High-contrast grating resonators for label-free detection of disease biomarkers

    NASA Astrophysics Data System (ADS)

    Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

    2016-06-01

    A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI.

  14. Label-free biosensing using cascaded double-microring resonators integrated with microfluidic channels

    NASA Astrophysics Data System (ADS)

    Chen, Yangqing; Yu, Fang; Yang, Chang; Song, Jinyan; Tang, Longhua; Li, Mingyu; He, Jian-Jun

    2015-06-01

    Fast and accurate quantitative measurement of biologically relevant molecules has been demonstrated for medical diagnostics and drug applications in photonic integrated circuits. Herein, we reported a highly-sensitive optical biosensor based on cascaded double-microring resonators. The sensor was integrated with microfluidic channels and investigated with its label-free detection capability. With a wavelength resolution of 0.47 nm, the measured binding capacity of the antibody on the surface exhibits reliable detection limit down to 7.10 μg/mL using human immunoglobulin G (hIgG).

  15. Intensity interrogation near cutoff resonance for label-free cellular profiling

    PubMed Central

    Nazirizadeh, Yousef; Behrends, Volker; Prósz, Aurél; Orgovan, Norbert; Horvath, Robert; Ferrie, Ann M.; Fang, Ye; Selhuber-Unkel, Christine; Gerken, Martina

    2016-01-01

    We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems. PMID:27086879

  16. Label-free optical quantification of structural alterations in Alzheimer’s disease

    PubMed Central

    Lee, Moosung; Lee, Eeksung; Jung, JaeHwang; Yu, Hyeonseung; Kim, Kyoohyun; Yoon, Jonghee; Lee, Shinhwa; Jeong, Yong; Park, YongKeun

    2016-01-01

    We present a wide-field quantitative label-free imaging of mouse brain tissue slices with sub-micrometre resolution, employing holographic microscopy and an automated scanning platform. From the measured light field images, scattering coefficients and anisotropies are quantitatively retrieved by using the modified the scattering-phase theorem, which enables access to structural information about brain tissues. As a proof of principle, we demonstrate that these scattering parameters enable us to quantitatively address structural alteration in the brain tissues of mice with Alzheimer’s disease. PMID:27485313

  17. Nanopore Biosensor for Label-Free and Real-Time Detection of Anthrax Lethal Factor

    PubMed Central

    2015-01-01

    We report a label-free real-time nanopore sensing method for the detection of anthrax lethal factor, a component of the anthrax toxin, by using a complementary single-stranded DNA as a molecular probe. The method is rapid and sensitive: sub-nanomolar concentrations of the target anthrax lethal factor DNA could be detected in ∼1 min. Further, our method is selective, which can differentiate the target DNA from other single-stranded DNA molecules at the single-base resolution. This sequence-specific detection approach should find useful application in the development of nanopore sensors for the detection of other pathogens. PMID:24806593

  18. Fiber optic label-free biophotonic diagnostic tool for cardiovascular disease

    NASA Astrophysics Data System (ADS)

    Rius, Cristina; Ackermann, Tobias N.; Dorado, Beatriz; Muñoz-Berbel, Xavier; Andrés, Vicente; Llobera, Andreu

    2015-06-01

    A label-free compact method for performing photonic characterization of "healthy" versus "diseased" arteries has been developed. It permits the detection of atherosclerotic lesion in living mouse arteries. Using this prototype, we observed that the spectral response (photonic fingerprint, PIN) obtained from aortas of wild-type mice differs from the response of ApoE-KO mice fed with high-fat diet (an atheroprone mouse model). Benchmark of the results against gold standard was performed by staining the aortas with Oil-Red-O to visualize atherosclerotic plaques.

