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Sample records for label-free cell discrimination

  1. 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).

  2. 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

  3. Deep Learning in Label-free Cell Classification

    DOE PAGES

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; ...

    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

  4. 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

  5. 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.

  6. Deep Learning in Label-free Cell Classification

    SciTech Connect

    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 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. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  7. 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

    2016-03-15

    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 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. Label-free density difference amplification-based cell sorting.

    PubMed

    Song, Jihwan; Song, Minsun; Kang, Taewook; Kim, Dongchoul; Lee, Luke P

    2014-11-01

    The selective cell separation is a critical step in fundamental life sciences, translational medicine, biotechnology, and energy harvesting. Conventional cell separation methods are fluorescent activated cell sorting and magnetic-activated cell sorting based on fluorescent probes and magnetic particles on cell surfaces. Label-free cell separation methods such as Raman-activated cell sorting, electro-physiologically activated cell sorting, dielectric-activated cell sorting, or inertial microfluidic cell sorting are, however, limited when separating cells of the same kind or cells with similar sizes and dielectric properties, as well as similar electrophysiological phenotypes. Here we report a label-free density difference amplification-based cell sorting (dDACS) without using any external optical, magnetic, electrical forces, or fluidic activations. The conceptual microfluidic design consists of an inlet, hydraulic jump cavity, and multiple outlets. Incoming particles experience gravity, buoyancy, and drag forces in the separation chamber. The height and distance that each particle can reach in the chamber are different and depend on its density, thus allowing for the separation of particles into multiple outlets. The separation behavior of the particles, based on the ratio of the channel heights of the inlet and chamber and Reynolds number has been systematically studied. Numerical simulation reveals that the difference between the heights of only lighter particles with densities close to that of water increases with increasing the ratio of the channel heights, while decreasing Reynolds number can amplify the difference in the heights between the particles considered irrespective of their densities.

  9. Label-free classification of cultured cells through diffraction imaging.

    PubMed

    Dong, Ke; Feng, Yuanming; Jacobs, Kenneth M; Lu, Jun Q; Brock, R Scott; Yang, Li V; Bertrand, Fred E; Farwell, Mary A; Hu, Xin-Hua

    2011-06-01

    Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells.

  10. A scalable label-free approach to separate human pluripotent cells from differentiated derivatives.

    PubMed

    Willoughby, N A; Bock, H; Hoeve, M A; Pells, S; Williams, C; McPhee, G; Freile, P; Choudhury, D; De Sousa, P A

    2016-01-01

    The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells.

  11. A scalable label-free approach to separate human pluripotent cells from differentiated derivatives

    PubMed Central

    Willoughby, N. A.; Hoeve, M. A.; Pells, S.; Williams, C.; McPhee, G.

    2016-01-01

    The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 106–107 cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells. PMID:26858819

  12. 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.

  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 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

    Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample giving insight into the vibration eigenmodes of the excited molecules. For these reasons, Raman spectroscopy has been used as a powerful tool to investigate different biological tissues and living cells. In this paper, we present a Raman spectroscopy-based method for sensitive biochemical characterization of bovine sperm cells. Importantly, by analysing separate Raman spectra from the nucleus, acrosomale vesicle and tail of single sperm cells, we are able to identify characteristic Raman features associated with DNA, protein and lipid molecular vibrations for discriminating among different locations inside the cell with sub-micrometric resolution (˜0.3 μm). We demonstrate that our Raman spectroscopy facilitates spectral assignment and increases detection sensitivity, opening the way for novel bio-imaging platforms.

  16. Label-free detection and discrimination of poly-brominated diphenylethers using molecularly imprinted photonic cross-reactive sensor arrays.

    PubMed

    Xu, Dan; Zhu, Wei; Wang, Chen; Tian, Tian; Li, Jian; Lan, Yue; Zhang, Guanxin; Zhang, Deqing; Li, Guangtao

    2014-11-25

    Molecularly imprinted photonic polymers can serve as ideal sensing elements for efficiently creating cross-reactive sensor arrays. Based on this concept, a new method for sensitive and label-free detection of challenging PBDEs was developed, by which the direct detection and discrimination of trace levels of PBDEs against a high-background of interferents was achieved with 100% accuracy.

  17. 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.

  18. 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.

  19. Rapid and label-free classification of human glioma cells by infrared spectroscopic imaging.

    PubMed

    Steiner, Gerald; Küchler, Saskia; Hermann, Andreas; Koch, Edmund; Salzer, Reiner; Schackert, Gabriele; Kirsch, Matthias

    2008-12-01

    The discrimination of cell types is a crucial task in cell biology. Available techniques, based on an irreversible treatment of the cells, do not allow a sensitive label-free characterization under in situ conditions. Infrared spectroscopic imaging is a new and useful tool for studying individual cells. It has established itself as a powerful method to probe the molecular composition and to indicate the biochemistry of cells. Monolayers of cultivated U343, T1115 and T508 human glioma cells were characterized using infrared spectroscopic imaging. A classification algorithm based on linear discriminant analysis was developed to distinguish different cells without labeling. The classification is based upon spectral features which mainly arise from proteins, nucleic acids, and cholesterol. An accuracy of 91% and 84% was obtained for cells of U343 and T1115, respectively. Cells of the T508 cell line exhibit some misclassifications resulting in a lower accuracy rate of 73%. As the results demonstrate, the potential of infrared spectroscopic imaging method to assess the overall molecular composition of cells in a non-destructive manner opens the possibility to characterize cells on a molecular level without labels or an irreversible treatment.

  20. 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

  1. 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

  2. Label-free optical sensor based on red blood cells laser tweezers Raman spectroscopy analysis for ABO blood typing.

    PubMed

    Lin, Duo; Zheng, Zuci; Wang, Qiwen; Huang, Hao; Huang, Zufang; Yu, Yun; Qiu, Sufang; Wen, Cuncheng; Cheng, Min; Feng, Shangyuan

    2016-10-17

    The clinical significance of ABO blood typing extends beyond transfusion medicine and is demonstrated to be associated with susceptibility to various diseases, even including cancer. In this study, a home-made laser tweezers Raman spectroscopy (LTRS) system was applied to detect red blood cells (RBCs) with the aim to develop a label-free, simple and objective blood typing method for the first time. High-quality Raman spectra of RBCs in the fingerprint region of 420-1700 cm-1 can be obtained, meanwhile exciting blood typing results can be achieved, especially with an accuracy of 100% for identifying Type AB from other blood types with the use of multivariate statistical analysis based on principal component analysis (PCA) combined with linear discriminant analysis (LDA). This primary work demonstrates that the label-free RBCs LTRS analysis in conjunction with PCA-LDA diagnostic algorithms has great potential as a biosensor for ABO blood typing.

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

    PubMed

    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.

  4. Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives

    PubMed Central

    Cima, Igor; Wen Yee, Chay; Iliescu, Florina S.; Min Phyo, Wai; Hon Lim, Kiat; Iliescu, Ciprian; Han Tan, Min

    2013-01-01

    This review will cover the recent advances in label-free approaches to isolate and manipulate circulating tumor cells (CTCs). In essence, label-free approaches do not rely on antibodies or biological markers for labeling the cells of interest, but enrich them using the differential physical properties intrinsic to cancer and blood cells. We will discuss technologies that isolate cells based on their biomechanical and electrical properties. Label-free approaches to analyze CTCs have been recently invoked as a valid alternative to “marker-based” techniques, because classical epithelial and tumor markers are lost on some CTC populations and there is no comprehensive phenotypic definition for CTCs. We will highlight the advantages and drawbacks of these technologies and the status on their implementation in the clinics. PMID:24403992

  5. Label-free Optofluidic Cell Classifier Utilizing Support Vector Machines.

    PubMed

    Wu, Tsung-Feng; Mei, Zhe; Lo, Yu-Hwa

    2013-09-01

    A unique optofluidic lab-on-a-chip device that can measure optically encoded forward scattering signals has been demonstrated. From the design of the spatial pattern, the position and velocity of each cell in the flow can be detected and then a spatial cell distribution over the cross section of the channel can be generated. According to the forward scattering intensity and position information of cells, a data-mining method, support vector machines (SVMs), is applied for cell classification. With the help of SVMs, the multi-dimensional analysis can be performed to significantly increase all figures of merit for cell classification.

  6. 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).

  7. Label-free discrimination of normal and pulmonary cancer tissues using multiphoton fluorescence ratiometric microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Wu, Ruei-Jr; Lin, Sung-Jan; Chen, Yang-Fang; Dong, Chen-Yuan

    2010-07-01

    We performed multiphoton excited autofluorescence and second harmonic generation microscopy for the distinction of normal, lung adenocarcinoma (LAC), and squamous cell carcinoma (SCC) specimens. In addition to morphological distinction, we derived quantitative metrics of cellular redox ratios for cancer discrimination. Specifically, the redox ratios of paired normal/SCC and normal/LAC specimens were found to be 0.53±0.05/0.41±0.06 and 0.56±0.02/0.35±0.06, respectively. The lower redox ratios in cancer specimens, indicating an increase in metabolic activity. These results show that the combination of morphological multiphoton imaging along with redox ratio indices can be used for the discrimination of normal and pulmonary cancer tissues.

  8. 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.

  9. Label-free fingerprinting of tumor cells in bulk flow using inline digital holographic microscopy

    PubMed Central

    Singh, Dhananjay Kumar; Ahrens, Caroline C.; Li, Wei; Vanapalli, Siva A.

    2017-01-01

    Large-scale and label-free phenotyping of cells holds great promise in medicine, especially in cancer diagnostics and prognosis. Here, we introduce inline digital holography microscopy for volumetric imaging of cells in bulk flow and fingerprinting of flowing tumor cells based on two metrics, in-focus scattered intensity and cell diameter. Using planar distribution of immobilized particles, we identify the optimal recording distance and microscope objective magnification that minimizes the error in measurement of particle position, size and scattered intensity. Using the optimized conditions and the two metrics, we demonstrate the capacity to enumerate and fingerprint more than 100,000 cells. Finally, we highlight the power of our label-free and high throughput technology by characterizing breast tumor cell lines with different metastatic potentials and distinguishing drug resistant ovarian cancer cells from their parental cell line. PMID:28270966

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

    PubMed

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

    2015-11-23

    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.

  11. 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.

  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. 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

  14. 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

  15. 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.

  16. 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

  17. Original Research: Label-free detection for radiation-induced apoptosis in glioblastoma cells.

    PubMed

    Qi, Dandan; Feng, Jingwen; Yang, Chengwen; Jin, Changrong; Sa, Yu; Feng, Yuanming

    2016-10-01

    Current flow cytometry (FCM) requires fluorescent dyes labeling cells which make the procedure costly and time consuming. This manuscript reports a feasibility study of detecting the cell apoptosis with a label-free method in glioblastoma cells. A human glioma cell line M059K was exposed to 8 Gy dose of radiation, which enables the cells to undergo radiation-induced apoptosis. The rates of apoptosis were studied at different time points post-irradiation with two different methods: FCM in combination with Annexin V-FITC/PI staining and a newly developed technique named polarization diffraction imaging flow cytometry. Totally 1000 diffraction images were acquired for each sample and the gray level co-occurrence matrix (GLCM) algorithm was used in morphological characterization of the apoptotic cells. Among the feature parameters extracted from each image pair, we found that the two GLCM parameters of angular second moment (ASM) and sum entropy (SumEnt) exhibit high sensitivities and consistencies as the apoptotic rates (Pa) measured with FCM method. In addition, no significant difference exists between Pa and ASM_S, Pa and SumEnt_S, respectively (P > 0.05). These results demonstrated that the new label-free method can detect cell apoptosis effectively. Cells can be directly used in the subsequent biochemical experiments as the structure and function of cells and biomolecules are well-preserved with this new method.

  18. Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.

    PubMed

    Seidel, Susanne A I; Wienken, Christoph J; Geissler, Sandra; Jerabek-Willemsen, Moran; Duhr, Stefan; Reiter, Alwin; Trauner, Dirk; Braun, Dieter; Baaske, Philipp

    2012-10-15

    Look, no label! Microscale thermophoresis makes use of the intrinsic fluorescence of proteins to quantify the binding affinities of ligands and discriminate between binding sites. This method is suitable for studying binding interactions of very small amounts of protein in solution. The binding of ligands to iGluR membrane receptors, small-molecule inhibitorss to kinase p38, aptamers to thrombin, and Ca(2+) ions to synaptotagmin was quantified.

  19. 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.

  20. Analysis of Cell Surface Proteome Changes via Label-free, Quantitative Mass Spectrometry*S⃞

    PubMed Central

    Schiess, Ralph; Mueller, Lukas N.; Schmidt, Alexander; Mueller, Markus; Wollscheid, Bernd; Aebersold, Ruedi

    2009-01-01

    We present a mass spectrometry-based strategy for the specific detection and quantification of cell surface proteome changes. The method is based on the label-free quantification of peptide patterns acquired by high mass accuracy mass spectrometry using new software tools and the cell surface capturing technology that selectively enriches glycopeptides exposed to the cell exterior. The method was applied to monitor dynamic protein changes in the cell surface glycoproteome of Drosophila melanogaster cells. The results led to the construction of a cell surface glycoprotein atlas consisting of 202 cell surface glycoproteins of D. melanogaster Kc167 cells and indicated relative quantitative changes of cell surface glycoproteins in four different cellular states. Furthermore we specifically investigated cell surface proteome changes upon prolonged insulin stimulation. The data revealed insulin-dependent cell surface glycoprotein dynamics, including insulin receptor internalization, and linked these changes to intracellular signaling networks. PMID:19036722

  1. 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.

  2. 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.

  3. Label-free optical detection of cells grown in 3D silicon microstructures.

    PubMed

    Merlo, Sabina; Carpignano, Francesca; Silva, Gloria; Aredia, Francesca; Scovassi, A Ivana; Mazzini, Giuliano; Surdo, Salvatore; Barillaro, Giuseppe

    2013-08-21

    We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells

    NASA Astrophysics Data System (ADS)

    Su, Xuantao; Qiu, Yuanyuan; Marquez-Curtis, Leah; Gupta, Manisha; Capjack, Clarence E.; Rozmus, Wojciech; Janowska-Wieczorek, Anna; Tsui, Ying Y.

    2011-06-01

    A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

  9. 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.

  10. 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

  11. 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.

  12. 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

    2015-03-03

    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.

  13. 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.

  14. 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

  15. Direct high-resolution label-free imaging of cellular nanostructure dynamics in living cells

    NASA Astrophysics Data System (ADS)

    Heo, Chaejeong; Lee, Sohee; Lee, Si Young; Jeong, Mun Seok; Lee, Young Hee; Suh, Minah

    2013-06-01

    We report the application of an optical microscope equipped with a high-resolution dark-field condenser for detecting dynamic responses of cellular nanostructures in real time. Our system provides an easy-to-use technique to visualize biological specimens without any staining. This system can visualize the dynamic behavior of nanospheres and nanofibers, such as F-actin, at the leading edges of adjacent neuronal cells. We confirmed that the nanofibers imaged with this high-resolution optical microscopic technique are F-actin by using fluorescence microscopy after immunostaining the F-actin of fixed cells. Furthermore, cellular dynamics are enhanced by applying noncontact electric field stimulation through a transparent graphene electric field stimulator. High-resolution label-free optical microscopy enables the visualization of nanofiber dynamics initiated by filopodial nanofiber contacts. In conclusion, our optical microscopy system allows the visualization of nanoscale cellular dynamics under various external stimuli in real time without specific staining.

  16. 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

  17. Label-free detection of DNA with interdigitated micro-electrodes in a fluidic cell.

    PubMed

    Berdat, Daniel; Martin Rodríguez, Ana C; Herrera, Fernando; Gijs, Martin A M

    2008-02-01

    We investigate the analytical performance of an interdigitated electrode sensor for the label-free detection of DNA, by monitoring the complex impedance of 5 microm wide interdigitated Pt microelectrodes on a glass substrate. We detect the hybridization of unlabeled 38-mer target ssDNA with a complementary probe that is bound on the glass in between the electrodes by a disuccinimidyl terephtalate and aminosilane immobilization procedure. The sensor is mounted in a microfluidic flow cell, in which hybridization is monitored and in situ compared with a reference. After hybridization, the cell is perfused with deionised water and the dependence of the measured conductance due to the immobilized target DNA layer, to target DNA concentrations down to 1 nM is demonstrated. Subsequently, we apply our sensor to the detection of pathogen DNA from Salmonella choleraesuis in dairy food.

  18. 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.

  19. 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

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

    PubMed

    Spencer, Adrianne; Baker, Aaron B

    2016-01-27

    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.

  1. Microfluidic devices for label-free separation of cells through transient interaction with asymmetric receptor patterns

    NASA Astrophysics Data System (ADS)

    Bose, S.; Singh, R.; Hollatz, M. H.; Lee, C.-H.; Karp, J.; Karnik, R.

    2012-02-01

    Cell sorting serves an important role in clinical diagnosis and biological research. Most of the existing microscale sorting techniques are either non-specific to antigen type or rely on capturing cells making sample recovery difficult. We demonstrate a simple; yet effective technique for isolating cells in an antigen specific manner by using transient interactions of the cell surface antigens with asymmetric receptor patterned surface. Using microfluidic devices incorporating P-selectin patterns we demonstrate separation of HL60 cells from K562 cells. We achieved a sorting purity above 90% and efficiency greater than 85% with this system. We also present a mathematical model incorporating flow mediated and adhesion mediated transport of cells in the microchannel that can be used to predict the performance of these devices. Lastly, we demonstrate the clinical significance of the method by demonstrating single step separation of neutrophils from whole blood. When whole blood is introduced in the device, the granulocyte population gets separated exclusively yielding neutrophils of high purity (<10% RBC contamination). To our knowledge, this is the first ever demonstration of continuous label free sorting of neutrophils from whole blood. We believe this technology will be useful in developing point-of-care diagnostic devices and also for a host of cell sorting applications.

  2. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  4. Chronic label-free volumetric photoacoustic microscopy of melanoma cells in scaffolds in vitro

    NASA Astrophysics Data System (ADS)

    Cai, Xin; Zhang, Yu; Kim, Chulhong; Choi, Sung-Wook; Xia, Younan; Wang, Lihong V.

    2011-03-01

    Visualizing cells in three-dimensional (3D) scaffolds has been one of the major challenges in tissue engineering. Current imaging modalities have limitations. Microscopy, including confocal microscopy, cannot penetrate deeply (> 300 μm) into the scaffolds; X-ray micro-computed tomography (micro-CT) requires staining of the structure with a toxic agent such as osmium tetroxide. Here, we demonstrate photoacoustic microscopy (PAM) of the spatial distribution and temporal proliferation of melanoma cells inside three-dimensionally porous scaffolds with thicknesses over 1 mm. Melanoma cells have a strong intrinsic contrast which is easily imaged by label-free PAM with high sensitivity. Spatial distributions of the cells in the scaffold were well-resolved in PAM images. Moreover, we chronically imaged the same cell/scaffold constructs at different time points over 2 weeks. The number of cells in the scaffold was quantitatively measured from the PAM volumetric information. The cell proliferation profile obtained from PAM correlated well with that obtained using the traditional 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We believe that PAM will become a useful imaging modality for tissue engineering applications, especially when thick scaffold constructs are involved, and that this modality can also be extended to image other cell types labeled with contrast agents.

  5. Highly sensitive and selective detection of cancer cell with a label-free electrochemical cytosensor.

    PubMed

    Liu, Jiyang; Qin, Yinan; Li, Dan; Wang, Tianshu; Liu, Yaqing; Wang, Jin; Wang, Erkang

    2013-03-15

    Electrochemical methods have attracted considerable attention for developing cytosensing system since they can decrease the cost and time requirement for cell detection with simple instrumentation. Herein, a label-free electrochemical cytosensor with surface-confined ferrocene as signal indicator was developed for highly sensitive and selective detection of cancer cell. With layer-by-layer (LBL) self-assembly technique, positively charged poly(ethylene imine) functionalized with ferrocene (Fc-PEI) and negatively charged single-wall carbon nanotubes (SWNTs) were alternately assembled on 3-mercaptopropionic acid (MPA) modified gold substrate. Folic acid (FA) was covalently bonded onto SWNTs surface to specifically recognize cancer cells according to the high affinity of FA for folate receptor (FR) on cellular surface. The developed cytosensor presented high sensitivity and selectivity for the detection of human cervical carcinoma (HeLa) cell. By using fast-response differential pulse voltammetry (DPV) method, a wide detection range from 10 to 10(6) cells/mL with a detection limit as low as 10 cells/mL was reached even in the presence of a large amount of non-cancerous cells.

  6. Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

    PubMed Central

    Votteler, Miriam; Carvajal Berrio, Daniel A.; Pudlas, Marieke; Walles, Heike; Schenke-Layland, Katja

    2012-01-01

    Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10 Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization. PMID:22688496

  7. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

    PubMed Central

    Paesen, Rik; Gyselaers, Wilfried; Stinissen, Piet

    2016-01-01

    In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin. PMID:27746820

  8. 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.

  9. 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.

  10. Label-free imaging and identification of typical cells of acute myeloid leukaemia and myelodysplastic syndrome by Raman microspectroscopy.

    PubMed

    Vanna, R; Ronchi, P; Lenferink, A T M; Tresoldi, C; Morasso, C; Mehn, D; Bedoni, M; Picciolini, S; Terstappen, L W M M; Ciceri, F; Otto, C; Gramatica, F

    2015-02-21

    In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) start from the manual examination of stained smears of bone marrow (BM) and peripheral blood (PB) by using an optical microscope. This step is subjective and scarcely reproducible. Therefore, the development of subjective and potentially automatable methods for the recognition of typical AML/MDS cells is necessary. Here we have used Raman spectroscopy for distinguishing myeloblasts, promyelocytes, abnormal promyelocytes and erhytroblasts, which have to be counted for a correct diagnosis and morphological classification of AML and MDS. BM samples from patients affected by four different AML subtypes, mostly characterized by the presence of the four subpopulations selected for this study, were analyzed. First, each cell was scanned by acquiring 4096 spectra, thus obtaining Raman images which demonstrate an accurate description of morphological features characteristic of each subpopulation. Raman imaging coupled with hierarchical cluster analysis permitted the automatic discrimination and localization of the nucleus, the cytoplasm, myeloperoxidase containing granules and haemoglobin. Second, the averaged Raman fingerprint of each cell was analysed by multivariate analysis (principal component analysis and linear discriminant analysis) in order to study the typical vibrational features of each subpopulation and also for the automatic recognition of cells. The leave-one-out cross validation of a Raman-based classification model demonstrated the correct classification of myeloblasts, promyelocytes (normal/abnormal) and erhytroblasts with an accuracy of 100%. Normal and abnormal promyelocytes were distinguished with 95% accuracy. The overall classification accuracy considering the four subpopulations was 98%. This proof-of-concept study shows that Raman micro-spectroscopy could be a valid approach for developing label-free, objective and automatic

  11. 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.

  12. 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.

  13. Immersion Mirau interferometry for label-free live cell imaging in an epi-illumination geometry

    PubMed Central

    Lyulko, Oleksandra V.; Randers-Pehrson, Gerhard; Brenner, David J.