  19. High-contrast grating resonators for label-free detection of disease biomarkers.

    PubMed

    Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

    2016-01-01

    A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI. PMID:27265624

  20. A symmetric metamaterial element-based RF biosensor for rapid and label-free detection

    NASA Astrophysics Data System (ADS)

    Lee, Hee-Jo; Lee, Jung-Hyun; Jung, Hyo-Il

    2011-10-01

    A symmetric metamaterial element-based RF biosensing scheme is experimentally demonstrated by detecting biomolecular binding between a prostate-specific antigen (PSA) and its antibody. The metamaterial element in a high-impedance microstrip line shows an intrinsic S21 resonance having a Q-factor of 55. The frequency shift with PSA concentration, i.e., 100 ng/ml, 10 ng/ml, and 1 ng/ml, is observed and the changes are Δf ≈ 20 MHz, 10 MHz, and 5 MHz, respectively. The proposed biosensor offers advantages of label-free detection, a simple and direct scheme, and cost-efficient fabrication.

  1. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  2. Label-free monitoring of interaction between DNA and oxaliplatin in aqueous solution by terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Wu, Xiaojun; E, Yiwen; Xu, Xinlong; Wang, Li

    2012-07-01

    We demonstrated the feasibility of applying terahertz time-domain spectroscopy (THz-TDS) to monitor the molecular reactions in aqueous solutions of anticancer drug oxaliplatin with λ-DNA and macrophages DNA. The reaction time dependent refractive index and absorption coefficient were extracted and analyzed. The reaction half-decaying time of about 4.0 h for λ-DNA and 12.9 h for M-DNA was established. The results suggest that the THz-TDS detection could be an effective label-free technique to sense the molecular reaction in aqueous solutions and could be very useful in biology, medicine, and pharmacy industry.

  3. Intensity interrogation near cutoff resonance for label-free cellular profiling

    NASA Astrophysics Data System (ADS)

    Nazirizadeh, Yousef; Behrends, Volker; Prósz, Aurél; Orgovan, Norbert; Horvath, Robert; Ferrie, Ann M.; Fang, Ye; Selhuber-Unkel, Christine; Gerken, Martina

    2016-04-01

    We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

  4. High-contrast grating resonators for label-free detection of disease biomarkers

    PubMed Central

    Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

    2016-01-01

    A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI. PMID:27265624

  5. Biosensor for label-free DNA quantification based on functionalized LPGs.

    PubMed

    Gonçalves, Helena M R; Moreira, Luis; Pereira, Leonor; Jorge, Pedro; Gouveia, Carlos; Martins-Lopes, Paula; Fernandes, José R A

    2016-10-15

    A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62±2nM and the limit of quantification was 209±7nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity. PMID:26456729

  6. Disinhibition of olfactory bulb granule cells accelerates odour discrimination in mice

    PubMed Central

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

    Granule cells are the dominant cell type of the olfactory bulb inhibiting mitral and tufted cells via dendrodendritic synapses; yet the factors regulating the strength of their inhibitory output, and, therefore, their impact on odour discrimination, remain unknown. Here we show that GABAAR β3-subunits are distributed in a somatodendritic pattern, mostly sparing the large granule cell spines also known as gemmules. Granule cell-selective deletion of β3-subunits nearly abolishes spontaneous and muscimol-induced currents mediated by GABAA receptors in granule cells, yet recurrent inhibition of mitral cells is strongly enhanced. Mice with disinhibited granule cells require less time to discriminate both dissimilar as well as highly similar odourants, while discrimination learning remains unaffected. Hence, granule cells are controlled by an inhibitory drive that in turn tunes mitral cell inhibition. As a consequence, the olfactory bulb inhibitory network adjusts the speed of early sensory processing. PMID:26592770

  7. Disinhibition of olfactory bulb granule cells accelerates odour discrimination in mice.

    PubMed

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

    Granule cells are the dominant cell type of the olfactory bulb inhibiting mitral and tufted cells via dendrodendritic synapses; yet the factors regulating the strength of their inhibitory output, and, therefore, their impact on odour discrimination, remain unknown. Here we show that GABAAR β3-subunits are distributed in a somatodendritic pattern, mostly sparing the large granule cell spines also known as gemmules. Granule cell-selective deletion of β3-subunits nearly abolishes spontaneous and muscimol-induced currents mediated by GABAA receptors in granule cells, yet recurrent inhibition of mitral cells is strongly enhanced. Mice with disinhibited granule cells require less time to discriminate both dissimilar as well as highly similar odourants, while discrimination learning remains unaffected. Hence, granule cells are controlled by an inhibitory drive that in turn tunes mitral cell inhibition. As a consequence, the olfactory bulb inhibitory network adjusts the speed of early sensory processing. PMID:26592770

  8. Hairpin assembly-triggered cyclic activation of a DNA machine for label-free and ultrasensitive chemiluminescence detection of DNA.

    PubMed

    Chen, Jia; Qiu, Hongdeng; Zhang, Mingliang; Gu, Tongnian; Shao, Shijun; Huang, Yong; Zhao, Shulin

    2015-06-15

    DNA plays important regulatory roles in many life activities. Here, we have developed a novel label-free, ultrasensitive and specific chemiluminescence (CL) assay protocol for DNA detection based on hairpin assembly-triggered cyclic activation of a DNA machine. The system involves two hairpin structures, H1 and H2. Firstly, a target DNA binds with and opens the hairpin structure of H1. Then, H2 hybridizes with H1 and displaces the target DNA, which is used to trigger another new hybridization cycle between H1 and H2, leading to the generation of numerous H1-H2 complexes. The generated H1-H2 complexes are further activated with the help of polymerase and nicking enzyme, continuously yielding a large amount of G-riched DNA fragments. The G-riched DNA fragment products interact with hemin to form the activated HRP-mimicking DNAzymes that can catalyze the oxidation of luminol by H2O2 to produce strong CL signal resulting in an amplified sensing process. Our newly proposed homogeneous assay enables the quantitative measurement of p53 DNA (as a model) with a detection limit of 0.85 fM, which is at least 5 orders of magnitude lower than that of traditional unamplified homogeneous optical approaches. Moreover, this assay exhibits high discrimination ability even against a single base mismatch. In addition, this strategy is also capable of detecting p53 DNA in complex biological samples. The proposed sensing approach might hold a great promise for further applications in biomedical research and early clinical diagnosis. PMID:25638797

  9. Label-free and ultrasensitive microRNA detection based on novel molecular beacon binding readout and target recycling amplification.

    PubMed

    Dong, Haifeng; Hao, Kaihong; Tian, Yaping; Jin, Shi; Lu, Huiting; Zhou, Shu-Feng; Zhang, Xueji

    2014-03-15

    A label-free and high-sensitive microRNA (miRNA) detection approach by coupling a metal ion-meditated conformational molecular beacon (MB), using novel fluorescent Ag nanocluster (AgNCs) as fluorophore, with endonuclease-assisted target recycling amplification was developed. The assay comprised an Hg(2+) ion-meditated conformational MB probe and an assistant probe that do not hybridize with each other at a specific temperature and can be annealed to each other in the presence of the target to form a Y-shape junction structure and released Hg(2+). The target-MB hybridization event with the help of assistant probe can readily be read out based on the efficient fluorescence quenching of AgNCs by released Hg(2+), while the Y-shape junction structure consisting of the probe MB, assistant probe and target miRNA could be recognized by the endonuclease Nt.BbvCI. The MB probe was then effectively cleaved by the endonuclease, and the regenerated assistant probe and the target further attended another cleavage cycle to implement the signal amplification. The competition displacing interaction between the target and the Hg(2+) endows the biosensor with high sequence discrimination capability, while the high signal-to-noise ratio and target recycling amplification allows the biosensor to detect the target with high sensitivity. Under the optimal conditions, the concentration of target miRNA could be conveniently read out with a linear range from 10 pM to 1 fM. The proposed approach, avoiding any laborious label, possessing high sensitivity and selectivity, provided significant potential applications in future clinical analysis. PMID:24185256

  10. Rapid label-free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome.

    PubMed

    Papanastasiou, Malvina; Orfanoudaki, Georgia; Kountourakis, Nikos; Koukaki, Marina; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Tsolis, Konstantinos C; Karamanou, Spyridoula; Economou, Anastassios