    2013-01-01

    In cell biology studies it is often important to avoid the damaging effects caused by fluorescent stains or UV-light. Immersion Mirau Interferometry (IMI) is an epi-illumination label-free imaging technique developed at the Columbia University Radiological Research Accelerator Facility. It is based on the principles of phase-shifting interferometry (PSI) and represents a novel approach for interferometric imaging of living cells in medium. To accommodate the use of medium, a custom immersion Mirau interferometric attachment was designed and built in-house. The space between the reference mirror and the beam splitter is filled with liquid to ensure identical optical paths in the test and reference arms. The interferometer is mountable onto a microscope objective. The greatest limitation of standard PSI is the sensitivity to environmental vibrations, because it requires consecutive acquisition of several interferograms. We are developing Simultaneous Immersion Mirau Interferometry (SIMI), which facilitates simultaneous acquisition of all interferograms and eliminates the effects of vibration. Polarization optics, incorporated into the design, introduces a phase delay to one of the components of the test beam. This enables simultaneous creation and spatial separation of two interferograms, which are combined with the background image to reconstruct the intensity map of the specimen. Our results of imaging live and fixed cells with IMI and SIMI show that this system produces images of a quality that is sufficient to perform targeted cellular irradiation experiments. PMID:24392197

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

    PubMed Central

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

    2014-01-01

    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

  15. 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.

  16. 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

  17. 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.

  18. Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

    PubMed Central

    Elvington, Elizabeth S.; Salmanzadeh, Alireza; Stremler, Mark A.; Davalos, Rafael V.

    2013-01-01

    Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process. cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles. Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing

  19. Label-free isolation and enrichment of cells through contactless dielectrophoresis.

    PubMed

    Elvington, Elizabeth S; Salmanzadeh, Alireza; Stremler, Mark A; Davalos, Rafael V

    2013-09-03

    Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process. cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles. Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing

  20. Label-free quantification proteomics reveals novel calcium binding proteins in matrix vesicles isolated from mineralizing Saos-2 cells.

    PubMed

    Zhou, Xiaoying; Cui, Yazhou; Luan, Jing; Zhou, Xiaoyan; Zhang, Genglin; Zhang, Xiumei; Han, Jinxiang

    2013-06-01

    Matrix vesicles (MVs) involved in the initiation of mineralization by deposition of hydroxyapatite (HA) in their lumen are released by the budding of mineralization-competent cells during skeletogenesis and bone development. To identify additional mineralization-related proteins, MVs were isolated from non-stimulated and stimulated Saos-2 cells in culture via an Exoquick™ approach and the corresponding proteomes were identified and quantified with label-free quantitative proteome technology. The isolated MVs were confirmed by electron microscopy, alkaline phosphatase activity (ALP), biomarkers, and mineral formation analyses. Label-free quantitative proteome analysis revealed that 19 calcium binding proteins (CaBPs), including Grp94, calnexin, calreticulin, calmodulin, and S100A4/A10, were up-regulated in MVs of Saos-2 cells upon stimulation of mineralization. This result provides new clues to study the mechanism of the initiation of MV-mediated mineralization.

  1. Sensitive imaging of organelles in label-free cells by surface plasmon resonance in deep-ultraviolet region (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kawata, Yoshimasa; Kikawada, Masakazu; Ono, Atsushi; Inami, Wataru

    2016-09-01

    In this research, we demonstrate the enhanced autofluorescence and high-sensitivity bioimaging of intracellular organelles using DUV-SPR. The Kretschmann configuration is used for excitation of DUV-SPR. We used an aluminum thickness of 24 nm. The alumina surface was estimated to be 6 nm by comparison between the experimental and calculated results. Reflectance after culturing of cells was measured. DUV-SPR is excited at an incident angle of 52° after the biological samples are cultured. MC3T3-E1 cells as Label-free cells are directly cultured on an aluminum and glass surfaces, and they were cultured on the both substrates in an incubator. Autofluorescence spectra excited of the label-free MC3T3-E1 cells was measured by 266-nm exictation. The autofluorescence intensity for the aluminum is higher than that for the glass. In the autofluorescence spectra, MC3T3-E1 cells exhibited two fluorescence peaks, which were located around 330 and 500 nm. These 330 and 500 nm emissions indicate aromatic amino acid and mitochondria, respectively. Both of the ehnahcement factors were 8 times. We also observed autofluorescence of aromatic amino acid and mitochondrial NADH in the label-free MC3T3-E1 cells cultured on the aluminum and glass surfaces. In the autofluorescence image with DUV-SPR, organelles can be clearly observed in the MC3T3-E1 cells. On the other hand, the autofluorescence intensity is very weak in the image without DUV-SPR. Accordingly, DUV-SPR can facilitate the observation of proteins, DNA in nucleus, and other structures that cannot be excited by visible light. DUV-SPR is shown to be a powerful technique for acquiring high-sensitivity label-free observation of biological samples.

  2. Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel

    PubMed Central

    Sun, Haiyan; Wei, Ying; Deng, Huayun; Xiong, Qiaojie; Li, Min; Lahiri, Joydeep; Fang, Ye

    2014-01-01

    Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel. PMID:24816792

  3. 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.

  4. 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.

  5. 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

  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. 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

  8. Label-free in situ imaging of lignification in plant cell walls.

    PubMed

    Schmidt, Martin; Perera, Pradeep; Schwartzberg, Adam M; Adams, Paul D; Schuck, P James

    2010-11-01

    Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion. Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non

  9. Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress.

    PubMed

    Lee, Myung Gwon; Shin, Joong Ho; Bae, Chae Yun; Choi, Sungyoung; Park, Je-Kyun

    2013-07-02

    We report a contraction-expansion array (CEA) microchannel device that performs label-free high-throughput separation of cancer cells from whole blood at low Reynolds number (Re). The CEA microfluidic device utilizes hydrodynamic field effect for cancer cell separation, two kinds of inertial effects: (1) inertial lift force and (2) Dean flow, which results in label-free size-based separation with high throughput. To avoid cell damages potentially caused by high shear stress in conventional inertial separation techniques, the CEA microfluidic device isolates the cells with low operational Re, maintaining high-throughput separation, using nondiluted whole blood samples (hematocrit ~45%). We characterized inertial particle migration and investigated the migration of blood cells and various cancer cells (MCF-7, SK-BR-3, and HCC70) in the CEA microchannel. The separation of cancer cells from whole blood was demonstrated with a cancer cell recovery rate of 99.1%, a blood cell rejection ratio of 88.9%, and a throughput of 1.1 × 10(8) cells/min. In addition, the blood cell rejection ratio was further improved to 97.3% by a two-step filtration process with two devices connected in series.

  10. 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

  11. In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

    DOE PAGES

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; ...

    2016-11-22

    Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials. Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have provenmore » to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall. We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. As a result, we believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.« less

  12. In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

    SciTech Connect

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

    2016-11-22

    Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials. Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall. We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. As a result, we believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography.

    PubMed

    Mölder, A; Sebesta, M; Gustafsson, M; Gisselson, L; Wingren, A Gjörloff; Alm, K

    2008-11-01

    Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label-free and completely non-invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non-invasive automatic cell counting as precisely as conventional manual cell counting.

  18. Label-free determination of the cell cycle phase in human embryonic stem cells by Raman microspectroscopy.

    PubMed

    Konorov, Stanislav O; Schulze, H Georg; Piret, James M; Blades, Michael W; Turner, Robin F B

    2013-10-01

    The cell cycle is a series of integrated and coordinated physiological events that results in cell growth and replication. Besides observing the event of cell division it is not feasible to determine the cell cycle phase without fatal and/or perturbing invasive procedures such as cell staining, fixing, and/or dissociation. Raman microspectroscopy (RMS) is a chemical imaging technique that exploits molecular vibrations as a contrast mechanism; it can be applied to single living cells noninvasively to allow unperturbed analysis over time. We used RMS to determine the cell cycle phase based on integrating the composite 783 cm(-1) nucleic acid band intensities across individual cell nuclei. After correcting for RNA contributions using the RNA 811 cm(-1) band, the measured intensities essentially reflected DNA content. When quantifying Raman images from single cells in a population of methanol-fixed human embryonic stem cells, the histogram of corrected 783 cm(-1) band intensities exhibited a profile analogous to that obtained using flow-cytometry with nuclear stains. The two population peaks in the histogram occur at Raman intensities corresponding to a 1-fold and 2-fold diploid DNA complement per cell, consistent with a distribution of cells with a population peak due to cells at the end of G1 phase (1-fold) and a peak due to cells entering M phase (2-fold). When treated with EdU to label the replicating DNA and block cell division, cells with higher EdU-related fluorescence generally had higher integrated Raman intensities. This provides proof-of-principle of an analytical method for label-free RMS determination in situ of cell cycle phase in adherent monolayers or even single adherent cells.

  19. Cell and brain tissue imaging of the flavonoid fisetin using label-free two-photon microscopy.

    PubMed

    Krasieva, Tatiana B; Ehren, Jennifer; O'Sullivan, Thomas; Tromberg, Bruce J; Maher, Pamela

    2015-10-01

    Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids.

  20. Cell and Brain Tissue Imaging of the Flavonoid Fisetin Using Label-Free Two-Photon Microscopy

    PubMed Central

    Krasieva, Tatiana B.; Ehren, Jennifer; O’Sullivan, Thomas; Tromberg, Bruce J.; Maher, Pamela

    2015-01-01

    Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their byproducts. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids. PMID:26271433

  1. 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.

  2. 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.

  3. Label-Free Molecular Imaging of Biological Cells and Tissues by Linear and Nonlinear Raman Spectroscopic Approaches.

    PubMed

    Krafft, Christoph; Schmitt, Michael; Schie, Iwan W; Cialla-May, Dana; Matthäus, Christian; Bocklitz, Thomas; Popp, Jürgen

    2016-11-15

    Raman spectroscopy is an emerging technique in bioanalysis and imaging of biomaterials owing to its unique capability of generating spectroscopic fingerprints. Imaging cells and tissues by Raman microspectroscopy represents a nondestructive and label-free approach. All components of cells or tissues contribute to the Raman signals, giving rise to complex spectral signatures. Resonance Raman scattering and surface-enhanced Raman scattering can be used to enhance the signals and reduce the spectral complexity. Raman-active labels can be introduced to increase specificity and multimodality. In addition, nonlinear coherent Raman scattering methods offer higher sensitivities, which enable the rapid imaging of larger sampling areas. Finally, fiber-based imaging techniques pave the way towards in vivo applications of Raman spectroscopy. This Review summarizes the basic principles behind medical Raman imaging and its progress since 2012.

  4. Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay.

    PubMed

    Li, Yan; Sun, Shao-kai; Yang, Jia-lin; Jiang, Yan

    2011-12-07

    Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.

  5. The xCELLigence system for real-time and label-free monitoring of cell viability.

    PubMed

    Ke, Ning; Wang, Xiaobo; Xu, Xiao; Abassi, Yama A

    2011-01-01

    We describe here the use of the xCELLigence system for label-free and real-time monitoring of cell -viability. The xCELLigence system uses specially designed microtiter plates containing interdigitated gold microelectrodes to noninvasively monitor the viability of cultured cells using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system makes it possible to distinguish between different perturbations of cell viability, such as senescence, cell toxicity (cell death), and reduced proliferation (cell cycle arrest). In addition, the time resolution of the xCELLigence system allows for the determination of optimal time points to perform standard cell viability assays as well as other end-point assays to understand the mode of action. We have used the WST-1 assay (end-point viability readout), the cell index determination (continuous monitoring of viability by xCELLigence), and the DNA fragmentation assay (end-point apoptosis assay) to systematically examine cytotoxic effects triggered by two cytotoxic compounds with different cell-killing kinetics. Good correlation was observed for viability readouts between WST-1 and cell index. The significance of time resolution by xCELLigence readout is exemplified by its ability to pinpoint the optimal time points for conducting end point viability and apoptosis assays.

  6. Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye

    PubMed Central

    Guevara-Torres, A.; Joseph, A.; Schallek, J. B.

    2016-01-01

    Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level. PMID:27867728

  7. A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders.

    PubMed

    Pesciotta, Esther N; Sriswasdi, Sira; Tang, Hsin-Yao; Mason, Philip J; Bessler, Monica; Speicher, David W

    2012-12-05

    Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated the removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis that included the identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell was obtained. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders. This article is part of a Special Issue entitled: Integrated omics.

  8. In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells

    PubMed Central

    He, Yun; Wang, Lidai; Shi, Junhui; Yao, Junjie; Li, Lei; Zhang, Ruiying; Huang, Chih-Hsien; Zou, Jun; Wang, Lihong V.

    2016-01-01

    Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma. Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis. Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism. We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice. Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage. A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature. PMID:28000788

  9. In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells

    NASA Astrophysics Data System (ADS)

    He, Yun; Wang, Lidai; Shi, Junhui; Yao, Junjie; Li, Lei; Zhang, Ruiying; Huang, Chih-Hsien; Zou, Jun; Wang, Lihong V.

    2016-12-01

    Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma. Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis. Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism. We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice. Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage. A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature.

  10. 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.

  11. 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.

  12. Exploiting serum interactions with cationic biomaterials enables label-free circulating tumor cell isolation

    NASA Astrophysics Data System (ADS)

    Castellanos, Carlos

    Herein we investigate the role charged biomaterials and fluid dielectric properties have on microfluidic capture and isolation of circulating tumor cells. We determine that heparan sulfate proteoglycans on cancer cell surfaces are responsible for elevated electric charge of cancer cells compared with white blood cells and that these proteoglycans help mediate adhesive interactions between cells and charged surfaces in albumin-containing fluids. Cancer cell firm adhesion to charged surfaces persists when cells are bathed in up to 1% (w/v) human albumin solution, while white blood cell adhesion is nearly abrogated. As many protocols rely on electrical interactions between cells and biomaterials, our study could reveal a new determinant of efficient adhesion and targeting of specific tissue types in the context of a biological fluid environment.

  13. Enzyme-Activated G-Quadruplex Synthesis for in Situ Label-Free Detection and Bioimaging of Cell Apoptosis.

    PubMed

    Liu, Zhuoliang; Luo, Xingyu; Li, Zhu; Huang, Yan; Nie, Zhou; Wang, Hong-Hui; Yao, Shouzhuo

    2017-02-07

    Fluorogenic probes targeting G-quadruplex structures have emerged as the promising toolkit for functional research of G-quadruplex and biosensor development. However, their biosensing applications are still largely limited in in-tube detection. Herein, we proposed a fluorescent bioimaging method based on enzyme-generated G-quadruplexes for detecting apoptotic cells at the cell and tissue level, namely, terminal deoxynucleotidyl transferase (TdT)-activated de novo G-quadruplex synthesis (TAGS) assay. The detection target is genomic DNA fragmentation, a biochemical hallmark of apoptosis. The TAGS assay can efficiently "tag" DNA fragments via using their DNA double-strand breaks (DSBs) to initiate the de novo synthesis of G-quadruplexes by TdT with an unmodified G-rich dNTP pool, followed by a rapid fluorescent readout upon the binding of thioflavin T (ThT), a fluorogenic dye highly specific for G-quadruplex. The feasibility of the TAGS assay was proved by in situ sensitive detection of individual apoptotic cells in both cultured cells and tissue sections. The TAGS assay has notable advantages, including being label-free and having quick detection, high sensitivity and contrast, mix-and-read operation without tedious washing, and low cost. This method not only shows the feasibility of G-quadruplex in tissue bioanalysis but also provides a promising tool for basic research of apoptosis and drug evaluation for antitumor therapy.

  14. Label-free cell-substrate adhesion imaging on plasmonic nanocup arrays

    PubMed Central

    Hackett, L. P.; Seo, S.; Kim, S.; Goddard, L. L.; Liu, G. L.

    2017-01-01

    Cell adhesion is a crucial biological and biomedical parameter defining cell differentiation, cell migration, cell survival, and state of disease. Because of its importance in cellular function, several tools have been developed in order to monitor cell adhesion in response to various biochemical and mechanical cues. However, there remains a need to monitor cell adhesion and cell-substrate separation with a method that allows real-time measurements on accessible equipment. In this article, we present a method to monitor cell-substrate separation at the single cell level using a plasmonic extraordinary optical transmission substrate, which has a high sensitivity to refractive index changes at the metal-dielectric interface. We show how refractive index changes can be detected using intensity peaks in color channel histograms from RGB images taken of the device surface with a brightfield microscope. This allows mapping of the nonuniform refractive index pattern of a single cell cultured on the plasmonic substrate and therefore high-throughput detection of cell-substrate adhesion with observations in real time. PMID:28271009

  15. Optofluidic device for label-free cell classification from whole blood.

    PubMed

    Wu, Tsung-Feng; Lo, Yu-Hwa

    2015-01-01

    A unique optofluidic lab-on-a-chip device that can detect optically encoded forward scattering signals is demonstrated. With a unique design of a spatial mask that patterns the intensity distribution of the illuminating light, the position and velocity of each travelling cell in the flow can be measured with submicrometer resolution, which enables the generation of a cell distribution plot over the cross section of the channel. The distribution of cells is highly sensitive to its size and stiffness, both being important biomarkers for cell classification without cell labelling. The optical-coding technique offers an easy route to classify cells based on their size and stiffness. Because the stiffness and size of neutrophils are distinct from other types of white blood cells, the number of neutrophils can be detected from other white blood cells and red blood cells. Above all, the enumeration of neutrophil concentration can be obtained from only 5 μL of human blood with a simple blood preparation process saving the usual steps of anticoagulation, centrifugation, antibody labelling, or filtering. The optofluidic system is compact, inexpensive, and simple to fabricate and operate. The system uses a commodity laser diode and a Si PIN photoreceiver and digital signal processing to extract vital information about cells and suppress the noise from the encoded optical scattering signals. The optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy.

  16. 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.

  17. 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.

  18. Chronic Label-free Volumetric Photoacoustic Microscopy of Melanoma Cells in Three-Dimensional Porous Scaffolds

    PubMed Central

    Zhang, Yu; Cai, Xin; Choi, Sung-Wook; Kim, Chulhong; Wang, Lihong V.; Xia, Younan

    2010-01-01

    Visualizing cells in three-dimensional (3D) scaffolds has been one of the major challenges in tissue engineering. Most current imaging modalities either suffer from poor penetration depth or require exogenous contrast agents. Here, we demonstrate photoacoustic microscopy (PAM) of the spatial distribution and temporal proliferation of cells inside three-dimensional porous scaffolds with thicknesses over 1 mm. Specifically, we evaluated the effects of seeding and culture methods on the spatial distribution of melanoma cells. Spatial distribution of the cells in the scaffold was well-resolved in PAM images. Moreover, the number of cells in the scaffold was quantitatively measured from the as-obtained volumetric information. The cell proliferation profile obtained from PAM correlated well with what was obtained using the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PMID:20727581

  19. 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.

  20. 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

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

    PubMed

    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

    2012-09-07

    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.

  2. Label-free enumeration, collection and downstream cytological and cytogenetic analysis of circulating tumor cells

    PubMed Central

    Dhar, Manjima; Pao, Edward; Renier, Corinne; Go, Derek E.; Che, James; Montoya, Rosita; Conrad, Rachel; Matsumoto, Melissa; Heirich, Kyra; Triboulet, Melanie; Rao, Jianyu; Jeffrey, Stefanie S.; Garon, Edward B.; Goldman, Jonathan; Rao, Nagesh P.; Kulkarni, Rajan; Sollier-Christen, Elodie; Di Carlo, Dino

    2016-01-01

    Circulating tumor cells (CTCs) have a great potential as indicators of metastatic disease that may help physicians improve cancer prognostication, treatment and patient outcomes. Heterogeneous marker expression as well as the complexity of current antibody-based isolation and analysis systems highlights the need for alternative methods. In this work, we use a microfluidic Vortex device that can selectively isolate potential tumor cells from blood independent of cell surface expression. This system was adapted to interface with three protein-marker-free analysis techniques: (i) an in-flow automated image processing system to enumerate cells released, (ii) cytological analysis using Papanicolaou (Pap) staining and (iii) fluorescence in situ hybridization (FISH) targeting the ALK rearrangement. In-flow counting enables a rapid assessment of the cancer-associated large circulating cells in a sample within minutes to determine whether standard downstream assays such as cytological and cytogenetic analyses that are more time consuming and costly are warranted. Using our platform integrated with these workflows, we analyzed 32 non-small cell lung cancer (NSCLC) and 22 breast cancer patient samples, yielding 60 to 100% of the cancer patients with a cell count over the healthy threshold, depending on the detection method used: respectively 77.8% for automated, 60–100% for cytology, and 80% for immunostaining based enumeration. PMID:27739521

  3. 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.

  4. Label-free assessment of endothelial cell metabolic state using autofluorescent microscopy

    NASA Astrophysics Data System (ADS)

    Pullen, Benjamin J.; Nguyen, Tam; Gosnell, Martin; Anwer, Ayad G.; Goldys, Ewa; Nicholls, Stephen J.; Psaltis, Peter J.

    2016-12-01

    To examine the process of endothelial cell aging we utilised hyperspectral imaging to collect broad autofluorescence emission at the individual cellular level and mathematically isolate the characteristic spectra of nicotinamide and flavin adenine dinucleotides (NADH and FAD, respectively). Quantitative analysis of this data provides the basis for a non-destructive spatial imaging method for cells and tissue. FAD and NADH are important factors in cellular metabolism and have been shown to be involved with the redox state of the cell; with the ratio between the two providing the basis for an `optical redox ratio'.

  5. Label-Free Imaging of Dynamic and Transient Calcium Signaling in Single Cells.

    PubMed

    Lu, Jin; Li, Jinghong

    2015-11-09

    Cell signaling consists of diverse events that occur at various temporal and spatial scales, ranging from milliseconds to hours and from single biomolecules to cell populations. The pathway complexities require the development of new techniques that detect the overall signaling activities and are not limited to quantifying a single event. A plasmonic-based electrochemical impedance microscope (P-EIM) that can provide such data with excellent temporal and spatial resolution and does not require the addition of any labels for detection has now been developed. The highly dynamic and transient calcium signaling activities at the early stage of G-protein-coupled receptor (GPCR) stimulation were thus studied. It could be shown that a subpopulation of cells is more responsive towards agonist stimulation, and the heterogeneity of the local distributions and the transient activities of the ion channels during agonist-activated calcium flux in single HeLa cells were investigated.

  6. 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.

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

    PubMed

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

    2012-08-31

    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.

  8. 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

  9. 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.

  10. Impact of label-free technologies in head and neck cancer circulating tumour cells

    PubMed Central

    Kulasinghe, Arutha; Kenny, Liz; Perry, Chris; Thiery, Jean-Paul; Jovanovic, Lidija; Vela, Ian; Nelson, Colleen; Punyadeera, Chamindie

    2016-01-01

    Background The ability to identify high risk head and neck cancer (HNC) patients with disseminated disease prior to presenting with clinically detectable metastases holds remarkable potential. A fraction of circulating tumour cells (CTCs) are invasive cancer cells which mediate metastasis by intravasation, survival and extravasation from the blood stream to metastatic sites. CTCs have been cleared by the FDA for use as surrogate markers of overall survival and progression free survival for breast, prostate and colorectal cancers using the CellSearch® system. However, the clinical significance of CTCs in head and neck cancer patients has yet to be determined. There has been a significant shift in CTC enrichment platforms, away from exclusively single marker selection, to epitope-independent systems. Methods The aim of this study was to screen advanced stage HNC patients by the CellSearch® platform and utilise two other epitope-independent approaches, ScreenCell® (microfiltration device) and RosetteSep™ (negative enrichment), to determine how a shift to such methodologies would enable CTC enrichment and detection. Results In advanced stage HNC patients, single CTCs were detected in 8/43 (18.6%) on CellSearch®, 13/28 (46.4%) on ScreenCell® and 16/25 (64.0%) by RosetteSep™ (the latter could also detect CTC clusters). Notably, in patients with suspicious lung nodules, too small to biopsy, CTCs were found upon presentation. Moreover, CTCs were readily detected in advanced stage HNC patients. Conclusion The epitope-independent platforms detected higher CTC numbers and clusters. Further studies are needed to ascertain whether CTCs can be used as independent prognostic markers for HNCs. PMID:27655722

  11. 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.