    2016-01-01

    Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria. PMID:26466526

  11. Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

    PubMed

    Shangguan, Jingfang; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Liu, Jinquan; Tang, Jinlu; Yang, Xue; Huang, Jin

    2016-08-01

    Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels. PMID:27334762

  12. Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

    PubMed Central

    Uckermann, Ortrud; Galli, Roberta; Beiermeister, Rudolf; Sitoci-Ficici, Kerim-Hakan; Later, Robert; Leipnitz, Elke; Neuwirth, Ales; Chavakis, Triantafyllos; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2015-01-01

    Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes. PMID:26355949

  13. Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord.

    PubMed

    Uckermann, Ortrud; Galli, Roberta; Beiermeister, Rudolf; Sitoci-Ficici, Kerim-Hakan; Later, Robert; Leipnitz, Elke; Neuwirth, Ales; Chavakis, Triantafyllos; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2015-01-01

    Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes. PMID:26355949

  14. Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

    PubMed Central

    Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

    2016-01-01

    In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

  15. Label-Free Nanometer-Resolution Imaging of Biological Architectures through Surface Enhanced Raman Scattering

    PubMed Central

    Ayas, Sencer; Cinar, Goksu; Ozkan, Alper Devrim; Soran, Zeliha; Ekiz, Oner; Kocaay, Deniz; Tomak, Aysel; Toren, Pelin; Kaya, Yasin; Tunc, Ilknur; Zareie, Hadi; Tekinay, Turgay; Tekinay, Ayse Begum; Guler, Mustafa Ozgur; Dana, Aykutlu

    2013-01-01

    Label free imaging of the chemical environment of biological specimens would readily bridge the supramolecular and the cellular scales, if a chemical fingerprint technique such as Raman scattering can be coupled with super resolution imaging. We demonstrate the possibility of label-free super-resolution Raman imaging, by applying stochastic reconstruction to temporal fluctuations of the surface enhanced Raman scattering (SERS) signal which originate from biomolecular layers on large-area plasmonic surfaces with a high and uniform hot-spot density (>1011/cm2, 20 to 35 nm spacing). A resolution of 20 nm is demonstrated in reconstructed images of self-assembled peptide network and fibrilated lamellipodia of cardiomyocytes. Blink rate density is observed to be proportional to the excitation intensity and at high excitation densities (>10 kW/cm2) blinking is accompanied by molecular breakdown. However, at low powers, simultaneous Raman measurements show that SERS can provide sufficient blink rates required for image reconstruction without completely damaging the chemical structure. PMID:24022059

  16. Label-free fluorescence detection of melamine with a truncated aptamer.

    PubMed

    Gu, Chunmei; Xiang, Yu; Guo, Hongli; Shi, Hanchang

    2016-07-21

    The 2008 Chinese milk scandal caused by the adulteration of melamine encouraged the public to pay attention to melamine detection in milk products and other food stuffs. To allow simple and rapid detection of melamine, we previously isolated an 88 nt melamine aptamer (called Rd29C33) using the structure-switching SELEX. However, this 88 nt oligonucleotide is costly to synthesize, and may also complicate the rational design of biosensors for melamine detection. To overcome this obstacle, we truncated Rd29C33 at several sites, and a 34 nt Rd29C33-T7 melamine aptamer was finally found to show comparable binding affinity and better selectivity to melamine compared to the original 88 nt Rd29C33. Furthermore, a label-free bioassay method for melamine detection was designed by using Rd29C33-T7 and thiazole orange (TO). The addition of melamine to a mixture of Rd29C33-T7 and TO caused the release of TO from Rd29C33-T7, resulting in a decrease of the fluorescence intensity of the solution. A detection limit of 0.12 μM for melamine was achieved using this label-free method. Good recovery ranging from 82.6% to 97.2% for melamine detection in whole milk samples suggested the promise of this bioassay method for application in monitoring melamine in real food stuffs. PMID:27171923

  17. MIRG Survey 2011: snapshot of rapidly evolving label-free technologies used for characterizing molecular interactions.

    PubMed

    Yadav, Satya P; Bergqvist, Simon; Doyle, Michael L; Neubert, Thomas A; Yamniuk, Aaron P

    2012-09-01

    The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions. PMID:22942789

  18. Silicon-based optoelectronic integrated circuit for label-free bio/chemical sensor.

    PubMed

    Song, Junfeng; Luo, Xianshu; Kee, Jack Sheng; Han, Kyungsup; Li, Chao; Park, Mi Kyoung; Tu, Xiaoguang; Zhang, Huijuan; Fang, Qing; Jia, Lianxi; Yoon, Yong-Jin; Liow, Tsung-Yang; Yu, Mingbin; Lo, Guo-Qiang

    2013-07-29

    We demonstrate a silicon-based optoelectronic integrated circuit (OEIC) for label-free bio/chemical sensing application. Such on-chip OEIC sensor system consists of optical grating couplers for vertical light coupling into silicon waveguides, a thermal-tunable microring as a tunable filter, an exposed microring as an optical label-free sensor, and a Ge photodetector for a direct electrical readout. Different from the conventional wavelength-scanning method, we adopt low-cost broadband ASE light source, together with the on-chip tunable filter to generate sliced light source. The effective refractive index change of the sensing microring induced by the sensing target is traced by scanning the supplied electrical power applied onto the tracing microring, and the detected electrical signal is read out by the Ge photodetector. For bulk refractive index sensing, we demonstrate using such OEIC sensing system with a sensitivity of ~15 mW/RIU and a detection limit of 3.9 μ-RIU, while for surface sensing of biotin-streptavidin, we obtain a surface mass sensitivity of S(m) = ~192 µW/ng·mm(-2) and a surface detection limit of 0.3 pg/mm(2). The presented OEIC sensing system is suitable for point-of-care applications. PMID:23938665

  19. Zwitterionic polymer-modified silicon microring resonators for label-free biosensing in undiluted human plasma

    PubMed Central

    Kirk, James T.; Brault, Norman D.; Baehr-Jones, Tom; Hochberg, Michael; Jiang, Shaoyi; Ratner, Daniel M.

    2013-01-01

    A widely acknowledged goal in personalized medicine is to radically reduce the costs of highly parallelized, small fluid volume, point-of-care and home-based diagnostics. Recently, there has been a surge of interest in using complementary metal-oxide-semiconductor (CMOS)-compatible silicon photonic circuits for biosensing, with the promise of producing chip-scale integrated devices containing thousands of orthogonal sensors, at minimal cost on a per-chip basis. A central challenge in biosensor translation is to engineer devices that are both sensitive and specific to a target analyte within unprocessed biological fluids. Despite advances in the sensitivity of silicon photonic biosensors, poor biological specificity at the sensor surface remains a significant factor limiting assay performance in complex media (i.e. whole blood, plasma, serum) due to the non-specific adsorption of proteins and other biomolecules. Here, we chemically modify the surface of silicon microring resonator biosensors for the label-free detection of an analyte in undiluted human plasma. This work highlights the first application of a non-fouling zwitterionic surface coating to enable silicon photonic-based label-free detection of a protein analyte at clinically relevant sensitivities in undiluted human plasma. PMID:23202337

  20. Label-free and non-contact optical biosensing of glucose with quantum dots.

    PubMed

    Khan, Saara A; Smith, Gennifer T; Seo, Felix; Ellerbee, Audrey K

    2015-02-15

    We present a label-free, optical sensor for biomedical applications based on changes in the visible photoluminescence (PL) of quantum dots in a thin polymer film. Using glucose as the target molecule, the screening of UV excitation due to pre-absorption by the product of an enzymatic assay leads to quenching of the PL of quantum dots (QDs) in a non-contact scheme. The irradiance changes in QD PL indicate quantitatively the level of glucose present. The non-contact nature of the assay prevents surface degradation of the QDs, which yields an efficient, waste-free, cost-effective, portable, and sustainable biosensor with attractive market features. The limit of detection of the demonstrated biosensor is ~3.5 µm, which is competitive with existing contact-based bioassays. In addition, the biosensor operates over the entire clinically relevant range of glucose concentrations of biological fluids including urine and whole blood. The comparable results achieved across a range of cost-affordable detectors, including a spectrophotometer, portable spectrometer, and iPhone camera, suggest that label-free and visible quantification of glucose with QD films can be applied to low-cost, point-of-care biomedical sensing as well as scientific applications in the laboratory for characterizing glucose or other analytes. PMID:25189097

  1. Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex.