  12. Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

    PubMed Central

    Khamenehfar, A.; Beischlag, T. V.; Russell, P. J.; Ling, M. T. P.; Nelson, C.; Li, P. C. H.

    2015-01-01

    Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients. PMID:26594265

  13. 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.

  14. 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.

  15. Polymer photonic crystal dye lasers as label free evanescent cell sensors

    NASA Astrophysics Data System (ADS)

    Christiansen, Mads B.; Lopacinska, Joanna M.; Jakobsen, Mogens H.; Mortensen, Niels A.; Dufva, Martin; Kristensen, Anders

    2009-08-01

    Dye doped polymer photonic crystal band edge lasers are applied for evanescent wave sensing of cells. The lasers are rectangular shaped slab waveguides of dye doped polymer on a glass substrate, where a photonic crystal is formed by 100 nm deep air-holes in the surface of the 375 nm high waveguides. The lasers are fabricated by combined nanoimprint and photolithography (CNP) in Ormocore hybrid polymer doped with the laser dye Pyrromethene 597. The lasers emit in the chip plane at a wavelength around 595 nm when pumped with 5 ns pulses from a compact frequency doubled Nd:YAG laser. We investigate the sensitivity of photonic crystal band-edge lasers to partial coverage with HeLa cells. The lasers are chemically activated with a flexible UV activated anthraquinone based linker molecule, which enables selective binding of cells and molecules. When measuring in Phosphate Buffered Saline (PBS), which has a refractive index close to that of the cells, the emission wavelength depends linearly on the cell density on the sensor surface. Our results demonstrate that nanostructured hybrid polymer lasers, which are cheap to fabricate and very simple to operate, can be selectively chemically activated with UV sensitive photolinkers for further bioanalytical applications. This opens the possibility to functionalize arrays of optofluidic laser sensors with different bio-recognition molecules for multiplexed sensing. The linear relationship between cell coverage and wavelength indicates that the slight refractive index perturbation from the partial coverage of the sensor influences the entire optical mode, rather than breaking down the photonic crystal feedback.

  16. 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.

  17. 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.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26901310

  20. 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

  1. 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.

  2. 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

  3. Label-free mass spectrometry exploits dozens of detected peptides to quantify lamins in wildtype and knockdown cells.

    PubMed

    Swift, Joe; Harada, Takamasa; Buxboim, Amnon; Shin, Jae-Won; Tang, Hsin-Yao; Speicher, David W; Discher, Dennis E

    2013-01-01

    Label-free quantitation and characterization of proteins by mass spectrometry (MS) is now feasible, especially for moderately expressed structural proteins such as lamins that typically yield dozens of tryptic peptides from tissue cells. Using standard cell culture samples, we describe general algorithms for quantitative analysis of peptides identified in liquid chromatography tandem mass spectrometry (LC-MS/MS). The algorithms were foundational to the discovery that the absolute stoichiometry of A-type to B-type lamins scales with tissue stiffness (Swift et al., Science 2013). Isoform dominance helps make sense of why mutations and changes with age of mechanosensitive lamin-A,C only affect "stiff" tissues such as heart, muscle, bone, or even fat, but not brain. A Peak Ratio Fingerprinting (PRF) algorithm is elaborated here through its application to lamin-A,C knockdown. After demonstrating the large dynamic range of PRF using calibrated mixtures of human and mouse lysates, we validate measurements of partial knockdown with standard cell biology analyses using quantitative immunofluorescence and immunoblotting. Optimal sets of MS-detected peptides as determined by PRF demonstrate that the strongest peptide signals are not necessarily the most reliable for quantitation. After lamin-A,C knockdown, PRF computes an invariant set of "housekeeping" proteins as part of a broader proteomic analysis that also shows the proteome of mesenchymal stem cells (MSCs) is more broadly perturbed than that of a human epithelial cancer line (A549s), with particular variation in nuclear and cytoskeletal proteins. These methods offer exciting prospects for basic and clinical studies of lamin-A,C as well as other MS-detectable proteins.

  4. 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

  5. Label-free enumeration of colorectal cancer cells from lymphocytes performed at a high cell-loading density by using interdigitated ring-array microelectrodes.

    PubMed

    Xing, Xiaoxing; Poon, Randy Y C; Wong, Cesar S C; Yobas, Levent

    2014-11-15

    We report the label-free enumeration of human colorectal-carcinoma cells from blood lymphocytes by using interdigitated ring-array microelectrodes; this enumeration was based on the dielectrophoretic selection of cells. Because of the novel design of the device, a continuous flow of cells is uniformly distributed into parallel streams through 300 rings (~40 μm in diameter each) that are integrated into the electrode digits. Using this array, 82% of cancer cells were recovered and 99% of blood lymphocytes were removed. Most of the cancer cells recovered were viable (94%) and could be cultivated for >8 days, during which period they retained their normal cell morphology and proliferation rates. The recovery rate correlated closely with cancer-cell loadings in spiked samples and this relationship was linear over a range of at least 2 orders of magnitude. Importantly, because of the 3D structure of the rings, these results were obtained at a high cell-loading concentration (10(7)cells/mL). The rings could be further optimized for use in accurate label-free identification and measurement of circulating tumor cells in cancer research and disease management.

  6. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.

    PubMed

    Peper, Janet Kerstin; Schuster, Heiko; Löffler, Markus W; Schmid-Horch, Barbara; Rammensee, Hans-Georg; Stevanović, Stefan

    2014-03-01

    The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

  7. A novel electrochemical biosensor based on polyadenine modified aptamer for label-free and ultrasensitive detection of human breast cancer cells.

    PubMed

    Wang, Kun; He, Meng-Qi; Zhai, Fu-Heng; He, Rong-Huan; Yu, Yong-Liang

    2017-05-01

    Simple, rapid, sensitive, and specific detection of cancer cells plays a pivotal role in the diagnosis and prognosis of cancer. A sandwich electrochemical biosensor was developed based on polyadenine (polydA)-aptamer modified gold electrode (GE) and polydA-aptamer functionalized gold nanoparticles/graphene oxide (AuNPs/GO) hybrid for the label-free and selective detection of breast cancer cells (MCF-7) via a differential pulse voltammetry (DPV) technique. Due to the intrinsic affinity between multiple consecutive adenines of polydA sequences and gold, polydA modified aptamer instead of thiol terminated aptamer was immobilized on the surface of GE and AuNPs/GO. The label-free MCF-7 cells could be recognized by polydA-aptamer and self-assembled onto the surface of GE. The polydA-aptamer functionalized AuNPs/GO hybrid could further bind to MCF-7 cells to form a sandwich sensing system. Characterization of the surface modified GE was carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using Fe(CN)6(3-/4-) as a redox probe. Under the optimized experimental conditions, a detection limit of 8 cellsmL(-1) (3σ/slope) was obtained for MCF-7 cells by the present electrochemical biosensor, along with a linear range of 10-10(5) cellsmL(-1). By virtue of excellent sensitivity, specificity and repeatability, the present electrochemical biosensor provides a potential application in point-of-care cancer diagnosis.

  8. Multiplexed specific label-free detection of NCI-H358 lung cancer cell line lysates with silicon based photonic crystal microcavity biosensors.

    PubMed

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Drabkin, Harry A; Gemmill, Robert M; Simon, George R; Chin, Steve H; Chen, Ray T

    2013-05-15

    We experimentally demonstrate label-free photonic crystal (PC) microcavity biosensors in silicon-on-insulator (SOI) to detect the epithelial-mesenchymal transition (EMT) transcription factor, ZEB1, in minute volumes of sample. Multiplexed specific detection of ZEB1 in lysates from NCI-H358 lung cancer cells down to an estimated concentration of 2 cells per micro-liter is demonstrated. L13 photonic crystal microcavities, coupled to W1 photonic crystal waveguides, are employed in which resonances show high Q in the bio-ambient phosphate buffered saline (PBS). When the sensor surface is derivatized with a specific antibody, the binding of the corresponding antigen from a complex whole-cell lysate generates a change in refractive index in the vicinity of the photonic crystal microcavity, leading to a change in the resonance wavelength of the resonance modes of the photonic crystal microcavity. The shift in the resonance wavelength reveals the presence of the antigen. The sensor cavity has a surface area of ∼11μm(2). Multiplexed sensors permit simultaneous detection of many binding interactions with specific immobilized antibodies from the same bio-sample at the same instant of time. Specificity was demonstrated using a sandwich assay which further amplifies the detection sensitivity at low concentrations. The device represents a proof-of-concept demonstration of label-free, high throughput, multiplexed detection of cancer cells with specificity and sensitivity on a silicon chip platform.

  9. Detection of soluble ERBB2 in breast cancer cell lysates using a combined label-free/fluorescence platform based on Bloch surface waves.

    PubMed

    Sinibaldi, Alberto; Sampaoli, Camilla; Danz, Norbert; Munzert, Peter; Sibilio, Leonardo; Sonntag, Frank; Occhicone, Agostino; Falvo, Elisabetta; Tremante, Elisa; Giacomini, Patrizio; Michelotti, Francesco

    2017-06-15

    We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of ERBB2/neu/Her2 in cell lysates. ERBB2 is a pivotal breast cancer biomarker and targetable oncogenic driver associated with aggressive breast cancer subtypes. To quantitate soluble ERBB2, we developed an optical platform that combines label-free and fluorescence detection modes. Such platform makes use of a sandwich assay in which the one-dimensional photonic crystals sustaining Bloch surface waves are tailored with a monoclonal antibody for highly specific biological recognition (BSW biochip). In a second step, a second antibody to ERBB2 quantitatively detects the bound analyte. The strategy of the present approach takes advantage of the combination of label-free and fluorescence techniques, making bio-recognition more robust and sensitive. In the fluorescence operation mode, the platform can attain the limit of detection 0.3ng/mL (1.5pM) for ERBB2 in cell lysates. Such resolution meets the international guidelines and recommendations (15ng/mL) for diagnostic ERBB2 assays that in the future may help to more precisely assign therapies counteracting cancer cell proliferation and metastatic spread.

  10. 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.

  11. Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4+ T Cells from Patients with Vogt-Koyanagi-Harada Syndrome

    PubMed Central

    Mao, Liming; Yang, Peizeng; Hou, Shengping; Li, Fuzhen; Kijlstra, Aize

    2011-01-01

    Vogt-Koyanagi-Harada (VKH) syndrome is a systemic autoimmune disease. CD4+ T cells have been shown to be involved in autoimmune diseases including VKH syndrome. To screen aberrantly expressed membrane proteins in CD4+ T cell from patients with active VKH syndrome, blood samples were taken from five patients with active VKH syndrome and five healthy individuals. A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the two groups. The results revealed that the expression of 102 peptides was significantly altered (p<0.05) between two groups and matched amino acid sequences of proteins deposited in the international protein index (ipi.HUMAN.v3.36.fasta). The identified peptides corresponded to 64 proteins, in which 30 showed more than a 1.5-fold difference between the two groups. The decreased expression of CD18 and AKNA transcription factor (AKNA), both being three-fold lower than controls in expression identified by the label-free method, was further confirmed in an additional group of five active VKH patients and six normal individuals using the Western blot technique. A significantly decreased expression of CD18 and AKNA suggests a role for both proteins in the pathogenesis of this syndrome. PMID:21297967

  12. High-Throughput, Label-Free Isolation of Cancer Stem Cells on the Basis of Cell Adhesion Capacity.

    PubMed

    Zhang, Yuanqing; Wu, Minhao; Han, Xin; Wang, Ping; Qin, Lidong

    2015-09-07

    Herein we report a microfluidics method that enriches cancer stem cells (CSCs) or tumor-initiating cells on the basis of cell adhesion properties. In our on-chip enrichment system, cancer cells were driven by hydrodynamic forces to flow through microchannels coated with basement membrane extract. Highly adhesive cells were captured by the functionalized microchannels, and less adhesive cells were collected from the outlets. Two heterogeneous breast cancer cell lines (SUM-149 and SUM-159) were successfully separated into enriched subpopulations according to their adhesive capacity, and the enrichment of the cancer stem cells was confirmed by flow cytometry biomarker analysis and tumor-formation assays. Our findings show that the less adhesive phenotype is associated with a higher percentage of CSCs, higher cancer-cell motility, and higher resistance to chemotherapeutic drugs.

  13. 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.

  14. 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.

  15. Biopatterning for label-free detection

    PubMed Central

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

    2009-01-01

    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

  16. Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

    PubMed

    Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

    2015-02-15

    We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers.

  17. A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification.

    PubMed

    Xiao, Kunyi; Liu, Juan; Chen, Hui; Zhang, Song; Kong, Jilie

    2017-05-15

    A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 10(5) cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis.

  18. 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

    2016-03-02

    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.

  19. Integrating cell phone imaging with magnetic levitation (i-LEV) for label-free blood analysis at the point-of-living

    PubMed Central

    Durmus, Naside Gozde; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

    2016-01-01

    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, we present a magnetic levitation-based diagnosis system 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, we introduce an easy-to-use, smartphone incorporated levitation system for cell analysis. Using our portable imaging magnetic levitation (i-LEV) system, we show 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

  20. Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

    PubMed Central

    Heaton, Richard J.; Peterson, Alexander W.; Georgiadis, Rosina M.

    2001-01-01

    We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum. PMID:11259682

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

    PubMed

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

    2014-12-01

    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.

  2. Mechanisms of kidney repair by human mesenchymal stromal cells after ischemia: a comprehensive view using label-free MS(E).

    PubMed

    da Costa, Milene R; Pizzatti, Luciana; Lindoso, Rafael S; Sant'Anna, Julliana Ferreira; DuRocher, Barbara; Abdelhay, Eliana; Vieyra, Adalberto

    2014-06-01

    Acute kidney injury (AKI) is one of the more frequent and lethal pathological conditions seen in intensive care units. Currently available treatments are not totally effective but stem cell-based therapies are emerging as promising alternatives, especially the use of mesenchymal stromal cells (MSC), although the signaling pathways involved in their beneficial actions are not fully understood. The objective of this study was to identify signaling networks and key proteins involved in the repair of ischemia by MSC. Using an in vitro model of AKI to investigate paracrine interactions and label-free high definition 2D-NanoESI-MS(E) , differentially expressed proteins were identified in a human renal proximal tubule cell lineage (HK-2) exposed to human MSC (hMSC) after an ischemic insult. In silico analysis showed that hMSC stimulated antiapoptotic activity, normal ROS handling, energy production, cytoskeleton organization, protein synthesis, and cell proliferation. The proteomic data were validated by parallel experiments demonstrating reduced apoptosis in HK-2 cells and recovery of intracellular ATP levels. qRT-PCR for proteins implicated in the above processes revealed that hMSC exerted their effects by stimulating translation, not transcription. Western blotting of proteins associated with ROS and energy metabolism confirmed their higher abundance in HK-2 cells exposed to hMSC.

  3. Label-free electrochemiluminescence biosensor for ultrasensitive detection of telomerase activity in HeLa cells based on extension reaction and intercalation of Ru(phen)3 (2.).

    PubMed

    Lin, Yue; Yang, Linlin; Yue, Guiyin; Chen, Lifen; Qiu, Bin; Guo, Longhua; Lin, Zhenyu; Chen, Guonan

    2016-10-01

    Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 (2+) has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were <5 % at 100 HeLa cells. The proposed method provides a convenient approach for telomerase-related cancer screening or diagnosis.

  4. 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.

  5. A signal-on fluorescent aptasensor based on single-stranded DNA-sensitized luminescence of terbium (III) for label-free detection of breast cancer cells.

    PubMed

    Cai, Shuxian; Li, Guangwen; Zhang, Xi; Xia, Yaokun; Chen, Mei; Wu, Dongzhi; Chen, Qiuxiang; Zhang, Jing; Chen, Jinghua

    2015-06-01

    Breast cancer is the most common type of malignant tumor in women. Recently, it has been shown that detection of breast cancer tumor cells outside the primitive tumor is an effective early diagnosis with great prognostic and clinical utility. For this purpose, we developed a signal-on fluorescence aptasensor for label-free, facile and sensitive detection of MCF-7 breast cancer cells. Due to target-aptamer specific recognition and single-stranded DNA-sensitized luminescence of terbium (III), the proposed aptasensor exhibits excellent sensitivity with detection limit as low as 70 cells mL(-1). Compared with common organic dyes and the emerging nano-technological probes, the combination of terbium (III) and single-stranded DNA signal probe (Tb(3+)-SP) serves as a more powerful bio-probe because of its stable optical property, good biocompatibility and free from complex synthesis. The feasibility investigations have illustrated the potential applicability of this aptasensor for selective and sensitive detection of MCF-7 breast cancer cells. Moreover, this proposed aptasensor can be also extended for the determination of other tumor cancers or bio-molecules by altering corresponding aptamers. Taken together, this easy-to-perform aptasensor may represent a promising way for early screening and detection of tumor cancers or other bio-molecules in clinical diagnosis.

  6. 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.

  7. 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 Central

    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

  8. A lab-on-chip cell-based biosensor for label-free sensing of water toxicants.

    PubMed

    Liu, F; Nordin, A N; Li, F; Voiculescu, I

    2014-04-07

    This paper presents a lab-on-chip biosensor containing an enclosed fluidic cell culturing well seeded with live cells for rapid screening of toxicants in drinking water. The sensor is based on the innovative placement of the working electrode for the electrical cell-substrate impedance sensing (ECIS) technique as the top electrode of a quartz crystal microbalance (QCM) resonator. Cell damage induced by toxic water will cause a decrease in impedance, as well as an increase in the resonant frequency. For water toxicity tests, the biosensor's unique capabilities of performing two complementary measurements simultaneously (impedance and mass-sensing) will increase the accuracy of detection while decreasing the false-positive rate. Bovine aortic endothelial cells (BAECs) were used as toxicity sensing cells. The effects of the toxicants, ammonia, nicotine and aldicarb, on cells were monitored with both the QCM and the ECIS technique. The lab-on-chip was demonstrated to be sensitive to low concentrations of toxicants. The responses of BAECs to toxic samples occurred during the initial 5 to 20 minutes depending on the type of chemical and concentrations. Testing the multiparameter biosensor with aldicarb also demonstrated the hypothesis that using two different sensors to monitor the same cell monolayer provides cross validation and increases the accuracy of detection. For low concentrations of aldicarb, the variations in impedance measurements are insignificant in comparison with the shifts of resonant frequency monitored using the QCM resonator. A highly linear correlation between signal shifts and chemical concentrations was demonstrated for each toxicant.

  9. 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

  10. Real-Time, Label-Free Detection of Local Exocytosis Outside Pancreatic β Cells Using Laser Tweezers Raman Spectroscopy.

    PubMed

    Luo, Rui-Qiong; Wei, Fang; Huang, Shu-Shi; Jiang, Yue-Ming; Zhang, Shan-Lei; Mo, Wen-Qing; Liu, Hong; Rong, Xi

    2016-12-09

    The examination of insulin (Ins) exocytosis at the single-cell level by conventional methods, such as electrophysiological approaches, total internal reflection imaging, and two-photon imaging technology, often requires an invasive microelectrode puncture or label. In this study, high concentrations of glucose and potassium chloride were used to stimulate β cell Ins exocytosis, while low concentrations of glucose and calcium channel blockers served as the blank and negative control, respectively. Laser tweezers Raman spectroscopy (LTRS) was used to capture the possible Raman scattering signal from a local zone outside of the cell edge. The results show that the frequencies of the strong signals from the local zones outside the cellular edge in the stimulated groups are greater than those of the control. The Raman spectra from the cellular edge, Ins and cell membrane were compared. Thus, local Ins exocytosis activity outside pancreatic β cells might be observed indirectly using LTRS, a non-invasive optical method.

  11. High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

    PubMed

    Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

    2017-04-11

    The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry.

  12. High-resolution, label-free imaging of living cells with direct electron-beam-excitation-assisted optical microscopy.

    PubMed

    Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-06-01

    High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.

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

    PubMed

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

    2014-10-24

    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 60min 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 250nm diameter), flow cytometry, showing high phosphatidylserine exposition and by fluorescent (Annexin V Alexa Fluor® 488-positive) and electron microscopy. Over a period of 1000s (16.7min) during which early apoptosis increased from 4% plateauing at 10% and late apoptosis rose to 2%, the Δf increased 20Hz, 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(6)MVs, was calculated to be 23ng. We therefore estimated the mass of an MV to be 0.24pg. With the deposition on the QCM-D of 3.5×10(7)MVs over 200s, the decrease in Δf (Hz) gave an estimate of 0.235pg per MV.

  14. Filamin C, a dysregulated protein in cancer revealed by label-free quantitative proteomic analyses of human gastric cancer cells.

    PubMed

    Qiao, Jie; Cui, Shu-Jian; Xu, Lei-Lei; Chen, Si-Jie; Yao, Jun; Jiang, Ying-Hua; Peng, Gang; Fang, Cai-Yun; Yang, Peng-Yuan; Liu, Feng

    2015-01-20

    Gastric cancer (GC) is the fourth and fifth most common cancer in men and women, respectively. We identified 2,750 proteins at false discovery rates of 1.3% (protein) and 0.03% (spectrum) by comparing the proteomic profiles of three GC and a normal gastric cell lines. Nine proteins were significantly dysregulated in all three GC cell lines, including filamin C, a muscle-specific filamin and a large actin-cross-linking protein. Downregulation of filamin C in GC cell lines and tissues were verified using quantitative PCR and immunohistochemistry. Data-mining using public microarray datasets shown that filamin C was significantly reduced in many human primary and metastasis cancers. Transient expression or silencing of filamin C affected the proliferation and colony formation of cancer cells. Silencing of endogenous filamin C enhanced cancer cell migration and invasion, whereas ectopic expression of filamin C had opposing effects. Silencing of filamin C increased the expression of matrix metallopeptidase 2 and improved the metastasis of prostate cancer in a zebrafish model. High filamin C associated with better prognosis of prostate cancer, leukemia and breast cancer patients. These findings establish a functional role of filamin C in human cancers and these data will be valuable for further study of its mechanisms.

  15. Label-free measurement of cell-electrode cleft gap distance with high spatial resolution surface plasmon microscopy.

    PubMed

    Toma, Koji; Kano, Hiroshi; Offenhäusser, Andreas

    2014-12-23

    Understanding the interface between cells or tissues and artificial materials is of critical importance for a broad range of areas. For example, in neurotechnology, the interfaces between neurons and external devices create a link between technical and the nervous systems by stimulating or recording from neural tissue. Here, a more effective interface is required to enhance the electrical characteristics of neuronal recordings and stimulations. Up to now, the lack of a systematic characterization of cell-electrode interaction turns out to be the major bottleneck. In this work, we employed a recently developed surface plasmon microscope (SPM) to monitor in real-time the cell-metal interface and to measure in situ the gap distance of the cleft with the spatial resolution reaching to the optical diffraction limit. The SPM allowed determination of the distance of human embryonic kidney 293 cells cultured on gold surfaces coated with various peptides or proteins without any labeling. This method can dramatically simplify the interaction investigation at metal-living cell interface and should be incorporated into systematic characterization methods.

  16. 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

    2016-02-05

    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.

  17. 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.