    PubMed

    Wang, Sai; Liu, Jiahui; Wu, Yanxia; You, Yawen; He, Jingyi; Zhang, Jichuan; Zhang, Liqun; Dong, Yiyang

    2016-04-15

    Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy-immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content. PMID:26844871

  2. Label-free, multiplexed, molecular sensing and imaging by stamping SERS

    NASA Astrophysics Data System (ADS)

    Li, Ming; Zhao, Fusheng; Zeng, Jianbo; Santos, Greggy M.; Shih, Wei-Chuan

    2015-03-01

    Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic technique, where Raman scattering is boosted primarily by enhanced electric field due to localized surface plasmon resonance (LSPR). With advances in nanofabrication techniques, SERS has attracted great attention for label-free molecular sensing and imaging. However, the practical use of SERS has often encountered an inherent issues regarding a molecule transfer step where target molecules need to be within the close proximity of a SERS-active surface by either mixing with nanoparticles or coating onto surface-bound nanostructures. To address this issue, we have developed stamping surface-enhanced Raman spectroscopy (S-SERS) for label-free, multiplexed, molecular sensing and large-area, high-resolution molecular imaging on a flexible, non-plasmonic surface without solution-phase molecule transfer. In this technique, a polydimethylsiloxane (PDMS) thin film and nanoporous gold disk SERS substrate play the roles as molecule carrier and Raman signal enhancer, respectively. After stamping the SERS substrate onto the PDMS film, SERS measurements can be directly taken from the "sandwiched" target molecules. The performance of S-SERS is evaluated by the detection of Rhodamine 6G (R6G), urea, and its mixture with acetaminophen (APAP), in physiologically relevant concentration range, along with corresponding SERS spectroscopic maps. S-SERS features simple sample preparation, low cost, and high reproducibility, which could lead to SERS-based sensing and imaging for point-of-care and forensics applications.

  3. Label-free Detection of Cardiac Troponin I with a Photonic Crystal Biosensor

    PubMed Central

    Zhang, Bailin; Morales, Andres W.; Peterson, Ralph; Tang, Liang; Ye, Jing Yong

    2014-01-01

    A biosensor has been developed with a photonic crystal structure used in a total-internal-reflection (PC-TIR) configuration for label-free detection of a cardiac biomarker: Troponin I (cTnI). In contrast to a conventional optical microcavity that has a closed structure with its cavity layer sandwiched between two high-reflection surfaces, the PC-TIR configuration creates a unique open microcavity, which allows its cavity layer (sensing layer) to be easily functionalized and directly exposed to analyte molecules for bioassays. In this study, a PC-TIR sensor has been used for the label-free measurements of cardiac biomarkers by monitoring the changes in the resonant condition of the cavity due to biomolecular binding processes. Antibodies against cTnI are immobilized on the sensor surface for specific detection of cTnI with a wide range of concentrations. Detection limit of cTnI with a concentration as low as 0.1 ng mL−1 has been achieved. PMID:24632136

  4. Novel homogeneous label-free electrochemical aptasensor based on functional DNA hairpin for target detection.

    PubMed

    Zhang, De-Wen; Nie, Ji; Zhang, Fang-Ting; Xu, Li; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2013-10-01