  18. Label-free imaging of trabecular meshwork cells using Coherent Anti-Stokes Raman Scattering (CARS) microscopy

    PubMed Central

    Lei, Tim C.; Ammar, David A.; Masihzadeh, Omid; Gibson, Emily A.

    2011-01-01

    Purpose To image the human trabecular meshwork (TM) using a non-invasive, non-destructive technique without the application of exogenous label. Methods Flat-mounted TM samples from a human cadaver eye were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). In TPAF, two optical photons are simultaneously absorbed and excite molecules in the sample that then emit a higher energy photon. The signal is predominately from collagen and elastin. The CARS technique uses two laser frequencies to specifically excite carbon-hydrogen bonds, allowing the visualization of lipid-rich cell membranes. Multiple images were taken along an axis perpendicular to the surface of the TM for subsequent analysis. Results Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes. Simultaneous CARS imaging revealed elliptical structures of ~7×10 µm in diameter populating the meshwork which were consistent with TM cells. Irregularly shaped objects of ~4 µm diameter appeared in both the TPAF and CARS channels, and are consistent with melanin granules. Conclusions CARS techniques were successful in imaging live TM cells in freshly isolated human TM samples. Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols. CARS imaging of the TM represents a new avenue for exploring details of aqueous outflow and TM cell physiology. PMID:22025898

  19. Label-free, high-throughput measurements of dynamic changes in cell nuclei using angle-resolved low coherence interferometry.

    PubMed

    Chalut, Kevin J; Chen, Sulin; Finan, John D; Giacomelli, Michael G; Guilak, Farshid; Leong, Kam W; Wax, Adam

    2008-06-01

    Accurate measurements of nuclear deformation, i.e., structural changes of the nucleus in response to environmental stimuli, are important for signal transduction studies. Traditionally, these measurements require labeling and imaging, and then nuclear measurement using image analysis. This approach is time-consuming, invasive, and unavoidably perturbs cellular systems. Light scattering, an emerging biophotonics technique for probing physical characteristics of living systems, offers a promising alternative. Angle-resolved low-coherence interferometry (a/LCI), a novel light scattering technique, was developed to quantify nuclear morphology for early cancer detection. In this study, a/LCI is used for the first time to noninvasively measure small changes in nuclear morphology in response to environmental stimuli. With this new application, we broaden the potential uses of a/LCI by demonstrating high-throughput measurements and by probing aspherical nuclei. To demonstrate the versatility of this approach, two distinct models relevant to current investigations in cell and tissue engineering research are used. Structural changes in cell nuclei due to subtle environmental stimuli, including substrate topography and osmotic pressure, are profiled rapidly without disrupting the cells or introducing artifacts associated with traditional measurements. Accuracy > or = 3% is obtained for the range of nuclear geometries examined here, with the greatest deviations occurring for the more complex geometries. Given the high-throughput nature of the measurements, this deviation may be acceptable for many biological applications that seek to establish connections between morphology and function.

  20. 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.

  1. Label-free study of the function of ion channel protein on a microfluidic optical sensor integrated with artificial cell membrane.

    PubMed

    Li, Zhen; Tang, Yanyan; Zhang, Ling; Wu, Jianmin

    2014-01-21

    A label-free optical sensor was constructed by integrating pH sensing material and supported phospholipid bilayers (SPBs) in a microfluidic chip. The pH sensing material was composed of a double layer structure consisting of chitosan hydrogel and electrochemically etched porous silicon. The pH change in the microchip could induce a reversible swelling of the chitosan hydrogel layer and consequently caused a shift in effective optical thickness (EOT) of the double layer, which could be observed by Fourier transformed reflectometric interference spectroscopy (FT-RIS). After phospholipid bilayers (PLBs) were self-assembled on the sensing layer, the EOT almost remained constant during the cycling of pH from 7.4 to 6.2, indicating the blockage of H(+) translocation by the PLBs. For studying the behavior of ion channel protein, gramicidin A, a typical ion channel protein, was inserted in the SPBs for mimicking the ion transportation function of cell membrane. Due to the H(+) transportation capability of gramicidin A, the optical response to pH change could partially recover. In the presence of Ca(2+), the pore of the ion channel protein was blocked, causing a significant decrease in the EOT response upon pH change. The bio-functionalized microfluidic sensor fabricated in this work will provide a reliable platform for studying the function of ion channel protein, which is an important class of drug targets.

  2. 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.

  3. Label-free multiphoton fluorescence imaging monitors metabolism in living primary human cells used for tissue engineering

    NASA Astrophysics Data System (ADS)

    Chen, Leng-Chun; Lloyd, William R.; Kuo, Shiuhyang; Marcelo, Cynthia L.; Feinberg, Stephen E.; Mycek, Mary-Ann

    2012-03-01

    Fluorescence redox imaging was employed to monitor the metabolic activity of primary human oral keratinocytes prior to the development of tissue-engineered constructs. Keratinocytes with controlled culture conditions were treated with varying levels of chemical stimuli, resulting in differing cellular morphology, growth rate, and metabolic activity. Fluorescence images of keratinocytes were noninvasively acquired from endogenous intracellular metabolic fluorophores NAD(P)H and FAD. A redox ratio quantitatively analyzed each pair of images, showing that fluorescence redox imaging may be a novel technique to characterize live cell viability

  4. 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.

  5. 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

  6. Reproducible E. coli detection based on label-free SERS and mapping.

    PubMed

    Yang, Danting; Zhou, Haibo; Haisch, Christoph; Niessner, Reinhard; Ying, Yibin

    2016-01-01

    The biosensing for rapid detection of bacteria based on surface-enhanced Raman scattering (SERS) has been widely explored for recent years. It is still a challenge to achieve a high sensitive, reproducible label free detection method for bacteria. In this work, a label-free SERS detection method of Escherichia coli based on incubation with silver colloid was reported. Optimized incubation conditions including shaking speed, time and temperature were used to help construct a rapid SERS method for E. coli analysis. It was found that the enhancement of the Raman signal of E. coli could be achieved to 1.8×10(4) cps (counts per second) with high reproducibility. Three strains of E. coli DSM 1116/498/5695 could be successfully discriminated using such SERS method combining discriminant analysis. Finally, the lowest concentration of E. coli at 1×10(5) cell/mL can be detected by SERS mapping. Thus, our detection method offers higher sensitivity and reproducibility compared to previously reported label free simple-mixing methods, opening an avenue for developing various SERS-based biosensor.

  7. Label-Free Raman Imaging to Monitor Breast Tumor Signatures.

    PubMed

    Manciu, Felicia S; Ciubuc, John D; Parra, Karla; Manciu, Marian; Bennet, Kevin E; Valenzuela, Paloma; Sundin, Emma M; Durrer, William G; Reza, Luis; Francia, Giulio

    2016-07-04

    Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for

  8. 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.

  9. 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.

  10. Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy

    PubMed Central

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

    2013-01-01

    Abstract 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

  11. Label-free functional selectivity assays.

    PubMed

    Ferrie, Ann M; Goral, Vasiliy; Wang, Chaoming; Fang, Ye

    2015-01-01

    G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the β2-adrenergic receptor (β2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of β2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the β2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map.

  12. Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression

    PubMed Central

    Schmitt, Anna; Riabinska, Arina; Thelen, Lisa; Peifer, Martin; Leeser, Uschi; Nuernberg, Peter; Altmueller, Janine; Gaestel, Matthias; Dieterich, Christoph; Reinhardt, H. Christian

    2015-01-01

    Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G1/S transition through the suppression of Cdkn1aP21 transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme. PMID:25993413

  13. 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

  14. 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

  15. 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

  16. Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

    2013-03-01

    We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called "membrane nanotubes" or "tunneling nanotubes." However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

  17. Multimodal discrimination of immune cells using a combination of Raman spectroscopy and digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    McReynolds, Naomi; Cooke, Fiona G. M.; Chen, Mingzhou; Powis, Simon J.; Dholakia, Kishan

    2017-03-01

    The ability to identify and characterise individual cells of the immune system under label-free conditions would be a significant advantage in biomedical and clinical studies where untouched and unmodified cells are required. We present a multi-modal system capable of simultaneously acquiring both single point Raman spectra and digital holographic images of single cells. We use this combined approach to identify and discriminate between immune cell populations CD4+ T cells, B cells and monocytes. We investigate several approaches to interpret the phase images including signal intensity histograms and texture analysis. Both modalities are independently able to discriminate between cell subsets and dual-modality may therefore be used a means for validation. We demonstrate here sensitivities achieved in the range of 86.8% to 100%, and specificities in the range of 85.4% to 100%. Additionally each modality provides information not available from the other providing both a molecular and a morphological signature of each cell.

  18. Issues and Applications in Label-Free Quantitative Mass Spectrometry

    PubMed Central

    Lai, Xianyin; Wang, Lianshui; Witzmann, Frank A.

    2013-01-01

    To address the challenges associated with differential expression proteomics, label-free mass spectrometric protein quantification methods have been developed as alternatives to array-based, gel-based, and stable isotope tag or label-based approaches. In this paper, we focus on the issues associated with label-free methods that rely on quantitation based on peptide ion peak area measurement. These issues include chromatographic alignment, peptide qualification for quantitation, and normalization. In addressing these issues, we present various approaches, assembled in a recently developed label-free quantitative mass spectrometry platform, that overcome these difficulties and enable comprehensive, accurate, and reproducible protein quantitation in highly complex protein mixtures from experiments with many sample groups. As examples of the utility of this approach, we present a variety of cases where the platform was applied successfully to assess differential protein expression or abundance in body fluids, in vitro nanotoxicology models, tissue proteomics in genetic knock-in mice, and cell membrane proteomics. PMID:23401775

  19. 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

    2016-09-01

    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

  20. 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

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

    2015-06-01

    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.

  1. 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.

  2. 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

  3. Label-free identification and characterization of murine hair follicle stem cells located in thin tissue sections with Raman micro-spectroscopy.

    PubMed

    Tsai, Tsung-Hua; Short, Michael A; McLean, David I; Zeng, Haishan; McElwee, Kevin; Lui, Harvey

    2014-06-07

    Stem cells offer tremendous opportunities for regenerative medicine. Over the past decade considerable research has taken place to identify and characterize the differentiation states of stem cells in culture. Raman micro-spectroscopy has emerged as an ideal technology since it is fast, nondestructive, and does not require potentially toxic dyes. Raman spectroscopy systems can also be incorporated into confocal microscope imaging systems allowing spectra to be obtained from below the tissue surface. Thus there is significant potential for monitoring stem cells in living tissue. Stem cells that reside in hair follicles are suitable for testing this possibility since they are close to the skin surface, and typically clustered around the bulge area. One of the first steps needed would be to obtain Raman micro-spectra from stem cells located in thin sections of tissue, and then see whether these spectra are clearly different from those of the surrounding differentiated cells. To facilitate this test, standard 5 μm thick sections of murine skin tissue were stained to identify the location of hair follicle stem cells and their progeny. Raman spectra were then obtained from adjacent cells in a subsequent unstained 10 μm thick section. The spectra revealed significant differences in peak intensities associated with nucleic acids, proteins, lipids and amino acids. Statistical analyses of the Raman micro-spectra identified stem cells with 98% sensitivity and 94% specificity, as compared with a CD34 immunostaining gold standard. Furthermore analyses of the spectral variance indicated differences in cellular dynamics between the two cell groups. This study shows that Raman micro-spectroscopy has a potential role in identifying adult follicle stem cells, laying the groundwork for future applications of hair follicle stem cells and other somatic stem cells in situ.

  4. 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.

  5. 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

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

    PubMed

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

    2015-09-15

    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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Label-Free Detection of Breast Masses Using Multiphoton Microscopy

    PubMed Central

    Lu, Jianping; Zhu, Weifeng; Qiu, Jingting; Chen, Jianxin; Xie, Shusen; Zhuo, Shuangmu; Yan, Jun

    2013-01-01

    Histopathology forms the gold standard for the diagnosis of breast cancer. Multiphoton microscopy (MPM) has been proposed to be a potentially powerful adjunct to current histopathological techniques. A label-free imaging based on two- photon excited fluorescence and second-harmonic generation is developed for differentiating normal breast tissues, benign, as well as breast cancer tissues. Human breast biopsies (including human normal breast tissues, benign as well as breast cancer tissues ) that are first imaged (fresh, unfixed, and unstained) with MPM and are then processed for routine H-E histopathology. Our results suggest that the MPM images, obtained from these unprocessed biopsies, can readily distinguish between benign lesions and breast cancers. In the tissues of breast cancers, MPM showed that the tumor cells displayed marked cellular and nuclear pleomorphism. The tumor cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, infiltrated into disrupted connective tissue, leading to the loss of second-harmonic generation signals. For breast cancer, MPM diagnosis was 100% correct because the tissues of breast cancers did not have second-harmonic generation signals in MPM imaging. On the contrary, in benign breast masses, second-harmonic generation signals could be seen easily in MPM imaging. These observations indicate that MPM could be an important potential tool to provide label-free noninvasive diagnostic impressions that can guide surgeon in biopsy and patient management. PMID:23755295

  13. Label-free, rapid and quantitative phenotyping of stress response in E. coli via ramanome

    PubMed Central

    Teng, Lin; Wang, Xian; Wang, Xiaojun; Gou, Honglei; Ren, Lihui; Wang, Tingting; Wang, Yun; Ji, Yuetong; Huang, Wei E.; Xu, Jian

    2016-01-01

    Rapid profiling of stress-response at single-cell resolution yet in a label-free, non-disruptive and mechanism-specific manner can lead to many new applications. We propose a single-cell-level biochemical fingerprinting approach named “ramanome”, which is the collection of Single-cell Raman Spectra (SCRS) from a number of cells randomly selected from an isogenic population at a given time and condition, to rapidly and quantitatively detect and characterize stress responses of cellular population. SCRS of Escherichia coli cells are sensitive to both exposure time (eight time points) and dosage (six doses) of ethanol, with detection time as early as 5 min and discrimination rate of either factor over 80%. Moreover, the ramanomes upon six chemical compounds from three categories, including antibiotics of ampicillin and kanamycin, alcohols of ethanol and n-butanol and heavy metals of Cu2+ and Cr6+, were analyzed and 31 marker Raman bands were revealed which distinguish stress-responses via cytotoxicity mechanism and variation of inter-cellular heterogeneity. Furthermore, specificity, reproducibility and mechanistic basis of ramanome were validated by tracking stress-induced dynamics of metabolites and by contrasting between cells with and without genes that convey stress resistance. Thus ramanome enables rapid prediction and mechanism-based screening of cytotoxicity and stress-response programs at single-cell resolution. PMID:27756907

  14. 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.

  15. Extracting histones for the specific purpose of label-free MS.

    PubMed

    Govaert, Elisabeth; Van Steendam, Katleen; Scheerlinck, Ellen; Vossaert, Liesbeth; Meert, Paulien; Stella, Martina; Willems, Sander; De Clerck, Laura; Dhaenens, Maarten; Deforce, Dieter

    2016-12-01

    Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885.

  16. Spectroscopic Methods for Label-Free Optical Nanoscopy

    NASA Astrophysics Data System (ADS)

    Chandler, John E.

    It is becoming increasingly evident that the nanoscale organization and structure of macromolecules play a significant role in determining the function and properties of biological systems. To understand the relationships between biological structure and function at nanometer length scales, there is a need for methods which enable imaging of intact nanoscale biological structure. An ideal technique for these applications is sensitive to nanoscale structure below the resolution limit of conventional optical microscopy ( 200 nm), achieves label-free contrast, is non-perturbing to biological samples, is quantitative, is capable of molecular specificity, is high-throughput, and finally is simple, enabling widespread utilization. Existing techniques meet some of these criteria, but all have limitations. For example, super-resolution optical microscopy methods achieve molecular-specific nanoscale resolution well below the resolution limit of conventional optical microscopes, however, they rely on fluorescent labels often at high densities that can be toxic and can often require potentially damaging illumination intensities for imaging. As a result, there remains a need for label-free optical techniques to study the nanoscale structural properties of cells. To address this need, the development of instrumentation and algorithms for Partial Wave Spectroscopic (PWS) microscopy will be described. PWS is a spectroscopic, label-free, nanoscale sensitive microscope which, senses rather than resolves structure below the resolution limit of conventional microscopes ( 200nm). First, PWS has shown utility as a diagnostic screening tool for cancer due to nanoscale structural alterations that occur in cells as part of the earliest stages of carcinogenesis. Instrumentation and algorithms developed to enable high-throughput cancer screening applications will be described. Further enhancement of data acquisition and analysis speed will then be described through the development of new

  17. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization.

    PubMed

    Concepcion, Joy; Witte, Krista; Wartchow, Charles; Choo, Sae; Yao, Danfeng; Persson, Henrik; Wei, Jing; Li, Pu; Heidecker, Bettina; Ma, Weilei; Varma, Ram; Zhao, Lian-She; Perillat, Donald; Carricato, Greg; Recknor, Michael; Du, Kevin; Ho, Huddee; Ellis, Tim; Gamez, Juan; Howes, Michael; Phi-Wilson, Janette; Lockard, Scott; Zuk, Robert; Tan, Hong

    2009-09-01

    The analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.

  18. Label-free high-throughput imaging flow cytometry

    NASA Astrophysics Data System (ADS)

    Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

    2014-03-01

    Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

  19. Rapid Identification of Biotherapeutics with Label-Free Raman Spectroscopy.

    PubMed

    Paidi, Santosh Kumar; Siddhanta, Soumik; Strouse, Robert; McGivney, James B; Larkin, Christopher; Barman, Ishan

    2016-04-19

    Product identification is a critical and required analysis for biotheraputics. In addition to regulatory requirements for identity testing on final drug products, in-process identity testing is implemented to reduce business risks associated with fill operations and can also be used as a tool against counterfeiting. Biotherapeutics, in particular monoclonal antibodies, represent a challenging cohort for identity determination because of their similarity in chemical structure. Traditional methods used for product identification can be time and labor intensive, creating a need for quick, inexpensive and reliable methods of drug identification. Here, driven by its molecular-specific and nonperturbative nature, we present Raman spectroscopy as an alternate analytical tool for identity testing. By exploiting subtle differences in vibrational modes of the biologics, we have developed partial least-squares-discriminant analysis derived decision algorithms that offer excellent differentiation capability using spontaneous Raman spectra as well as label-free plasmon-enhanced Raman spectra. Coupled with the robustness to spurious correlations due to its high information content, our results highlight the potential of Raman spectroscopy as a powerful method for rapid, on-site biotherapeutic product identification.

  20. 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

  1. A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

    PubMed

    Nöll, Gilbert; Su, Qiang; Heidel, Björn; Yu, Yaming

    2014-01-01

    The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

  2. Label-free and high-sensitive detection for genetic point mutation based on hyperspectral interferometry

    NASA Astrophysics Data System (ADS)

    Fu, Rongxin; Li, Qi; Zhang, Junqi; Wang, Ruliang; Lin, Xue; Xue, Ning; Su, Ya; Jiang, Kai; Huang, Guoliang

    2016-10-01

    Label free point mutation detection is particularly momentous in the area of biomedical research and clinical diagnosis since gene mutations naturally occur and bring about highly fatal diseases. In this paper, a label free and high sensitive approach is proposed for point mutation detection based on hyperspectral interferometry. A hybridization strategy is designed to discriminate a single-base substitution with sequence-specific DNA ligase. Double-strand structures will take place only if added oligonucleotides are perfectly paired to the probe sequence. The proposed approach takes full use of the inherent conformation of double-strand DNA molecules on the substrate and a spectrum analysis method is established to point out the sub-nanoscale thickness variation, which benefits to high sensitive mutation detection. The limit of detection reach 4pg/mm2 according to the experimental result. A lung cancer gene point mutation was demonstrated, proving the high selectivity and multiplex analysis capability of the proposed biosensor.

  3. Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

    NASA Astrophysics Data System (ADS)

    Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

    2006-01-01

    We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

  4. Photonic crystal microcapsules for label-free multiplex detection.

    PubMed

    Ye, Baofen; Ding, Haibo; Cheng, Yao; Gu, Hongcheng; Zhao, Yuanjin; Xie, Zhuoying; Gu, Zhongze

    2014-05-28

    A novel suspension array, which possesses the joint advantages of photonic crystal encoded technology, bioresponsive hydrogels, and photonic crystal sensors with capability of full multiplexing label-free detection is developed.

  5. 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.

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

    PubMed

    Zhuo, Yue; Cunningham, Brian T

    2015-08-28

    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.

  7. 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

  8. 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.

  9. 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.

  10. Multimodal discrimination of immune cells using a combination of Raman spectroscopy and digital holographic microscopy

    PubMed Central

    McReynolds, Naomi; Cooke, Fiona G. M.; Chen, Mingzhou; Powis, Simon J.; Dholakia, Kishan

    2017-01-01

    The ability to identify and characterise individual cells of the immune system under label-free conditions would be a significant advantage in biomedical and clinical studies where untouched and unmodified cells are required. We present a multi-modal system capable of simultaneously acquiring both single point Raman spectra and digital holographic images of single cells. We use this combined approach to identify and discriminate between immune cell populations CD4+ T cells, B cells and monocytes. We investigate several approaches to interpret the phase images including signal intensity histograms and texture analysis. Both modalities are independently able to discriminate between cell subsets and dual-modality may therefore be used a means for validation. We demonstrate here sensitivities achieved in the range of 86.8% to 100%, and specificities in the range of 85.4% to 100%. Additionally each modality provides information not available from the other providing both a molecular and a morphological signature of each cell. PMID:28256551

  11. Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.

    PubMed

    Soderblom, Erik J; Philipp, Melanie; Thompson, J Will; Caron, Marc G; Moseley, M Arthur

    2011-05-15

    Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO(2) enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO(2) enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

  12. 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

  13. 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

  14. 2D light scattering label-free cytometry using light-sheet illumination

    NASA Astrophysics Data System (ADS)

    Lin, Meiai; Su, Xuantao

    2016-10-01

    Two-dimensional (2D) light scattering cytometry has been demonstrated as an effective label-free technology for cell analysis. Here we develop the light-sheet illumination in 2D light scattering static cytometry. In our cytometer, a cylindrical lens is used to form the light-sheet for better excitation of the static cells under an inverted microscope. The thickness of the light-sheet measured in fluorescent solution is about 13 μm. Two-dimensional light scattering patterns of standard microspheres and yeast cells are obtained by using a complementary metal oxide semiconductor (CMOS) detector via a low numerical aperture (NA 0.4) optical objective. The experimental patterns characterized with fringe structures agree well with Mie theory simulated ones. Our results suggest that the light-sheet illumination is an effective excitation method for 2D light scattering label-free cytometry.

  15. 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.

  16. Photonic crystal microring resonator for label-free biosensing.

    PubMed

    Lo, Stanley M; Hu, Shuren; Gaur, Girija; Kostoulas, Yiorgos; Weiss, Sharon M; Fauchet, Philippe M

    2017-03-20

    A label-free optical biosensor based on a one-dimensional photonic crystal microring resonator with enhanced light-matter interaction is demonstrated. More than a 2-fold improvement in volumetric and surface sensing sensitivity is achieved compared to conventional microring sensors. The experimental bulk detection sensitivity is ~248nm/RIU and label-free detection of DNA and proteins is reported at the nanomolar scale. With a minimum feature size greater than 100nm, the photonic crystal microring resonator biosensor can be fabricated with the same standard lithographic techniques used to mass fabricate conventional microring resonators.

  17. 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.

  18. Label-free characterization of biomembranes: from structure to dynamics.

    PubMed

    Mashaghi, Alireza; Mashaghi, Samaneh; Reviakine, Ilya; Heeren, Ron M A; Sandoghdar, Vahid; Bonn, Mischa

    2014-02-07

    We review recent progress in the study of the structure and dynamics of phospholipid membranes and associated proteins, using novel label-free analytical tools. We describe these techniques and illustrate them with examples highlighting current capabilities and limitations. Recent advances in applying such techniques to biological and model membranes for biophysical studies and biosensing applications are presented, and future prospects are discussed.

  19. 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

  20. 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

  1. Label-free detection of small-molecule binding to a GPCR in the membrane environment.

    PubMed

    Heym, Roland G; Hornberger, Wilfried B; Lakics, Viktor; Terstappen, Georg C

    2015-08-01

    Evaluation of drug-target interaction kinetics is becoming increasingly important during the drug-discovery process to investigate selectivity of a drug and predict in vivo target occupancy. To date, it remains challenging to obtain kinetic information for interactions between G-protein-coupled receptors (GPCRs) and small-molecule ligands in a label-free manner. Often GPCRs need to be solubilized or even stabilized by mutations which can be difficult and is time consuming. In addition, it is often unclear if the native conformation of the receptors is sustained. In this study, surface plasmon resonance (SPR) and surface acoustic wave (SAW) technologies have been used to detect ligand binding to the GPCR chemokine (C-X-C motif) receptor 4 (CXCR4) expressed in lipoparticles. We first evaluated different strategies to immobilize CXCR4-expressing lipoparticles. The highest small-molecule binding signal in SPR and SAW was achieved with a matrix-free carboxymethylated sensor chip coated with wheat germ agglutinin for lipoparticle capturing. Next, the binding kinetics of the anti-CXCR4 antibody 12G5 raised against a conformational epitope (k(on)=1.83×10(6)M(-1)s(-1), k(off)=2.79×10(-4) s(-1)) and the small molecule AMD3100 (k(on)=5.46×10(5)M(-1)s(-1), k(off)=1.01×10(-2)s(-1)) were assessed by SAW. Our kinetic and affinity data are consistent with previously published radioligand-binding experiments using cells and label-free experiments with solubilized CXCR4. This is the first study demonstrating label-free kinetic characterization of small-molecule binding to a GPCR in the membrane environment. The presented method holds the potential to greatly facilitate label-free assay development for GPRCs that can be expressed at high levels in lipoparticles.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Label-free intracellular transport measured by spatial light interference microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Zhuo; Millet, Larry; Chan, Vincent; Ding, Huafeng; Gillette, Martha U.; Bashir, Rashid; Popescu, Gabriel

    2011-02-01

    We show that applying the Laplace operator to a speckle-free quantitative phase image reveals an unprecedented level of detail in cell structure, without the gradient artifacts associated with differential interference contrast microscopy, or photobleaching and phototoxicity limitations common in fluorescence microscopy. This method, referred to as Laplace phase microscopy, is an efficient tool for tracking vesicles and organelles in living cells. The principle is demonstrated by tracking organelles in cardiomyocytes and vesicles in neurites of hippocampal neurons, which to our knowledge are the first label-free diffusion measurements of the organelles in such cells.

  7. Plasmonic nanorainbow for label-free biomolecule sensing

    NASA Astrophysics Data System (ADS)

    Liu, Gang L.; Anderson, Erik H.; Liddle, James A.; Lee, Luke P.

    2005-03-01

    A gold nanowire array that we call nanorainbow SPR sensor array can be chemically functionalized and used to capture biomolecules. The localized plasmon resonance wavelength of the gold nanowires shifts on the biomolecule binding and reaction sites. The plasmon resonance shift of the gold nanorainbow is sensitive to the biomolecule immobilization in sub-nM concentration. As an application example, label-free oligonucleotide hybridizations are detected on the nanorainbow sensor in a multiplexed microfluidic chip.

  8. Label-Free Biomarker Detection from Whole Blood

    DTIC Science & Technology

    2010-02-01

    David J. Mooney4, Mark A. Reed2,5* and Tarek M. Fahmy1,6* Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is...rapid translation to clinical settings. Biomarkers have emerged as potentially important diagnostic tools for cancer and many other diseases . Continuing... disease states. Thus, parallel detection of biomarker arrays is essential for translation from benchtop discovery to clinical vali- dation. Such a

  9. Real-Time Discrimination between Proliferation and Neuronal and Astroglial Differentiation of Human Neural Stem Cells

    PubMed Central

    Lee, Rimi; Kim, Il-Sun; Han, Nalae; Yun, Seokhwan; Park, Kook In; Yoo, Kyung-Hwa

    2014-01-01

    Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural lineages, all of which are considered to be promising components for neural regeneration. However, for cell-replacement therapies, it is essential to monitor the process of in vitro NSC differentiation and identify differentiated cell phenotypes. We report a real-time and label-free method that uses a capacitance sensor array to monitor the differentiation of human fetal brain-derived NSCs (hNSCs) and to identify the fates of differentiated cells. When hNSCs were placed under proliferation or differentiation conditions in five media, proliferating and differentiating hNSCs exhibited different frequency and time dependences of capacitance, indicating that the proliferation and differentiation status of hNSCs may be discriminated in real-time using our capacitance sensor. In addition, comparison between real-time capacitance and time-lapse optical images revealed that neuronal and astroglial differentiation of hNSCs may be identified in real-time without cell labeling. PMID:25204726

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. A label-free, impedance-based real time assay to identify drug-induced toxicities and differentiate cytostatic from cytotoxic effects.

    PubMed

    Kustermann, S; Boess, F; Buness, A; Schmitz, M; Watzele, M; Weiser, T; Singer, T; Suter, L; Roth, A

    2013-08-01

    Cell-based assays are key tools in drug safety assessment. However, they usually provide only limited information about time-kinetics of a toxic effect and implementing multiple measurements is often complex. To overcome these issues we established an impedance-based approach which is able to differentiate cytostatic from cytotoxic drugs by recording time-kinetics of compound-effects on cells. NIH 3T3 fibroblasts were seeded on xCELLigence® E-plates and impedance was continuously measured over 5 days. The obtained results reflected cytotoxicity and cell proliferation, as confirmed by neutral red uptake in vitro. Based on known toxicants, we established an algorithm able to discriminate cytostatic, cytotoxic and non-toxic compounds based on the shape of the impedance curves. Analyzing impedance curve patterns of additional 37 compounds allowed the identification and differentiation of these distinct effects as results correlated well with previous in vivo findings. We show that impedance-based real-time cell analysis is a convenient tool to characterize and discriminate effects of compounds on cells in a time-dependent and label-free manner. The presented impedance assay could be used to further characterize toxicities observed in vivo or in vitro. Due to the ease of performance it may also be a suitable screening tool.

  15. 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

  16. Label-Free Aptasensors for the Detection of Mycotoxins

    PubMed Central

    Rhouati, Amina; Catanante, Gaelle; Nunes, Gilvanda; Hayat, Akhtar; Marty, Jean-Louis

    2016-01-01

    Various methodologies have been reported in the literature for the qualitative and quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity, selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by exploring bio-recognition phenomena. However, a significant problem in the fabrication of these devices is that most of the biomolecules do not generate an easily measurable signal upon binding to the target analytes, and signal-generating labels are required to perform the measurements. In this context, aptamers have been emerged as a potential and attractive bio-recognition element to design label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches, the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on current designs in label-free conformational switchable design strategies, with a particular focus on applications in the detection of mycotoxins. PMID:27999353

  17. 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.

  18. Label-free quantitation, an extension to 2DB.

    PubMed

    Allmer, Jens

    2010-04-01

    Determining the differential expression of proteins under different conditions is of major importance in proteomics. Since mass spectrometry-based proteomics is often used to quantify proteins, several labelling strategies have been developed. While these are generally more precise than label-free quantitation approaches, they imply specifically designed experiments which also require knowledge about peptides that are expected to be measured and need to be modified. We recently designed the 2DB database which aids storage, analysis, and publication of data from mass spectrometric experiments to identify proteins. This database can aid identifying peptides which can be used for quantitation. Here an extension to the database application, named MSMAG, is presented which allows for more detailed analysis of the distribution of peptides and their associated proteins over the fractions of an experiment. Furthermore, given several biological samples in the database, label-free quantitation can be performed. Thus, interesting proteins, which may warrant further investigation, can be identified en passant while performing high-throughput proteomics studies.

  19. Label-free disposable immunosensor for detection of atrazine.

    PubMed

    Belkhamssa, Najet; Justino, Celine I L; Santos, Patrícia S M; Cardoso, Susana; Lopes, Isabel; Duarte, Armando C; Rocha-Santos, Teresa; Ksibi, Mohamed

    2016-01-01

    This work reports the construction of a fast, disposable, and label-free immunosensor for the determination of atrazine. The immunosensor is based on a field effect transistor (FET) where a network of single-walled carbon nanotubes (SWCNTs) acts as the conductor channel, constituting carbon nanotubes field effect transistors (CNTFETs). Anti-atrazine antibodies were adsorbed onto the SWCNTs and subsequently the SWCNTs were protected with Tween 20 to prevent the non-specific binding of bacteria or proteins. The principle of the immunoreaction consists in the direct adsorption of atrazine specific antibodies (anti-atrazine) to SWCNTs networks. After exposed to increasing concentrations of atrazine, the CNTFETs could be used as useful label-free platforms to detect atrazine. Under the optimal conditions, a limit of detection as low as 0.001 ng mL(-1) was obtained, which is lower than that of other methods for the atrazine detection, and in a working range between 0.001 and 10 ng mL(-1). The average recoveries obtained for real water samples spiked with atrazine varied from 87.3% to 108.0%. The results show that the constructed sensors display a high sensitivity and could be useful tools for detecting pesticides like atrazine at low concentrations. They could be also applied to the determination of atrazine in environmental aqueous samples, such as seawater and riverine water.

  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. 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.

  2. Multimodal label-free in vitro toxicity testing with digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Rommel, Christina E.; Dierker, Christian; Vollmer, Angelika; Ketelhut, Steffi; Kemper, Björn; Schnekenburger, Juergen

    2014-05-01

    Common in vitro toxicity tests of drugs, chemicals or nanomaterials involves the measurement of cellular endpoints like stress response, cell viability, proliferation or cell death. The assay systems determine enzyme activity or protein expression by optical read out of enzyme substrates or marker protein labeling. These standard procedures have several disadvantages. Cellular processes have to be stopped at a distinct time point for the read out, where usually only parts of the cells were affected by the treatment with substances. Typically, only one parameter is analyzed and detection of cellular processes requires several time consuming incubations and washing steps. Here we have applied digital holographic microscopy (DHM) for a multimodal label-free analysis of drug toxicity. NIH 3T3 cells were incubated with 1 μM Taxol for 24 h. The recorded quantitative phase images were analyzed for cell thickness, cell volume, dry mass and cell migration. Taxol treated cells showed rapidly decreasing cell motility as measure of cell viability. A short increase in cell thickness and dry mass indicated cell division and growth in control cells, whereas Taxol treatment resulted in a continuous increase in cell height followed by a rapid decrease and a decrease of dry mass as indicators of cell death. Multimodal DHM analysis of drug treatment by multiple parameters allows direct and label-free detection of several toxicity parameters in parallel. DHM can quantify cellular reactions minimally invasive over a long time period and analyze kinetics of delayed cellular responses. Our results demonstrate digital holographic microscopy as a valuable tool for multimodal toxicity testing.

  3. Label-free detection of telomerase activity using guanine electrochemical oxidation signal.

    PubMed

    Eskiocak, Ugur; Ozkan-Ariksoysal, Dilsat; Ozsoz, Mehmet; Oktem, Huseyin Avni

    2007-11-15

    Telomerase is an important biomarker for cancer cells and its activation in 85% of all cancer types confers a clinical diagnostic value. A label-free electrochemical assay based on guanine oxidation signal to measure telomerase activity is described. This developed technology combined with a disposable sensor, carbon graphite electrode (CGE), and differential pulse voltammetry (DPV) was performed by using PCR amplicons with/without telomeric repeats as the guanine oxidation signal observed at +1.0 V measured after the immobilization of PCR products. Guanine oxidation signal was chosen as a measure of telomerase activity because a substantial increase in the number of guanines was introduced by the action of telomerase which adds hexameric repeats (TTAGGG)n that contain 50% guanine. The developed assay was shown to specifically measure telomerase activity from cell extracts, and elongation rates increased linearly in a concentration dependent manner. Telomerase activity could be detected in cell extracts containing as low as 100 ng/microL of protein. All of the electrochemical measurements were also confirmed with the conventional TRAP-silver staining assay. Rapidity, simplicity, and the label-free nature of the developed assay make it suitable for practical use in quantitative determination of telomerase activity from clinical samples for diagnosis of cancer.

  4. Label-free optical imaging of lymphatic vessels within tissue beds in vivo

    PubMed Central

    Yousefi, Siavash; Zhi, Zhongwei; Wang, Ruikang K.

    2015-01-01

    Lymphatic vessels are a part of circulatory system in vertebrates that maintain tissue fluid homeostasis and drain excess fluid and large cells that cannot easily find their way back into venous system. Due to the lack of non-invasive monitoring tools, lymphatic vessels are known as forgotten circulation. However, lymphatic system plays an important role in diseases such as cancer and inflammatory conditions. In this paper, we start to briefly review the current existing methods for imaging lymphatic vessels, mostly involving dye/targeting cell injection. We then show the capability of optical coherence tomography (OCT) for label-free non-invasive in vivo imaging of lymph vessels and nodes. One of the advantages of using OCT over other imaging modalities is its ability to assess label-free blood flow perfusion that can be simultaneously observed along with lymphatic vessels for imaging the microcirculatory system within tissue beds. Imaging the microcirculatory system including blood and lymphatic vessels can be utilized for imaging and better understanding pathologic mechanisms and treatment technique development in some critical diseases such as inflammation, malignant cancer angiogenesis and metastasis. PMID:25642129

  5. Label-free optical imaging of lymphatic vessels within tissue beds in vivo.

    PubMed

    Yousefi, Siavash; Zhi, Zhongwei; Wang, Ruikang K

    2014-01-01

    Lymphatic vessels are a part of circulatory system in vertebrates that maintain tissue fluid homeostasis and drain excess fluid and large cells that cannot easily find their way back into venous system. Due to the lack of non-invasive monitoring tools, lymphatic vessels are known as forgotten circulation. However, lymphatic system plays an important role in diseases such as cancer and inflammatory conditions. In this paper, we start to briefly review the current existing methods for imaging lymphatic vessels, mostly involving dye/targeting cell injection. We then show the capability of optical coherence tomography (OCT) for label-free non-invasive in vivo imaging of lymph vessels and nodes. One of the advantages of using OCT over other imaging modalities is its ability to assess label-free blood flow perfusion that can be simultaneously observed along with lymphatic vessels for imaging the microcirculatory system within tissue beds. Imaging the microcirculatory system including blood and lymphatic vessels can be utilized for imaging and better understanding pathologic mechanisms and treatment technique development in some critical diseases such as inflammation, malignant cancer angiogenesis and metastasis.

  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. 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

  8. 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.

  9. Hybrid integrated label-free chemical and biological sensors.

    PubMed

    Mehrabani, Simin; Maker, Ashley J; Armani, Andrea M

    2014-03-26

    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.

  10. 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

  11. 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.

  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. 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.

  14. A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity.

    PubMed

    Du, Jiao; Xu, Qinfeng; Lu, Xiaoquan; Zhang, Chun-yang

    2014-08-19

    Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5'-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.

  15. Label-free detection of pathogenic bacteria via immobilized antimicrobial peptides.

    PubMed

    Dong, Zong-Mu; Zhao, Guang-Chao

    2015-05-01

    A novel label-free strategy for the detection of bacteria was developed by using a specific antimicrobial peptide (AMP)-functionalized quartz crystal microbalance (QCM) electrode. This electrode interface was successfully applied to detect pathogenic Escherichia coli O157:H7 based on the specific affinity between the small synthetic antimicrobial peptide and the bacterial cell of pathogenic E. coli O157:H7. The concentrations of pathogenic E. coli O157:H7 were sensitively measured by the frequency response of the QCM with a detection limit of 0.4 cfu μL(-1). The detection can be fulfilled within 10 min because it does not require germiculture process. On the other hand, if the specific antimicrobial peptides were immobilized on a gold electrode, this label-free strategy can also be performed by electrochemical impedance spectroscopy (EIS). Compared with QCM technique, the EIS measurement gives a lower sensitivity and needs a longer assay time. The combination of antimicrobial peptides with the real-time responses of QCM, as well as electronic read-out monitoring of EIS, may open a new way for the direct detection of bacteria.

  16. 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

  17. Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli.

    PubMed

    Arike, L; Valgepea, K; Peil, L; Nahku, R; Adamberg, K; Vilu, R

    2012-09-18

    Three different label-free proteome quantification methods--APEX, emPAI and iBAQ--were evaluated to measure proteome-wide protein concentrations in the cell. All the methods were applied to a sample from Escherichia coli chemostat culture. A Pearson squared correlation of approximately 0.6 among the three quantification methods was demonstrated. Importantly, the sum of quantified proteins by iBAQ and emPAI corresponded with the Lowry total protein quantification, demonstrating applicability of label-free methods for an accurate calculation of protein concentrations at the proteome level. The iBAQ method showed the best correlation between biological replicates, a normal distribution among all protein abundances, and the lowest variation among ribosomal protein abundances, which are expected to have equal amounts. Absolute quantitative proteome data enabled us to evaluate metabolic cost for protein synthesis and apparent catalytic activities of enzymes by integration with flux analysis. All the methods demonstrated similar ATP costs for protein synthesis for different cellular processes and that costs for expressing biomass synthesis related proteins were higher than those for energy generation. Importantly, catalytic activities of energy metabolism enzymes were an order or two higher than those of monomer synthesis. Interestingly, a staircase-like protein expression was demonstrated for most of the transcription units.

  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. 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.

  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. Noninvasive Label-Free Detection of Micrometastases in the Lymphatics with Ultrasound-Guided Photoacoustic Imaging

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-14-1-0356 TITLE: Noninvasive Label- Free Detection of Micrometastases in the Lymphatics With Ultrasound-Guided...Report 3. DATES COVERED 30 Sep 2014 - 29 Sep 2015 4. TITLE AND SUBTITLE Noninvasive Label- Free Detection of Micrometastases in the Lymphatics With...Journal publications. Geoffrey P. Luke and Stanislav Y. Emelianov; Label- free Detection of Lymph Node Metastases with US-guided Functional Photoacoustic

  2. 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.

  3. 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

  4. Photonic Crystal Surfaces as a General Purpose Platform for Label-Free and Fluorescent Assays.

    PubMed

    Cunningham, Brian T

    2010-04-01

    Photonic crystal surfaces can be designed to provide a wide range of functions that are used to perform biochemical and cell-based assays. Detection of the optical resonant reflections from photonic crystal surfaces enables high sensitivity label-free biosensing, while the enhanced electromagnetic fields that occur at resonant wavelengths can be used to enhance the detection sensitivity of any surface-based fluorescence assay. Fabrication of photonic crystals from inexpensive plastic materials over large surface areas enables them to be incorporated into standard formats that include microplates, microarrays, and microfluidic channels. This report reviews the design of photonic crystal biosensors, their associated detection instrumentation, and biological applications. Applications including small molecule high throughput screening, cell membrane integrin activation, gene expression analysis, and protein biomarker detection are highlighted. Recent results in which photonic crystal surfaces are used for enhancing the detection of Surface-Enhanced Raman Spectroscopy, and the development of high resolution photonic crystal-based laser biosensors are also described.

  5. Gold surface supported spherical liposome-gold nano-particle nano-composite for label free DNA sensing.

    PubMed

    Bhuvana, M; Narayanan, J Shankara; Dharuman, V; Teng, W; Hahn, J H; Jayakumar, K

    2013-03-15

    Immobilization of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposome-gold nano-particle (DOPE-AuNP) nano-composite covalently on 3-mercaptopropionic acid (MPA) on gold surface is demonstrated for the first time for electrochemical label free DNA sensing. Spherical nature of the DOPE on the MPA monolayer is confirmed by the appearance of sigmoidal voltammetric profile, characteristic behavior of linear diffusion, for the MPA-DOPE in presence of [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+) redox probes. The DOPE liposome vesicle fusion is prevented by electroless deposition of AuNP on the hydrophilic amine head groups of the DOPE. Immobilization of single stranded DNA (ssDNA) is made via simple gold-thiol linkage for DNA hybridization sensing in the presence of [Fe(CN)(6)](3-/4-). The sensor discriminates the hybridized (complementary target hybridized), un-hybridized (non-complementary target hybridized) and single base mismatch target hybridized surfaces sensitively and selectively without signal amplification. The lowest target DNA concentration detected is 0.1×10(-12)M. Cyclic voltammetry (CV), electrochemical impedance (EIS), differential pulse voltammetry (DPV) and quartz crystal microbalance (QCM) techniques are used for DNA sensing on DOPE-AuNP nano-composite. Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and Ultraviolet-Visible (UV) spectroscopic techniques are used to understand the interactions between the DOPE, AuNP and ssDNA. The results indicate the presence of an intact and well defined spherical DOPE-AuNP nano-composite on the gold surface. The method could be applied for fabrication of the surface based liposome-AuNP-DNA composite for cell transfection studies at reduced reagents and costs.

  6. Synergizing nucleic acid aptamers with 1-dimensional nanostructures as label-free field-effect transistor biosensors.

    PubMed

    Lung Khung, Yit; Narducci, Dario

    2013-12-15

    Since the introduction by Gold et al. in 1990, nucleic acid aptamers had evolved to become a true contender in biosensors for protein and cell detections. Aptamers are short strands of synthetically designed DNA or RNA oligonucleotides that can be self-assembled into unique 3-dimensional structures and can bind to different proteins, cells or even small molecules at a high level of specificity and affinity. In recent years, there had been many reports in literature in using aptamers in place of conventional antibodies as capture biomolecules on the surface. This is mainly due to the better thermal stability properties and ease in production. Consequently, also these characteristics allowed the aptamers to find use in field effect transistors (FETs) based upon 1D nanostructured (1D-NS) as label-free biosensing. In terms of designing label-free platforms for biosensors applications, 1D-NS FET had been an attractive option due to reported high sensitivities toward protein targets arising from the large surface area for detection as well as to their label-free nature. Since the first aptamer-based 1D-NS FET biosensor had surfaced in 2005, there had been many more improvements in the overall design and sensitivity in recent years. In this review, the latest developments in synergizing these two interesting areas of research (aptamers and 1D-NS FET) will be discussed for a range of different nanowire types as well as for the detection results.

  7. Detection of microRNA in clinical tumor samples by isothermal enzyme-free amplification and label-free graphene oxide-based SYBR Green I fluorescence platform.

    PubMed

    Zhu, Debin; Zhang, Lan; Ma, Wenge; Lu, Suqin; Xing, Xiaobo

    2015-03-15

    MicroRNAs (miRNAs) are a kind of small molecules that involve in many important life activities. They have higher expression levels in many kinds of cancers. In this study, we developed an isothermal enzyme-free amplification (EFA) and label-free graphene oxide (GO)-based SYBR Green I fluorescence platform for detection of miRNA. MiRNA-21 was used as an example to demonstrate the feasibility of the method. Results show that the sensitivity of miRNA-21 is 1pM, and the linearity range is from 1pM to 1nM. The method can specifically discriminate miRNA-21 from miRNA-210 and miRNA-214. Three tumor cell lines of A549, HepG2 and MCF7 were detected by the method. The sensitivities of them were 10(2) cells, 10(3) cells and 10(3) cells respectively. Clinical tumor samples were also tested by this method, and 29 of 40 samples gave out positive signals. The method holds great promise in miRNA detection due to its convenience, rapidness, inexpensive and specificity.