    We first developed a label-free and immobilization-free homogeneous electrochemical aptasensor, which combined a smart functional DNA hairpin and a designed miniaturized electrochemical device. Cocaine was chosen as a model target. The anticocaine aptamer and peroxidase-mimicking DNAzyme were integrated into one single-stranded DNA hairpin. Both aptamer and G-quadruplex were elaborately blocked by the stem region. The conformation switching induced by the affinity interaction between aptamer and cocaine released G-quadruplex part and turned on DNAzyme activity. The designed electrochemical device, constructed by a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode, was applied to the detection of homogeneous DNAzyme catalytic activity at the microliter level. The aptasensor realized the quantification of cocaine ranging from 1 to 500 μM with high specificity. The clever combination of the functional DNA hairpin and the novel device achieved an absolutely label-free electrochemical aptasensor, which showed excellent performance like low cost, easy operation, rapid detection, and high repeatability. PMID:23998357

  5. A Microfluidic Paper-Based Origami Nanobiosensor for Label-Free, Ultrasensitive Immunoassays.

    PubMed

    Li, Xiao; Liu, Xinyu

    2016-06-01

    Microfluidic paper-based analytical devices (μPADs) represent a promising platform technology for point-of-care diagnosis. Highly sensitive, rapid, and easy-to-perform immunoassays implemented on μPADs are desirable to fulfill the promise of the μPAD technology. This article reports the first microfluidic paper-based origami nanobiosensor (origami μPAD), which integrates zinc oxide nanowires (ZnO NWs) and electrochemical impedance spectroscopy (EIS) biosensing mechanism, for label-free, ultrasensitive immunoassays. The EIS mechanism features simple and label-free assay operations which take less than 25 min to be finished, while the ZnO NWs allow covalent bonding for immobilizing probe proteins and improve the biosensing performance with such features as high surface-area-to-volume ratios and high sensitivity to surface binding. The calibration of the device reveals an ultralow limit of detection (LOD) of 60 fg mL(-1) (>100 times lower than those of existing μPADs) for rabbit immunoglobulin G in phosphate-buffered saline. The detection of human immunodeficiency virus p24 antigen in human serum with a low LOD of 300 fg mL(-1) (>33 times lower than that of a commercial p24 antigen test kit) is also demonstrated. This novel μPAD design offers ultrahigh sensitivity, short assay time, and ease of operation, and thus possesses significant potential for low-cost, rapid molecular diagnosis of early-stage diseases. PMID:27122227

  6. A novel approach for assessing cardiac fibrosis using label-free second harmonic generation.

    PubMed

    Martin, Tamara P; Norris, Greg; McConnell, Gail; Currie, Susan

    2013-12-01

    To determine whether second harmonic generation (SHG) can be used as a novel and improved label-free technique for detection of collagen deposition in the heart. To verify whether SHG will allow accurate quantification of altered collagen deposition in diseased hearts following hypertrophic remodelling. Minimally invasive transverse aortic banding (MTAB) of mouse hearts was used to generate a reproducible model of cardiac hypertrophy. Physiological and functional assessment of hypertrophic development was performed using echocardiography and post-mortem analysis of remodelled hearts. Cardiac fibroblasts were isolated from sham-operated and hypertrophied hearts and proliferation rates compared. Multi-photon laser scanning microscopy was used to capture both two-photon excited autofluorescence (TPEF) and SHG images simultaneously in two channels. TPEF images were subtracted from SHG images and the resulting signal intensities from ventricular tissue sections were calculated. Traditional picrosirius red staining was used to verify the suitability of the SHG application. MTAB surgery induced significant hypertrophic remodelling and increased cardiac fibroblast proliferation. A significant increase in the density of collagen fibres between hypertrophic and control tissues (p < 0.05) was evident using SHG. Similar increases and patterns of staining were observed using parallel traditional picrosirius red staining of collagen. Label-free SHG microscopy provides a new alternative method for quantifying collagen deposition in fibrotic hearts. PMID:23921804

  7. In situ label-free imaging for visualizing the biotransformation of a bioactive polyphenol

    PubMed Central

    Kim, Yoon Hee; Fujimura, Yoshinori; Hagihara, Takatoki; Sasaki, Masako; Yukihira, Daichi; Nagao, Tatsuhiko; Miura, Daisuke; Yamaguchi, Shinichi; Saito, Kazunori; Ta