  8. Label-free bead-based metallothionein electrochemical immunosensor.

    PubMed

    Nejdl, Lukas; Nguyen, Hoai Viet; Richtera, Lukas; Krizkova, Sona; Guran, Roman; Masarik, Michal; Hynek, David; Heger, Zbynek; Lundberg, Karin; Erikson, Kristofer; Adam, Vojtech; Kizek, Rene

    2015-08-01

    A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.

  9. 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

  10. 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.

  11. 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.

  12. Label-free virus detection using silicon photonic microring resonators.

    PubMed

    McClellan, Melinda S; Domier, Leslie L; Bailey, Ryan C

    2012-01-15

    Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications.

  13. 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.

  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. 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.

  16. 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.

  17. Label-Free DNA Sequencing Using Millikan Detection

    PubMed Central

    Dettloff, Roger; Leiske, Danielle; Chow, Andrea; Farinas, Javier

    2015-01-01

    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 ~ 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

  18. 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.

  19. Symmetric curvature descriptors for label-free analysis of DNA

    PubMed Central

    Buzio, Renato; Repetto, Luca; Giacopelli, Francesca; Ravazzolo, Roberto; Valbusa, Ugo

    2014-01-01

    High-resolution microscopy techniques such as electron microscopy, scanning tunnelling microscopy and atomic force microscopy represent well-established, powerful tools for the structural characterization of adsorbed DNA molecules at the nanoscale. Notably, the analysis of DNA contours allows mapping intrinsic curvature and flexibility along the molecular backbone. This is particularly suited to address the impact of the base-pairs sequence on the local conformation of the strands and plays a pivotal role for investigations relating the inherent DNA shape and flexibility to other functional properties. Here, we introduce novel chain descriptors aimed to characterize the local intrinsic curvature and flexibility of adsorbed DNA molecules with unknown orientation. They consist of stochastic functions that couple the curvatures of two nanosized segments, symmetrically placed on the DNA contour. We show that the fine mapping of the ensemble-averaged functions along the molecular backbone generates characteristic patterns of variation that highlight all pairs of tracts with large intrinsic curvature or enhanced flexibility. We demonstrate the practical applicability of the method for DNA chains imaged by atomic force microscopy. Our approach paves the way for the label-free comparative analysis of duplexes, aimed to detect nanoscale conformational changes of physical or biological relevance in large sample numbers. PMID:25248631

  20. 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.

  1. 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.

  2. Coherent total internal reflection dark-field microscopy: label-free imaging beyond the diffraction limit.

    PubMed

    von Olshausen, Philipp; Rohrbach, Alexander

    2013-10-15

    Coherent imaging is barely applicable in life-science microscopy due to multiple interference artifacts. Here, we show how these interferences can be used to improve image resolution and contrast. We present a dark-field microscopy technique with evanescent illumination via total internal reflection that delivers high-contrast images of coherently scattering samples. By incoherent averaging of multiple coherent images illuminated from different directions we can resolve image structures that remain unresolved by conventional (incoherent) fluorescence microscopy. We provide images of 190 nm beads revealing resolution beyond the diffraction limit and slightly increased object distances. An analytical model is introduced that accounts for the observed effects and which is confirmed by numerical simulations. Our approach may be a route to fast, label-free, super-resolution imaging in live-cell microscopy.

  3. 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.

  4. 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

  5. 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.

  6. Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*

    PubMed Central

    Mosley, Amber L.; Sardiu, Mihaela E.; Pattenden, Samantha G.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

    2011-01-01

    The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. PMID

  7. Label-free chemically specific imaging in planta with stimulated Raman scattering microscopy.

    PubMed

    Mansfield, Jessica C; Littlejohn, George R; Seymour, Mark P; Lind, Rob J; Perfect, Sarah; Moger, Julian

    2013-05-21

    The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas. There is currently a lack of analytical tools that provide noninvasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade, these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis, it does not suffer from photobleaching, and it allows near real-time imaging in 3D with submicrometer spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll), the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored. In this paper, we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally, we demonstrate the potential of SRS for a range of in planta applications by presenting in situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.

  8. 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

  9. Label-Free, Electrochemical Quantitation of Potassium Ions from Femtomolar Levels.

    PubMed

    Zhu, Bicheng; Booth, Marsilea A; Woo, Han Young; Hodgkiss, Justin M; Travas-Sejdic, Jadranka

    2015-10-01

    In this communication, a label-free and sensitive electrochemical method to detect potassium ions is proposed. The conducting polymer polypyrrole was used as both an anchor for the probe and a transducer of the detection event. A K(+)-specific G-rich aptamer was applied as a recognition element, which folded into the G-quadruplex structure in the presence of K(+), and this resulted in an increase in the electrode impedance. The combination of the K(+)-selective aptamer and the porous conducting polymer as a signal transducer afforded a successful sensor platform. The sensor responded approximately logarithmically over a wide dynamic range of K(+) concentrations from 20 fM to 1 mM, with a very low detection limit of 14.7 fM and excellent discrimination against other ions. Additionally, electrochemical impedance spectroscopy was used to study the kinetics of K(+) binding at the conducting polymer-immobilized aptamer surface, which indicated strong binding between the two. This work demonstrates a powerful approach for the sensitive, selective, and direct electrochemical detection of metal ions based on the switching conformation of G-rich aptamers attached to a porous conducting polymer surface. This assay scheme can be expanded to the detection of a wide range of targets by modifying the aptamer structure as a recognizing moiety.

  10. A microarray chip for label-free detection of narcotics.

    PubMed

    Klenkar, Goran; Liedberg, Bo

    2008-07-01

    A protein array chip for label-free optical detection of low molecular weight compounds has been developed. As a proof of principle, the chip is proven capable of rapidly (approximately 1 min) determining hits from aqueous cocktails composed of four common narcotics, cocaine, ecstasy, heroin, and amphetamine, using imaging surface plasmon resonance (SPR) as the detection principle. The chip is produced by injecting a mixture of antibodies and letting them self-sort and bind to narcotic analog coupled proteins already present in a predefined pattern on the supporting substrate. An indirect detection method, where antibodies are displaced from the surface upon recognition of their corresponding narcotics, is used to obtain the optical contrast and thus a detectable SPR and/or ellipsometric signal. Two types of readouts are possible from the present setup: intensity SPR images and SPR/ellipsometric sensorgrams. Positive hits were routinely obtained for analyte concentrations of 50 pg/microL and the limit of detection, without any parameter optimizations, seems to fall in the range 0.5 pg/microL (1.4 nM) for heroin, 2.5 pg/microL (8.2 nM) for cocaine, and 5 pg/microL for the other two narcotics (26 nM for ecstasy and 37 nM for amphetamine). With improved readout possibilities (sampling frequency), signal evaluation algorithms, and antibody-antigen design strategies, we believe this limit can be further improved. The chip is shown to work for many measurement cycles with excellent reproducibility. Moreover, with a more advanced fluidic system, excess injected antibodies could be collected and reused for many cycles, which could make the running costs of the system very low. The chip is in no way limited to detection of narcotics. Other low molecular weight compounds could easily be detected on the same chip. For example, trinitrotoluene detection has already been demonstrated using our chip. Possible areas of application for the system are therefore envisaged in airport

  11. A label-free, fluorescence based assay for microarray

    NASA Astrophysics Data System (ADS)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  12. Graphene-Assisted Label-Free Homogeneous Electrochemical Biosensing Strategy based on Aptamer-Switched Bidirectional DNA Polymerization.

    PubMed

    Wang, Wenxiao; Ge, Lei; Sun, Ximei; Hou, Ting; Li, Feng

    2015-12-30

    In this contribution, taking the discrimination ability of graphene over single-stranded (ss) DNA/double-stranded (ds) DNA in combination with the electrochemical impedance transducer, we developed a novel label-free homogeneous electrochemical biosensor using graphene-modified glassy carbon electrode (GCE) as the sensing platform. To convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output, a novel aptamer-switched bidirectional DNA polymerization (BDP) strategy, capable of both target recycling and exponential signal amplification, was compatibly developed in this study. In this strategy, all the designed DNA structures could be adsorbed on the graphene/GCE and, thus, serve as the electrochemical impedance signal reporter, while the target acts as a trigger of this BDP reaction, in which these designed DNA structures are bound together and, then, converted to long dsDNA duplex. The distinct difference in electrochemical impedance spectroscopy between the designed structures and generated long dsDNA duplex on the graphene/GCE allows label-free and homogeneous detection of target down to femto-gram level. The target can be displaced from aptamer through the polymerization to initiate the next recognition-polymerization cycle. Herein, the design and signaling principle of aptamer-switched BDP amplification system were elucidated, and the working conditions were optimized. This method not only provides a universal platform for electrochemical biosensing but also shows great potential in biological process researches and clinic diagnostics.

  13. Label-free Raman imaging of the macrophage response to the malaria pigment hemozoin.

    PubMed

    Hobro, Alison J; Pavillon, Nicolas; Fujita, Katsumasa; Ozkan, Muge; Coban, Cevayir; Smith, Nicholas I

    2015-04-07

    Hemozoin, the 'malaria pigment', is engulfed by phagocytic cells, such as macrophages, during malaria infection. This biocrystalline substance is difficult to degrade and often accumulates in phagocytes. The macrophage response to hemozoin relates to the severity of the disease and the potential for malaria-related disease complications. In this study we have used Raman spectroscopy as a label-free method to investigate the biochemical changes occurring in macrophages during the first few hours of hemozoin uptake. We found a number of distinct spectral groups, spectrally or spatially related to the presence of the hemozoin inside the cell. Intracellular hemozoin was spectrally identical to extracellular hemozoin, regardless of the location in the cell. A small proportion of hemozoin was found to be associated with lipid-based components, consistent with the uptake of hemozoin into vesicles such as phagosomes and lysosomes. The spatial distribution of the hemozoin was observed to be inhomogeneous, and its presence largely excluded that of proteins and lipids, demonstrating that cells were not able to break down the biocrystals on the time scales studied here. These results show that Raman imaging can be used to answer some of the open questions regarding the role of hemozoin in the immune response. How different combinations of hemozoin and other molecules are treated by macrophages, whether hemozoin can be broken down by the cell, and more importantly, which co-factors or products are involved in the subsequent cell reaction are the expected issues to be elucidated by this technique.

  14. A quadruple wavelength IR sensor system for label-free tumour screening

    NASA Astrophysics Data System (ADS)

    van den Driesche, Sander; Witarski, Wojciech; Pastorekova, Silvia; Vellekoop, Michael J.

    2009-12-01

    In this contribution, we present a novel quadruple wavelength infrared sensor system for measuring the CH2-symmetric/CH2-antisymmetric stretch ratio of biological cells. This ratio can be used as a marker to distinguish between healthy and carcinoma cells. Compared to common techniques our system does not require specific labelling or staining, or the use of expensive liquid nitrogen cooled IR spectrometers. Two of the four wavelengths, 3.33 and 3.57 µm, are used as reference points and the other two represent CH2-symmetric and CH2-antisymmetric stretch absorption (respectively at 3.51 and 3.42 µm). IR absorbance spectra recordings of healthy (MDCK) and malignant (Caki-1) epithelial kidney cells with a conventional IR spectroscope showed significant differences in the absorbance ratio 3.51 µm/3.42 µm (CH2-symmetric/CH2-antisymmetric stretch). The sensor has been validated by measuring the CH2 stretch ratio of yeast samples both by IR spectroscopy and the sensor system. The methods yielded similar results. The application potential of our system is demonstrated by CH2 stretch measurements on healthy and carcinoma epithelial kidney cells. Measurements on both cell types yielded significant differences in CH2 stretch ratio. The sensor has the potential to be further developed into a fast, low-cost and label-free screening system for suspicious biopsy samples.

  15. 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).

  16. Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

    PubMed

    Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

    2015-11-06

    Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

  17. Advancing nanostructured porous si-based optical transducers for label free bacteria detection.

    PubMed

    Massad-Ivanir, Naama; Shtenberg, Giorgi; Segal, Ester

    2012-01-01

    Optical label-free porous Si-based biosensors for rapid bacteria detection are introduced. The biosensors are designed to directly capture the target bacteria cells onto their surface with no prior sample processing (such as cell lysis). Two types of nanostructured optical transducers based on oxidized porous Si (PSiO(2)) Fabry-Pérot thin films are synthesized and used to construct the biosensors. In the first system, we graft specific monoclonal antibodies (immunoglobulin G's) onto a neat electrochemically-machined PSiO(2) surface, based on well-established silanization chemistry. The second biosensor class consists of a PSiO(2)/hydrogel hybrid. The hydrogel, polyacrylamide, is synthesized in situ within the nanostructured PSiO(2) host and conjugated with specific monoclonal antibodies to provide the active component of the biosensor. Exposure of these modified-surfaces to the target bacteria results in "direct-cell-capture" onto the biosensor surface. These specific binding events induce predictable changes in the thin-film optical interference spectrum of the biosensor. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations, in the range of 10(3)-10(5) cell/ml, within minutes. The sensing performance of the two different platforms, in terms of their stability in aqueous media and sensitivity, are compared and discussed. This preliminary study suggests that biosensors based on PSiO(2)/hydrogel hybrid outperform the neat PSiO(2) system.

  18. 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.

  19. 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

  20. Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique.

    PubMed

    Laguna, Maríafe; Holgado, Miguel; Hernandez, Ana L; Santamaría, Beatriz; Lavín, Alvaro; Soria, Javier; Suarez, Tatiana; Bardina, Carlota; Jara, Mónica; Sanza, Francisco J; Casquel, Rafael

    2015-08-13

    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.

  1. Temporal proteomic analysis and label-free quantification of viral proteins of an invertebrate iridovirus.

    PubMed

    İnce, İkbal Agah; Boeren, Sjef; van Oers, Monique M; Vlak, Just M

    2015-01-01

    Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a ~212 kb linear dsDNA genome that encodes 215 putative ORFs. The IIV-6 virion-associated proteins consist of at least 54 virally encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicated that these structural proteins may not only function in the formation of the virion, but also in the initial stage of viral infection. In the current study, we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantification using a proteomic approach. A total of 95 virally encoded proteins were detected in infected cells, of which 37 were virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles: proteins with stably low expression levels during infection, proteins with exponentially increasing expression levels during infection and proteins that were initially highly abundant, but showed slightly reduced levels after 48 h post-infection. We thus provided novel information on the kinetics of virion and infected cell-specific protein levels that assists in our understanding of gene regulation in this lesser-known DNA virus model.

  2. Label-free imaging of rat spinal cords based on multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liao, Chenxi; Wang, Zhenyu; Zhou, Linquan; Zhu, Xiaoqin; Liu, Wenge; Chen, Jianxin

    2016-10-01

    As an integral part of the central nervous system, the spinal cord is a communication cable between the body and the brain. It mainly contains neurons, glial cells, nerve fibers and fiber tracts. The recent development of the optical imaging technique allows high-resolution imaging of biological tissues with the great potential for non-invasively looking inside the body. In this work, we evaluate the imaging capacity of multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) for the cells and extracellular matrix in the spinal cord at molecular level. Rat spinal cord tissues were sectioned and imaged by MPM to demonstrate that MPM is able to show the microstructure including white matter, gray matter, ventral horns, dorsal horns, and axons based on the distinct intrinsic sources in each region of spinal cord. In the high-resolution and high-contrast MPM images, the cell profile can be clearly identified as dark shadows caused by nuclei and encircled by cytoplasm. The nerve fibers in white matter region emitted both SHG and TPEF signals. The multiphoton microscopic imaging technique proves to be a fast and effective tool for label-free imaging spinal cord tissues, based on endogenous signals in biological tissue. It has the potential to extend this optical technique to clinical study, where the rapid and damage-free imaging is needed.

  3. Large-Scale Label-Free Quantitative Proteomics of the Pea aphid-Buchnera Symbiosis*

    PubMed Central

    Poliakov, Anton; Russell, Calum W.; Ponnala, Lalit; Hoops, Harold J.; Sun, Qi; Douglas, Angela E.; van Wijk, Klaas J.

    2011-01-01

    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

  4. 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

  5. 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.

  6. 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

  7. Photonic Crystal Surfaces as a General Purpose Platform for Label-Free and Fluorescent Assays

    PubMed Central

    Cunningham, Brian T.

    2009-01-01

    Photonic crystal surfaces can be designed to provide a wide range of functions that are used to perform biochemical and cell-based assays. Detection of the optical resonant reflections from photonic crystal surfaces enables high sensitivity label-free biosensing, while the enhanced electromagnetic fields that occur at resonant wavelengths can be used to enhance the detection sensitivity of any surface-based fluorescence assay. Fabrication of photonic crystals from inexpensive plastic materials over large surface areas enables them to be incorporated into standard formats that include microplates, microarrays, and microfluidic channels. This report reviews the design of photonic crystal biosensors, their associated detection instrumentation, and biological applications. Applications including small molecule high throughput screening, cell membrane integrin activation, gene expression analysis, and protein biomarker detection are highlighted. Recent results in which photonic crystal surfaces are used for enhancing the detection of Surface-Enhanced Raman Spectroscopy, and the development of high resolution photonic crystal-based laser biosensors are also described. PMID:20383277

  8. Rapid and label-free identification of normal spermatozoa based on image analysis and micro-Raman spectroscopy.

    PubMed

    Huang, Zufang; Chen, Guannan; Chen, Xiwen; Wang, Jing; Chen, Jinhua; Lu, Peng; Chen, Rong

    2014-09-01

    Semen analysis is performed for evaluation of fertility disorders, however it is susceptible to subjectivity of investigators, and lacking of objective criterion for sperm cell quality remains a problem. There is an ongoing debate on which criteria should be employed to define normal spermatozoa. Here, the aim of our study is to evaluate the possibility of label-free and rapid identification of normal sperm cell through the use of image analysis combined with micro-Raman spectroscopy. By using a smooth-surfaced and hydrophobic substrate, spermatozoa smear was rapidly prepared for microscopic imaging and acrosome area can be clearly visualized without any extra stains; then a self-written image analysis program was utilized to segment sperm head and acrosome area and automatically calculates morphological indices. Most important, intensity ratio of 1055 cm(-1) to 1095 cm(-1) from the obtained Raman spectra is found to indicate a potential biomarker for assessing the sperm DNA integrity. Our preliminary study demonstrates that micro-Raman spectroscopy combined with image analysis can be a potentially useful tool for rapid and label-free identification of normal sperm cell by providing both morphological and biochemical information.

  9. Development of a label-free assay for sodium-dependent phosphate transporter NaPi-IIb.

    PubMed

    Wong, Soo-Hang; Gao, Alice; Ward, Sabrina; Henley, Charles; Lee, Paul H

    2012-07-01

    The most widely used assay format for characterizing plasma membrane transporter activity measures accumulation of radiolabeled substrates in tissues or cells expressing the transporters. This assay format had limitations and disadvantages; therefore, there was an unmet need for development of a homogeneous, nonradioactive assay for membrane transporter proteins. In this report, the authors describe the development of a label-free homogeneous assay for the sodium-dependent phosphate transporter NaPi-IIb using the Epic system. The addition of phosphate stimulated a dynamic mass redistribution (DMR) profile unique to cells expressing NaPi-IIb but not on parental cells. This DMR profile was phosphate specific because sulfate or buffer alone did not elicit the same response. Furthermore, the DMR response observed was phosphate and sodium dependent, with Km values in the micromolar and millimolar range, respectively. A known NaPi-IIb noncompetitive inhibitor was shown to completely inhibit the phosphate-stimulated DMR response, suggesting that this observed DMR response is an NaPi-IIb-mediated cellular event. The results demonstrate that a novel label-free assay was developed for studying transporter-mediated cellular activity, and this DMR assay platform could be applicable to other membrane transporter proteins.

  10. Surface Plasmon Resonance Fiber Sensor for Real-Time and Label-Free Monitoring of Cellular Behavior

    PubMed Central

    Shevchenko, Yanina; Camci-Unal, Gulden; Cuttica, Davide F.; Dokmeci, Mehmet R.; Albert, Jacques; Khademhosseini, Ali

    2014-01-01

    This paper reports on the application of an optical fiber biosensor for real-time analysis of cellular behavior. Our findings illustrate that a fiber sensor manufactured from a traditional telecommunication fiber can be integrated into conventional cell culture equipment and used for real-time and label-free monitoring of cellular responses to chemical stimuli. The sensing mechanism used for the measurement of cellular responses is based on the excitation of Surface Plasmon Resonance (SPR) on the surface of the optical fiber. In this proof of concept study, the sensor was utilized to investigate the influence of a number of different stimuli on cells - we tested the effects of trypsin, serum and sodium azide. These stimuli induced detachment of cells from the sensor surface, uptake of serum and inhibition of cellular metabolism, accordingly. The effects of different stimuli were confirmed with alamar blue assay, phase contrast and fluorescence microscopy. The results indicated that the fiber biosensor can be successfully utilized for real-time and label-free monitoring of cellular response in the first 30 minutes following the introduction of a stimulus. Furthermore, we demonstrated that the optical fiber biosensors can be easily regenerated for repeated use, proving this platform as a versatile and cost-effective sensing tool. PMID:24549115

  11. Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy.

    PubMed

    Almassalha, Luay M; Bauer, Greta M; Chandler, John E; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K; Subramanian, Hariharan; Chandel, Navdeep S; Szleifer, Igal; Backman, Vadim

    2016-10-18

    The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.

  12. Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

    PubMed Central

    Almassalha, Luay M.; Bauer, Greta M.; Chandler, John E.; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K.; Subramanian, Hariharan; Chandel, Navdeep S.; Szleifer, Igal; Backman, Vadim

    2016-01-01

    The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells. PMID:27702891

  13. 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

  14. Progress of new label-free techniques for biosensors: a review.

    PubMed

    Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

    2016-01-01

    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.

  15. Label-free fast 3D coherent imaging reveals pancreatic islet micro-vascularization and dynamic blood flow

    PubMed Central

    Berclaz, Corinne; Szlag, Daniel; Nguyen, David; Extermann, Jérôme; Bouwens, Arno; Marchand, Paul J.; Nilsson, Julia; Schmidt-Christensen, Anja; Holmberg, Dan; Grapin-Botton, Anne; Lasser, Theo

    2016-01-01

    In diabetes, pancreatic β-cells play a key role. These cells are clustered within structures called islets of Langerhans inside the pancreas and produce insulin, which is directly secreted into the blood stream. The dense vascularization of islets of Langerhans is critical for maintaining a proper regulation of blood glucose homeostasis and is known to be affected from the early stage of diabetes. The deep localization of these islets inside the pancreas in the abdominal cavity renders their in vivo visualization a challenging task. A fast label-free imaging method with high spatial resolution is required to study the vascular network of islets of Langerhans. Based on these requirements, we developed a label-free and three-dimensional imaging method for observing islets of Langerhans using extended-focus Fourier domain Optical Coherence Microscopy (xfOCM). In addition to structural imaging, this system provides three-dimensional vascular network imaging and dynamic blood flow information within islets of Langerhans. We propose our method to deepen the understanding of the interconnection between diabetes and the evolution of the islet vascular network. PMID:27895996

  16. 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.

  17. Rapid and label-free detection and assessment of bacteria by terahertz time-domain spectroscopy.

    PubMed

    Yang, Xiang; Wei, Dongshan; Yan, Shihan; Liu, Yueping; Yu, Shu; Zhang, Mingkun; Yang, Zhongbo; Zhu, Xiaoyan; Huang, Qing; Cui, Hong-Liang; Fu, Weiling

    2016-10-01

    Here we demonstrated the potential and applicability of terahertz (THz) spectroscopy to detect four commonly found bacteria in the infectious diseases. Besides the different spectral characteristics between bacterial species, THz absorption differences for living bacteria, dead bacteria and bacterial powder of the same species were also investigated. Our results revealed that small differences in water contents between bacterial cells account for distinct discrepancies of the absorption coefficients, which can be used for bacterial species identification. Furthermore, living and dead bacteria showed different absorption coefficients as a result of their different hydration levels, suggesting that THz spectroscopy can be used to rapidly assess the living state of bacteria under test. Our results clearly demonstrated the ability of THz spectroscopy for time-saving and label-free detection of bacteria with minimal sample preparation, potentially to be utilized for point-of-care tests in the near future. Schematic representation of bacterial detection by THz spectroscopy. Different bacteria have distinctive absorption coefficients as a result of their different water contents.

  18. 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.

  19. 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.

  20. Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide.

    PubMed

    Song, Liang; Wang, Fei; Dong, Zhaoming; Hua, Xiaoting; Xia, Qingyou

    2017-02-10

    Paralytic peptide (PP) participates in diverse physiological processes as an insect cytokine, such as immunity control, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics. 2534 phosphosites were finally identified, of which the phosphorylation level of 620 phosphosites on 244 proteins was significantly up-regulated and 67 phosphosites on 43 proteins was down-regulated. Among those proteins, 13 were protein kinases (PKs), 13 were transcription factors (TFs) across 10 families and 17 were metabolism related enzymes. Meanwhile, Motif-X analysis of the phosphorylation sites showed that 16 motifs are significantly enriched, including 8 novel phosphorylation motifs. In addition, KEGG and functional interacting network analysis revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways, and highlighted the potential central position of the mitogen-activated protein kinases (MAPKs) in them. These analyses provide direct insights into the molecule mechanisms of cellular response induced by PP.

  1. Long-period gratings in photonic crystal fiber as an optofluidic label-free biosensor.

    PubMed

    He, Zonghu; Tian, Fei; Zhu, Yinian; Lavlinskaia, Nina; Du, Henry

    2011-08-15

    Using long-period gratings (LPG) inscribed in photonic crystal fiber (PCF) and coupling this structure with an optically aligned flow cell, we have developed an optofluidic refractive index transduction platform for label-free biosensing. The LPG-PCF scheme possesses extremely high sensitivity to the change in refractive index induced by localized binding event in different solution media. A model immunoassay experiment was carried out inside the air channels of PCF by a series of surface modification steps in sequence that include adsorption of poly(allylamine hydrochloride) monolayer, immobilization of anti-rat bone sialoprotein monoclonal primary antibody, and binding interactions with non-specific goat anti-rabbit IgG (H+L) and specific secondary goat anti-mouse IgG (H+L) antibodies. These adsorption and binding events were monitored in situ using the LPG-PCF by measuring the shift of the core-to-cladding mode coupling resonance wavelength. Steady and significant resonance changes, about 0.75 nm per nanometer-thick adsorbed/bound bio-molecules, have been observed following the sequence of the surface events with monolayer sensitivity, suggesting the promising potential of LPG-PCF for biological sensing and evaluation.

  2. 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

    2016-05-05

    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.

  3. Label-Free Raman Spectroscopy Detects Stromal Adaptations in Premetastatic Lungs Primed by Breast Cancer.

    PubMed

    Paidi, Santosh Kumar; Rizwan, Asif; Zheng, Chao; Cheng, Menglin; Glunde, Kristine; Barman, Ishan

    2017-01-15

    Recent advances in animal modeling, imaging technology, and functional genomics have permitted precise molecular observations of the metastatic process. However, a comprehensive understanding of the premetastatic niche remains elusive, owing to the limited tools that can map subtle differences in molecular mediators in organ-specific microenvironments. Here, we report the ability to detect premetastatic changes in the lung microenvironment, in response to primary breast tumors, using a combination of metastatic mouse models, Raman spectroscopy, and multivariate analysis of consistent patterns in molecular expression. We used tdTomato fluorescent protein expressing MDA-MB-231 and MCF-7 cells of high and low metastatic potential, respectively, to grow orthotopic xenografts in athymic nude mice and allow spontaneous dissemination from the primary mammary fat pad tumor. Label-free Raman spectroscopic mapping was used to record the molecular content of premetastatic lungs. These measurements show reliable distinctions in vibrational features, characteristic of the collageneous stroma and its cross-linkers as well as proteoglycans, which uniquely identify the metastatic potential of the primary tumor by recapitulating the compositional changes in the lungs. Consistent with histological assessment and gene expression analysis, our study suggests that remodeling of the extracellular matrix components may present promising markers for objective recognition of the premetastatic niche, independent of conventional clinical information. Cancer Res; 77(2); 247-56. ©2016 AACR.

  4. Direct, label-free, selective, and sensitive microbial detection using a bacteriorhodopsin-based photoelectric immunosensor.

    PubMed

    Chen, Hsiu-Mei; Jheng, Kai-Ru; Yu, An-Dih

    2017-05-15

    A photoelectric immunosensor using purple membranes (PM) as the transducer, which contains photoactive bacteriorhodopsin, is here first demonstrated for direct and label-free microbial detection. Biotinylated polyclonal antibodies against Escherichia coli were immobilized on a PM-coated electrode through further surface biotinylation and bridging avidin or NeutrAvidin. The photocurrent generated by the antibody-coated sensor was reduced after incubation with E. coli K-12 cultures, with the reduction level increased with the culture populations. The immunosensor prepared via NeutrAvidin exhibited much better selectivity than the one prepared via avidin, recognizing almost none of the tested Gram-positive bacteria. Cultures with populations ranging from 1 to 10(7)CFU/10mL were detected in a single step without any preprocessing. Both AFM and Raman analysis confirmed the layer-by-layer fabrication of the antibody-coated substrates as well as the binding of microorganisms. By investigating the effect of illumination orientation and simulating the photocurrent responses with an equivalent circuit model containing a chemical capacitance, we suggest that the photocurrent reduction was primarily caused by the light-shielding effect of the captured bacteria. Using the current fabrication technique, versatile bacteriorhodopsin-based photoelectric immunosensors can be readily prepared to detect a wide variety of biological cells.

  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.

  6. Label-free monitoring of tissue biochemistry following traumatic brain injury using Raman spectroscopy.

    PubMed

    Surmacki, Jakub Maciej; Ansel-Bollepalli, Laura; Pischiutta, Francesca; Zanier, Elisa R; Ercole, Ari; Bohndiek, Sarah Elizabeth

    2016-12-19

    Traumatic brain injury (TBI) constitutes a major cause of death and long-term disability. At present, we lack methods to non-invasively track tissue biochemistry and hence select appropriate interventions for patients. We hypothesized that detailed label-free vibrational chemical analysis of focal TBI could provide such information. We assessed the early spatial and temporal changes in tissue biochemistry that are associated with brain injury in mice. Numerous differences were observed in the spectra of the contusion core and pericontusional tissue between 2 and 7 days. For example, a strong signal from haem was seen in the contusion core at 2 days due to haemorrhage, which subsequently resolved. More importantly, elevated cholesterol levels were demonstrated by 7 days, which may be a marker of important cell repair processes. Principal component analysis revealed an early 'acute' component dominated by haemorrhage and a delayed component reflecting changes in protein and lipid composition. Notably we demonstrated changes in Raman signature with time even in the contralateral hemisphere when compared to sham control mice. Raman spectroscopy therefore shows promise as a probe that is sensitive to important pathobiological processes in TBI and could be applied in future both in the experimental setting, as well as in the clinic.

  7. Label-free cellular manipulation and sorting via biocompatible ferrofluids.

    PubMed

    Kose, Ayse R; Fischer, Birgit; Mao, Leidong; Koser, Hur

    2009-12-22

    We present a simple microfluidic platform that uses biocompatible ferrofluids for the controlled manipulation and rapid separation of both microparticles and live cells. This low-cost platform exploits differences in particle size, shape, and elasticity to achieve rapid and efficient separation. Using microspheres, we demonstrate size-based separation with 99% separation efficiency and sub-10-microm resolution in <45 s. We also show continuous manipulation and shape-based separation of live red blood cells from sickle cells and bacteria. These initial demonstrations reveal the potential of ferromicrofluidics in significantly reducing incubation times and increasing diagnostic sensitivity in cellular assays through rapid separation and delivery of target cells to sensor arrays.

  8. An adaptive alignment algorithm for quality-controlled label-free LC-MS.

    PubMed

    Sandin, Marianne; Ali, Ashfaq; Hansson, Karin; Månsson, Olle; Andreasson, Erik; Resjö, Svante; Levander, Fredrik

    2013-05-01

    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.

  9. A detection instrument for enhanced-fluorescence and label-free imaging on photonic crystal surfaces.

    PubMed

    Block, Ian D; Mathias, Patrick C; Ganesh, Nikhil; Jones, Sarah I; Dorvel, Brian R; Chaudhery, Vikram; Vodkin, Lila O; Bashir, Rashid; Cunningham, Brian T

    2009-07-20

    We report on the design and demonstration of an optical imaging system capable of exciting surface-bound fluorophores within the resonant evanescent electric field of a photonic crystal surface and gathering fluorescence emission that is directed toward the imaging objective by the photonic crystal. The system also has the ability to quantify shifts in the local resonance angle induced by the adsorption of biomolecules on the photonic crystal surface for label-free biomolecular imaging. With these two capabilities combined within a single detection system, we demonstrate label-free images self-registered to enhanced fluorescence images with 328x more sensitive fluorescence detection relative to a glass surface. This technique is applied to a DNA microarray where label-free quantification of immobilized capture DNA enables improved quality control and subsequent enhanced fluorescence detection of dye-tagged hybridized DNA yields 3x more genes to be detected versus commercially available microarray substrates.

  10. Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.

    PubMed

    Holt, Tom G; Choi, Bernard K; Geoghagen, Neil S; Jensen, Kristian K; Luo, Qi; LaMarr, William A; Makara, Gergely M; Malkowitz, Lorraine; Ozbal, Can C; Xiong, Yusheng; Dufresne, Claude; Luo, Ming-Juan

    2009-10-01

    Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

  11. Label-free Evaluation of Hepatic Microvesicular Steatosis with Multimodal Coherent Anti-Stokes Raman Scattering Microscopy

    PubMed Central

    Le, Thuc T.; Ziemba, Amy; Urasaki, Yasuyo; Brotman, Steven; Pizzorno, Giuseppe

    2012-01-01

    Hepatic microvesicular steatosis is a hallmark of drug-induced hepatotoxicity and early-stage fatty liver disease. Current histopathology techniques are inadequate for the clinical evaluation of hepatic microvesicular steatosis. In this paper, we explore the use of multimodal coherent anti-Stokes Raman scattering (CARS) microscopy for the detection and characterization of hepatic microvesicular steatosis. We show that CARS microscopy is more sensitive than Oil Red O histology for the detection of microvesicular steatosis. Computer-assisted analysis of liver lipid level based on CARS signal intensity is consistent with triglyceride measurement using a standard biochemical assay. Most importantly, in a single measurement procedure on unprocessed and unstained liver tissues, multimodal CARS imaging provides a wealth of critical information including the detection of microvesicular steatosis and quantitation of liver lipid content, number and size of lipid droplets, and lipid unsaturation and packing order of lipid droplets. Such information can only be assessed by multiple different methods on processed and stained liver tissues or tissue extracts using current standard analytical techniques. Multimodal CARS microscopy also permits label-free identification of lipid-rich non-parenchymal cells. In addition, label-free and non-perturbative CARS imaging allow rapid screening of mitochondrial toxins-induced microvesicular steatosis in primary hepatocyte cultures. With its sensitivity and versatility, multimodal CARS microscopy should be a powerful tool for the clinical evaluation of hepatic microvesicular steatosis. PMID:23226469

  12. 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.

  13. Implementation of Arithmetic and Nonarithmetic Functions on a Label-free and DNA-based Platform

    PubMed Central

    Wang, Kun; He, Mengqi; Wang, Jin; He, Ronghuan; Wang, Jianhua

    2016-01-01

    A series of complex logic gates were constructed based on graphene oxide and DNA-templated silver nanoclusters to perform both arithmetic and nonarithmetic functions. For the purpose of satisfying the requirements of progressive computational complexity and cost-effectiveness, a label-free and universal platform was developed by integration of various functions, including half adder, half subtractor, multiplexer and demultiplexer. The label-free system avoided laborious modification of biomolecules. The designed DNA-based logic gates can be implemented with readout of near-infrared fluorescence, and exhibit great potential applications in the field of bioimaging as well as disease diagnosis. PMID:27713517

  14. Label-free detection with the liquid core optical ring resonator sensing platform

    PubMed Central

    White, Ian M.; Zhu, Hongying; Suter, Jonathan D.; Fan, Xudong; Zourob, Mohammed

    2009-01-01

    Optical label-free detection avoids the cost and complexity of fluorescence and radio labeling while providing accurate quantitative and kinetic results. We have developed a new optical label-free sensor called the liquid core optical ring resonator (LCORR). The LCORR integrates optical ring resonator sensors into the microfluidic delivery system by using glass capillaries with a thin wall. The LCORR is capable of performing refractive index detection on liquid samples, as well as bio/chemical analyte detection down to detection limits on the scale of pg/mm2. PMID:19151939

  15. Implementation of Arithmetic and Nonarithmetic Functions on a Label-free and DNA-based Platform

    NASA Astrophysics Data System (ADS)

    Wang, Kun; He, Mengqi; Wang, Jin; He, Ronghuan; Wang, Jianhua

    2016-10-01

    A series of complex logic gates were constructed based on graphene oxide and DNA-templated silver nanoclusters to perform both arithmetic and nonarithmetic functions. For the purpose of satisfying the requirements of progressive computational complexity and cost-effectiveness, a label-free and universal platform was developed by integration of various functions, including half adder, half subtractor, multiplexer and demultiplexer. The label-free system avoided laborious modification of biomolecules. The designed DNA-based logic gates can be implemented with readout of near-infrared fluorescence, and exhibit great potential applications in the field of bioimaging as well as disease diagnosis.

  16. Comparison of principal component analysis and biochemical component analysis in Raman spectroscopy for the discrimination of apoptosis and necrosis in K562 leukemia cells.

    PubMed

    Ong, Yi Hong; Lim, Mayasari; Liu, Quan

    2012-09-24

    Raman spectroscopy has been explored as a promising label-free technique in discriminating apoptosis and necrosis induced cell death in leukemia cells. In addition to Principal component analysis (PCA) as commonly employed in Raman data analysis, another less commonly used but powerful method is Biochemical Component Analysis (BCA). In BCA, a Raman spectrum is decomposed into the contributions from several known basic biochemical components, such as proteins, lipid, nucleic acids and glycogen groups etc. The differences in terms of classification accuracy and interpretability of resulting data between these two methods in Raman spectroscopy have not been systematically investigated to our knowledge. In this study, we utilized both methods to analyze the Raman spectra measured from live cells, apoptotic and necrotic leukemia cells. The comparison indicates that two methods yield comparable accuracy in sample classification when the numbers of basic components are equal. The changes in the contributions of biochemical components in BCA can be interpreted by cell biology principles in apoptosis and necrosis. In contrast, the contributions of most principle components in PCA are difficult to interpret except the first one. The capability of BCA to unveil fine biochemical changes in cell spectra and excellent accuracy in classification can impel the broad application of Raman spectroscopy in biological research.

  17. Label-Free Characterization of Emerging Human Neuronal Networks

    PubMed Central

    Mir, Mustafa; Kim, Taewoo; Majumder, Anirban; Xiang, Mike; Wang, Ru; Liu, S. Chris; Gillette, Martha U.; Stice, Steven; Popescu, Gabriel

    2014-01-01

    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. PMID:24658536

  18. 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.

  19. 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

  20. Microfluidic microscopy-assisted label-free approach for cancer screening: automated microfluidic cytology for cancer screening.

    PubMed

    Jagannadh, Veerendra Kalyan; Gopakumar, G; Subrahmanyam, Gorthi R K Sai; Gorthi, Sai Siva

    2016-07-22

    Each year, about 7-8 million deaths occur due to cancer around the world. More than half of the cancer-related deaths occur in the less-developed parts of the world. Cancer mortality rate can be reduced with early detection and subsequent treatment of the disease. In this paper, we introduce a microfluidic microscopy-based cost-effective and label-free approach for identification of cancerous cells. We outline a diagnostic framework for the same and detail an instrumentation layout. We have employed classical computer vision techniques such as 2D principal component analysis-based cell type representation followed by support vector machine-based classification. Analogous to criminal face recognition systems implemented with help of surveillance cameras, a signature-based approach for cancerous cell identification using microfluidic microscopy surveillance is demonstrated. Such a platform would facilitate affordable mass screening camps in the developing countries and therefore help decrease cancer mortality rate.

  1. 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.

  2. Optimizing integrated optical chips for label-free (bio-)chemical sensing.

    PubMed

    Kunz, R E; Cottier, K

    2006-01-01

    Label-free sensing is an important method for many (bio-)chemical applications in fields such as biotechnology, medicine, pharma, ecology and food quality control. The broad range of applications includes liquid refractive index sensing, molecule detection, and the detection of particles or cells. Integrated optics based on the use of waveguide modes offers a great potential and flexibility to tailor the sensor properties to these applications. In this paper, the results of a numerical study are presented, showing that this flexibility is founded on the many degrees of freedom that can be used for the integrated optical chip design, in contrast to other technologies such as those based on surface plasmon resonance, for which the materials' properties limit the range of choices. The applications that are explicitly considered and discussed include (1) bulk refractometry, (2) thin-layer sensing, for example biosensors monitoring molecular adsorption processes occurring within some 10 nm of the chip's surface, (3) thick-layer sensing with processes involving molecules or ions to be monitored within a sensing matrix extending to some 100 nm from the chip's surface, for example hydrogel-based layers and chemo-optically sensitive membranes, and (4) particle sensing with particles or, for example, biological cells to be monitored within probe volumes extending to some 1,000 nm from the chip's surface. The peculiarities for the different types of applications will be discussed, and suitable modeling methods presented. Finally, the application-specific design guidelines supplied will enable the optimization of various types of integrated optical sensors, including interferometers and grating-based sensors.

  3. Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

    PubMed

    Lieb, S; Littmann, T; Plank, N; Felixberger, J; Tanaka, M; Schäfer, T; Krief, S; Elz, S; Friedland, K; Bernhardt, G; Wegener, J; Ozawa, T; Buschauer, A

    2016-12-01

    A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

  4. Nuclease stability of boron-modified nucleic acids: application to label-free mismatch detection.

    PubMed

    Reverte, Maëva; Vasseur, Jean-Jacques; Smietana, Michael

    2015-11-21

    5'-End boronic acid-modified oligonucleotides were evaluated against various nucleases at single and double stranded levels. The results show that these modifications induce a high resistance to degradation by calf-spleen and snake venom phosphodiesterases. More importantly, this eventually led to the development of a new label-free enzyme-assisted fluorescence-based method for single mismatch detection.

  5. 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…

  6. Surface immobilizable chelator for label-free electrical detection of pyrophosphate.

    PubMed

    Liu, David J; Credo, Grace M; Su, Xing; Wu, Kai; Lim, Hsiao C; Elibol, Oguz H; Bashir, Rashid; Varma, Madoo

    2011-08-07

    A new pyrophosphate (PPi) chelator was designed for surface-sensitive electrical detection of biomolecular reactions. This article describes the synthesis of the PPi-selective receptor, its surface immobilization and application to label-free electrical detection on a silicon-based field-effect transistor (FET) sensor.

  7. Real-time label-free biosensing with integrated planar waveguide ring resonators

    NASA Astrophysics Data System (ADS)

    Sohlström, Hans; Gylfason, Kristinn B.; Hill, Daniel

    2010-05-01

    We review the use of planar integrated optical waveguide ring resonators for label free bio-sensing and present recent results from two European biosensor collaborations: SABIO and InTopSens. Planar waveguide ring resonators are attractive for label-free biosensing due to their small footprint, high Q-factors, and compatibility with on-chip optics and microfluidics. This enables integrated sensor arrays for compact labs-on-chip. One application of label-free sensor arrays is for point-of-care medical diagnostics. Bringing such powerful tools to the single medical practitioner is an important step towards personalized medicine, but requires addressing a number of issues: improving limit of detection, managing the influence of temperature, parallelization of the measurement for higher throughput and on-chip referencing, efficient light-coupling strategies to simplify alignment, and packaging of the optical chip and integration with microfluidics. From the SABIO project we report refractive index measurement and label-free biosensing in an 8-channel slotwaveguide ring resonator sensor array, within a compact cartridge with integrated microfluidics. The sensors show a volume sensing detection limit of 5 x 10-6 RIU and a surface sensing detection limit of 0.9 pg/mm2. From the InTopSens project we report early results on silicon-on-insulator racetrack resonators.

  8. Label-free quantitation of a cancer biomarker in complex media using silicon photonic microring resonators.

    PubMed

    Washburn, Adam L; Gunn, L Cary; Bailey, Ryan C

    2009-11-15

    Recent advances in label-free biosensing techniques have shown the potential to simplify clinical analyses. With this motivation in mind, this paper demonstrates for the first time the use of silicon-on-insulator microring optical resonator arrays for the robust and label-free detection of a clinically important protein biomarker in undiluted serum, using carcinoembryonic antigen (CEA) as the test case. We utilize an initial-slope-based quantitation method to sensitively detect CEA at clinically relevant levels and to determine the CEA concentrations of unknown samples in both buffer and undiluted fetal bovine serum. Comparison with a commercial enzyme-linked immunosorbent assay (ELISA) kit reveals that the label-free microring sensor platform has a comparable limit of detection (2 ng/mL) and superior accuracy in the measurement of CEA concentration across a 3 order of magnitude dynamic range. Notably, we report the lowest limit of detection to date for a microring resonator sensor applied to a clinically relevant cancer biomarker. Although this report describes the robust biosensing capabilities of silicon photonic microring resonator arrays for a single parameter assay, future work will focus on utilizing the platform for highly multiplexed, label-free bioanalysis.

  9. Multiplex surface plasmon resonance imaging platform for label-free detection of foodborne pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonellae are among the leading causes of foodborne outbreaks in the United States, and more rapid and efficient detection methods are needed. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multiple targets simultaneous...

  10. Ultralow voltage, OTFT-based sensor for label-free DNA detection.

    PubMed

    Lai, S; Demelas, M; Casula, G; Cosseddu, P; Barbaro, M; Bonfiglio, A

    2013-01-04

    An organic ultralow voltage field effect transistor for DNA hybridization detection is presented. The transduction mechanism is based on a field-effect modulation due to the electrical charge of the oligonucleotides, so label-free detection can be performed. The device shows a sub-nanometer detection limit and unprecedented selectivity with respect to single nucleotide polymorphism.

  11. Combined enhanced fluorescence and label-free biomolecular detection with a photonic crystal surface.

    PubMed

    Mathias, Patrick C; Ganesh, Nikhil; Chan, Leo L; Cunningham, Brian T

    2007-04-20

    A 2D photonic crystal surface with a different period in each lateral direction is demonstrated to detect biomolecules using two distinct sensing modalities. The sensing mechanisms both rely on the generation of a resonant reflection peak at one of two specific wavelengths, depending on the polarization of light that is incident on the photonic crystal. One polarization results in a resonant reflection peak in the visible spectrum to coincide with the excitation wavelength of a fluorophore, while the orthogonal polarization results in a resonant reflection peak at an infrared wavelength which is used for label-free detection of adsorbed biomolecules. The photonic crystal resonance for fluorescence excitation causes enhanced near fields at the structure surface, resulting in increased signal from fluorophores within 100 nm of the device surface. Label-free detection is performed by illuminating the photonic crystal with white light and monitoring shifts in the peak reflected wavelength of the infrared resonance with a high-resolution imaging detection instrument. Rigorous coupled-wave analysis was used to determine optimal dimensions for the photonic crystal structure, and devices were fabricated using a polymer-based nanoreplica molding approach. Fluorescence-based and label-free detection were demonstrated using arrays of spots of dye-conjugated streptavidin. Quantification of the fluorescent signal showed that the fluorescence output from protein spots on the photonic crystal was increased by up to a factor of 35, and deposited spots were also imaged in the label-free detection mode.

  12. Coherent label-free imaging through turbidity: a holographic approach

    NASA Astrophysics Data System (ADS)

    Bianco, V.; Paturzo, M.; Marchesano, V.; Miccio, L.; Memmolo, P.; Ferraro, P.

    2016-03-01

    A challenging issue has to be faced in microscopy whenever samples are immersed inside a dynamic turbid medium, as occluding objects provoke severe light scattering or unpredictable time-variable phase delays that scramble the object information. In these cases, the transmission matrix of the medium cannot be fully characterized. Here we show a simple technique, named Multi-Look Digital Holography (MLDH), able to fully recover the useful signal of biological specimens dipped inside a turbid liquid phase. Multiple hologram recordings are incoherently combined to synthesize the whole complex field diffused by the sample, which is revealed through turbidity providing quantitative phase-contrast information. Moreover, we show that the presence of a turbid medium can have a positive effect on a coherent imaging system, helping to reduce the effect of speckle artifacts. In other words, occluding biological elements, like e.g. Red Blood Cells (RBCs), can be thought as useful optical components providing denoising capabilities.

  13. 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

  14. 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

  15. 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

  16. Label-free molecular beacons-based cascade amplification DNA machine for sensitive detection of telomerase activity.

    PubMed

    Li, Kan; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2017-05-15

    Sensitive detection of telomerase activity is critical to cancer diagnosis, screening of anticancer drugs and evaluation of cancer therapy. Herein, a label-free molecular beacons-based DNA machine was developed for sensitive detection of telomerase activity. The DNA machine consisted of T7 exonuclease (T7 Exo), label-free recognition molecular beacon (RMB) and signal molecular beacon (SMB) with projecting 5'-terminuses, which can protect RMB and SMB from being digested by T7 Exo. Firstly, telomerase elongated telomerase substrate (TS) primer, generating a telomerase elongation production (TEP) with tandem repeats (TTAGGG)n. Next, TEP activated the DNA machine by hybridizing with RMB, unfolding RMB with a recessed 5'-terminus, making RMB deprotection from T7 Exo. Then T7 Exo-assisted cycling cleavage was incurred, releasing intact TEP and numerous DNA fragments (trigger DNA), which got recycling I. Subsequently, trigger DNA specifically opened SMB and was recycled by T7 Exo, liberating multiple G-quadruplex (G4) structures, which got recycling II. Finally, TEP and the liberative G4 structures strongly interacted with N-methyl-mesoporphyrin IX (NMM), yielding a significantly enhanced fluorescence together. In this way, per telomerase-mediated elongation event was efficiently converted into the greatly amplified fluorescence signals. Telomerase activity in crude HeLa cells extracts equivalent to 50 cells/mL was successfully measured with a linear range from 50 cells/mL to 2000 cells/mL. Besides, the strategy was also successfully used to assay the inhibition effect of a telomerase-inhibiting drug, demonstrating the strategy holds the potential to screen telomerase inhibitors.

  17. 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.

  18. Label-free Raman monitoring of extracellular matrix formation in three-dimensional polymeric scaffolds

    PubMed Central

    Kunstar, Aliz; Leferink, Anne M.; Okagbare, Paul I.; Morris, Michael D.; Roessler, Blake J.; Otto, Cees; Karperien, Marcel; van Blitterswijk, Clemens A.; Moroni, Lorenzo; van Apeldoorn, Aart A.

    2013-01-01

    Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional scaffolds for regenerative medicine and clinical purposes. Raman spectroscopy can be used for non-invasive sensing of cellular and ECM biochemistry. We have investigated the use of conventional (confocal and semiconfocal) Raman microspectroscopy and fibre-optic Raman spectroscopy for in vitro monitoring of ECM formation in three-dimensional poly(ethylene oxide terephthalate)–poly(butylene terephthalate) (PEOT/PBT) scaffolds. Chondrocyte-seeded PEOT/PBT scaffolds were analysed for ECM formation by Raman microspectroscopy, biochemical analysis, histology and scanning electron microscopy. ECM deposition in these scaffolds was successfully detected by biochemical and histological analysis and by label-free non-destructive Raman microspectroscopy. In the spectra collected by the conventional Raman set-ups, the Raman bands at 937 and at 1062 cm−1 which, respectively, correspond to collagen and sulfated glycosaminoglycans could be used as Raman markers for ECM formation in scaffolds. Collagen synthesis was found to be different in single chondrocyte-seeded scaffolds when compared with microaggregate-seeded samples. Normalized band-area ratios for collagen content of single cell-seeded samples gradually decreased during a 21-day culture period, whereas collagen content of the microaggregate-seeded samples significantly increased during this period. Moreover, a fibre-optic Raman set-up allowed for the collection of Raman spectra from multiple pores inside scaffolds in parallel. These fibre-optic measurements could give a representative average of the ECM Raman signal present in tissue-engineered constructs. Results in this study provide proof-of-principle that Raman microspectroscopy is a promising non-invasive tool to monitor ECM production and remodelling in three-dimensional porous cartilage tissue-engineered constructs. PMID:23825118

  19. 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

  20. Label-free Raman monitoring of extracellular matrix formation in three-dimensional polymeric scaffolds.

    PubMed

    Kunstar, Aliz; Leferink, Anne M; Okagbare, Paul I; Morris, Michael D; Roessler, Blake J; Otto, Cees; Karperien, Marcel; van Blitterswijk, Clemens A; Moroni, Lorenzo; van Apeldoorn, Aart A

    2013-09-06

    Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional scaffolds for regenerative medicine and clinical purposes. Raman spectroscopy can be used for non-invasive sensing of cellular and ECM biochemistry. We have investigated the use of conventional (confocal and semiconfocal) Raman microspectroscopy and fibre-optic Raman spectroscopy for in vitro monitoring of ECM formation in three-dimensional poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) scaffolds. Chondrocyte-seeded PEOT/PBT scaffolds were analysed for ECM formation by Raman microspectroscopy, biochemical analysis, histology and scanning electron microscopy. ECM deposition in these scaffolds was successfully detected by biochemical and histological analysis and by label-free non-destructive Raman microspectroscopy. In the spectra collected by the conventional Raman set-ups, the Raman bands at 937 and at 1062 cm(-1) which, respectively, correspond to collagen and sulfated glycosaminoglycans could be used as Raman markers for ECM formation in scaffolds. Collagen synthesis was found to be different in single chondrocyte-seeded scaffolds when compared with microaggregate-seeded samples. Normalized band-area ratios for collagen content of single cell-seeded samples gradually decreased during a 21-day culture period, whereas collagen content of the microaggregate-seeded samples significantly increased during this period. Moreover, a fibre-optic Raman set-up allowed for the collection of Raman spectra from multiple pores inside scaffolds in parallel. These fibre-optic measurements could give a representative average of the ECM Raman signal present in tissue-engineered constructs. Results in this study provide proof-of-principle that Raman microspectroscopy is a promising non-invasive tool to monitor ECM production and remodelling in three-dimensional porous cartilage tissue-engineered constructs.

  1. 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

  2. Discrimination and quantification of autofluorescence spectra of human lung cells

    NASA Astrophysics Data System (ADS)

    Rahmani, Mahya; Khani, Mohammad Mehdi; Khazaei Koohpar, Zeinab; Molik, Paria

    2016-10-01

    To study laser-induced autofluorescence spectroscopy of the human lung cell line, we evaluated the native fluorescence properties of cancer QU-DB and normal MRC-5 human lung cells during continuous exposure to 405 nm laser light. Two emission bands centered at ~470 nm and ~560 nm were observed. These peaks are most likely attributable to mitochondrial fluorescent reduced nicotinamide adenine dinucleotide and riboflavin fluorophores, respectively. This article highlights lung cell autofluorescence characterization and signal discrimination by collective investigation of different spectral features. The absolute intensity, the spectral shape factor or redox ratio, the full width of half-maximum and the full width of quarter maximum was evaluated. Moreover, the intensity ratio, the area under the peak and the area ratio as a contrast factor for normal and cancerous cells were also calculated. Among all these features it seems that the contrast factor precisely and significantly discriminates the spectral differences of normal and cancerous lung cells. On the other hand, the relative quantum yield for both cell types were found by comparing the quantum yield of an unknown compound with known fluorescein sodium as a reference solution.

  3. 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.

  4. Label-free colorimetric detection of cadmium ions in rice samples using gold nanoparticles.

    PubMed

    Guo, Yongming; Zhang, Yi; Shao, Huawu; Wang, Zhuo; Wang, Xuefei; Jiang, Xingyu

    2014-09-02

    A simple and label-free colorimetric method for cadmium ions (Cd(2+)) detection using unmodified gold nanoparticles (AuNPs) is reported. The unmodified AuNPs easily aggregate in a high concentration of NaCl solution, but the presence of glutathione (GSH) can prevent the salt-induced aggregation of AuNPs. When Cd(2+) is added to the stable mixture of AuNPs, GSH, and NaCl, Cd(2+) can coordinate with 4× GSH as a spherical shaped complex, which decreases the amount of free GSH on the surface of gold nanoparticles to weaken the stability of AuNPs, and AuNPs will easily aggregate in high-salt conditions. On the basis of the mechanism, we design a simple, label-free colorimetric method using AuNPs accompanied by GSH in a high-salt environment to detect Cd(2+) in water and digested rice samples.

  5. In vivo label-free photoacoustic microscopy of the anterior segment of the mouse eye

    NASA Astrophysics Data System (ADS)

    Rao, Bin; Hu, Song; Li, Li; Maslov, Konstantin; Wang, Lihong V.

    2010-02-01

    Both iris fluorescein angiography (IFA) and indocyanine green angiography (ICGA) provide ophthalmologists imaging tools in studying the microvasculature structure and hemodynamics of the anterior segment of the eye in normal and diseased status. However, a non-invasive, endogenous imaging modality is preferable for the monitoring of hemodynamics of the iris microvasculature. We investigated the in vivo, label-free ocular anterior segment imaging with photo-acoustic microscopy (PAM) in mouse eyes. We demonstrated the unique advantage of endogenous contrast that is not available in both IFA and ICGA. The laser radiation was maintained within the ANSI laser safety limit. The in vivo, label-free nature of our imaging technology has the potential for ophthalmic applications.

  6. 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.

  7. Label-free identification of individual bacteria using Fourier transform light scattering.

    PubMed

    Jo, YoungJu; Jung, JaeHwang; Kim, Min-Hyeok; Park, HyunJoo; Kang, Suk-Jo; Park, YongKeun

    2015-06-15

    Rapid identification of bacterial species is crucial in medicine and food hygiene. In order to achieve rapid and label-free identification of bacterial species at the single bacterium level, we propose and experimentally demonstrate an optical method based on Fourier transform light scattering (FTLS) measurements and statistical classification. For individual rod-shaped bacteria belonging to four bacterial species (Listeria monocytogenes, Escherichia coli, Lactobacillus casei, and Bacillus subtilis), two-dimensional angle-resolved light scattering maps are precisely measured using FTLS technique. The scattering maps are then systematically analyzed, employing statistical classification in order to extract the unique fingerprint patterns for each species, so that a new unidentified bacterium can be identified by a single light scattering measurement. The single-bacterial and label-free nature of our method suggests wide applicability for rapid point-of-care bacterial diagnosis.

  8. Label-free amino acid detection based on nanocomposites of graphene oxide hybridized with gold nanoparticles.

    PubMed

    Zhang, Qian; Zhang, Diming; Lu, Yanli; Xu, Gang; Yao, Yao; Li, Shuang; Liu, Qingjun

    2016-03-15

    Nanocomposites of graphene oxide and gold nanoparticles (GO/GNPs) were synthesized for label-free detections of amino acids. Interactions between the composites and amino acids were investigated by both naked-eye observation and optical absorption spectroscopy. The GO/GNPs composites displayed apparent color changes and absorption spectra changes in presences of amino acids including glutamate, aspartate, and cysteine. The interaction mechanisms of the composites and amino acids were discussed and explored with sulfhydryl groups and non-α-carboxylic groups on the amino acids. Sensing properties of the composites were tested, while pure gold particles were used as the control. The results suggested that the GO/GNPs composites had better linearity and stability in dose-dependent responses to the amino acids than those of the particles, especially in detections for acidic amino acids. Therefore, the nanocomposites platform can provide a convenient and efficient approach for label-free optical detections of important molecules such as amino acids.

  9. 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

  10. 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.

  11. Advance ultra sensitive multi-layered nano plasmonic devices for label free biosensing targeting immunodiagnostics

    NASA Astrophysics Data System (ADS)

    Sharma, Divya; Dwivedi, R. P.

    2016-09-01

    The rapid advancement in technology has envisaged and drafted the use of optical bio-sensing units into label free and multiplexed bio-sensing, exploring the surface plasmon polaritons, which has turned into a gold standard on the commercial basis, but they are bulky and find difficulty in scaling up for the throughput detection. The integration of plasmonic crystals with microfluidics on the bio-sensing frontier offers a multi-level validation of results with the ease of real-time detection and imaging and holds a great promise to develop ultra-sensitive, fast, portable device for the point-of-care diagnostics. The paper describes the fast, low cost approach of designing and simulating label free biosensor using open source MEEP and other software tools targeting Immunodiagnostics.

  12. Label-Free Electrical Detection Using Carbon Nanotube-Based Biosensors

    PubMed Central

    Maehashi, Kenzo; Matsumoto, Kazuhiko

    2009-01-01

    Label-free detections of biomolecules have attracted great attention in a lot of life science fields such as genomics, clinical diagnosis and practical pharmacy. In this article, we reviewed amperometric and potentiometric biosensors based on carbon nanotubes (CNTs). In amperometric detections, CNT-modified electrodes were used as working electrodes to significantly enhance electroactive surface area. In contrast, the potentiometric biosensors were based on aptamer-modified CNT field-effect transistors (CNTFETs). Since aptamers are artificial oligonucleotides and thus are smaller than the Debye length, proteins can be detected with high sensitivity. In this review, we discussed on the technology, characteristics and developments for commercialization in label-free CNT-based biosensors. PMID:22346703

  13. 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.

  14. Microfluidics-integrated cascaded double-microring resonators for label-free biosensing

    NASA Astrophysics Data System (ADS)

    Chen, Yangqing; Yu, Fang; Yang, Chang; Li, Mingyu; Tang, Longhua; Song, Jinyan; He, Jian-Jun

    2014-11-01

    A highly-sensitive optical waveguide biosensor integrated with microfluidic channels based on silicon-on-insulator (SOI) was investigated in this paper. Experimental results of the label-free detection exhibits this novel biosensor with the superior reliability for quantitative and kinetic measurement of the interaction between biological molecules, dramatically improving the sensitivity due to the Vernier effect induced by cascaded double-microring resonators.

  15. 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.

  16. Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor.

    PubMed

    Zaffino, R L; Mir, M; Samitier, J

    2014-03-14

    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 50 nm, 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.

  17. 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

    2015-02-03

    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.

  18. 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.

  19. A silicon-based electrochemical sensor for highly sensitive, specific, label-free and real-time DNA detection.

    PubMed

    Guo, Yuanyuan; Su, Shao; Wei, Xinpan; Zhong, Yiling; Su, Yuanyuan; Huang, Qing; Fan, Chunhai; He, Yao

    2013-11-08

    We herein present a new kind of silicon-based electrochemical sensor using a gold nanoparticles-decorated silicon wafer (AuNPs@Si) as a high-performance electrode, which is facilely prepared via in situ AuNPs growth on a silicon wafer. Particularly significantly, the resultant electrochemical sensor is efficacious for label-free DNA detection with high sensitivity due to the unique merits of the prepared silicon-based electrode. Typically, DNA at remarkably low concentrations (1-10 fM) could be readily detected without requiring additional signal-amplification procedures, which is better than or comparable to the lowest DNA concentration ever detected via well-studied signal-amplification-assisted electrochemical sensors. Moreover, the silicon-based sensor features high specificity, allowing unambiguous discrimination of single-based mismatches. We further show that real-time DNA assembly is readily monitored via recording the intensity changes of current signals due to the robust thermal stability of the silicon-based electrode. The unprecedented advantages of the silicon-based electrochemical sensor would offer new opportunities for myriad sensing applications.

  20. 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.

  1. Label-free imaging of human breast tissues using coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Yaliang; Gao, Liang; Wang, Zhiyong; Thrall, Michael J.; Luo, Pengfei; Wong, Kelvin K.; Wong, Stephen T.

    2011-03-01

    Breast cancer is a common disease in women. Current imaging and diagnostic methods for breast cancer confront several limitations, like time-consuming, invasive and with a high cost. Alternative strategies are in high demand to alleviate patients' trauma and lower medical expenses. Coherent anti-Stokes Raman scattering (CARS) imaging technique offers many advantages, including label-free, sub-wavelength spatial resolution and video-rate imaging speed. Therefore, it has been demonstrated as a powerful tool for various biomedical applications. In this study, we present a label-free fast imaging method to identify breast cancer and its subtypes using CARS microscopy. Human breast tissues, including normal, benign and invasive carcinomas, were imaged ex vivo using a custom-built CARS microscope. Compared with results from corresponding hematoxylin and eosin (H&E) stains, the CARS technique has demonstrated its capability in identifying morphological features in a similar way as in H&E stain. These features can be used to distinguish breast cancer from normal and benign tissues, and further separate cancer subtypes from each other. Our pilot study suggests that CARS microscopy could be used as a routine examination tool to characterize breast cancer ex vivo. Moreover, its label-free and fast imaging properties render this technique as a promising approach for in vivo and real-time imaging and diagnosis of breast cancer.

  2. A multicenter study benchmarks software tools for label-free proteome quantification.

    PubMed

    Navarro, Pedro; Kuharev, Jörg; Gillet, Ludovic C; Bernhardt, Oliver M; MacLean, Brendan; Röst, Hannes L; Tate, Stephen A; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I; Aebersold, Ruedi; Tenzer, Stefan

    2016-11-01

    Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.

  3. freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

    PubMed

    Deng, Ning; 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.

  4. 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

  5. Label-free silicon nanodots featured ratiometric fluorescent aptasensor for lysosomal imaging and pH measurement.

    PubMed

    Zhang, Yanan; Guo, Shan; Cheng, Shibo; Ji, Xinghu; He, Zhike

    2017-03-22

    The homeostasis of lysosomal pH is crucial in cell physiology. Developing small fluorescent nanosensors for lysosome imaging and ratiometric measurement of pH is highly demanded yet challenging. Herein, a pH-sensitive fluorescein tagged aptamer AS1411 has been utilized to covalently modify the label-free fluorescent silicon nanodots via a crosslinker for construction of a ratiometric pH biosensor. The established aptasensor exhibits the advantages of ultrasmall size, hypotoxicity, excellent pH reversibility and good photostability, which favors its application in an intracellular environment. Using human breast MCF-7 cancer cells and MCF-10A normal cells as the model, this aptasensor shows cell specificity for cancer cells and displays a wide pH response range of 4.5-8.0 in living cells. The results demonstrate that the pH of MCF-7 cells is 5.1, which is the expected value for acidic organelles. Lysosome imaging and accurate measurement of pH in MCF-7 cells have been successfully conducted based on this nanosensor via fluorescent microscopy and flow cytometry.

  6. 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.

  7. Use of label-free optical biosensors to detect modulation of potassium channels by G-protein coupled receptors.

    PubMed

    Fleming, Matthew R; Shamah, Steven M; Kaczmarek, Leonard K

    2014-02-10

    Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane(1). To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex(2,3). Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein's behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

  8. 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.

  9. 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

  10. Label-free imaging of amyloids using their intrinsic linear and nonlinear optical properties

    PubMed Central

    Johansson, Patrik K.; Koelsch, Patrick

    2017-01-01

    The optical properties of amyloid fibers are often distinct from those of the source protein in its non-fibrillar form. These differences can be utilized for label-free imaging or characterization of such structures, which is particularly important for understanding amyloid fiber related diseases such as Alzheimer’s and Parkinson’s disease. We demonstrate that two amyloid forming proteins, insulin and β-lactoglobulin (β-LG), show intrinsic fluorescence with emission spectra that are dependent on the excitation wavelength. Additionally, a new fluorescence peak at about 430 nm emerges for β-LG in its amyloid state. The shift in emission wavelength is related to the red edge excitation shift (REES), whereas the additional fluorescence peak is likely associated with charge delocalization along the fiber backbone. Furthermore, the spherulitic amyloid plaque-like superstructures formed from the respective proteins were imaged label-free with confocal fluorescence, multiphoton excitation fluorescence (MPEF), and second-harmonic generation (SHG) microscopy. The latter two techniques in particular yield images with a high contrast between the amyloid fiber regions and the core of amorphously structured protein. Strong multiphoton absorption (MPA) for the amyloid fibers is a likely contributor to the observed contrast in the MPEF images. The crystalline fibrillar region provides even higher contrast in the SHG images, due to the inherently ordered non-centrosymmetric structure of the fibers together with their non-isotropic arrangement. Finally, we show that MPEF from the insulin spherulites exhibits a spectral dependence on the excitation wavelength. This behavior is consistent with the REES phenomenon, which we hypothesize is the origin of this observation. The presented results suggest that amyloid deposits can be identified and structurally characterized based on their intrinsic optical properties, which is important for probe-less and label-free identification

  11. 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.

  12. 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

  13. 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

  14. Highly Sensitive Colorimetric Detection of Ochratoxin A by a Label-Free Aptamer and Gold Nanoparticles.

    PubMed

    Luan, Yunxia; Chen, Jiayi; Li, Cheng; Xie, Gang; Fu, Hailong; Ma, Zhihong; Lu, Anxiang

    2015-12-10

    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.

  15. Label-free optical quantification of structural alterations in Alzheimer’s disease

    NASA Astrophysics Data System (ADS)

    Lee, Moosung; Lee, Eeksung; Jung, Jaehwang; Yu, Hyeonseung; Kim, Kyoohyun; Yoon, Jonghee; Lee, Shinhwa; Jeong, Yong; Park, Yongkeun

    2016-08-01

    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.

  16. Microneedle Biosensor: A Method for Direct Label-free Real Time Protein Detection

    PubMed Central

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

    2012-01-01

    Here we present the development of an array of electrical micro-biosensors in a microfluidic channel, called microneedle biosensors. A microneedle biosensor is a real-time, label-free, direct electrical detection platform, which is capable of high sensitivity detection, measuring the change in ionic current and impedance modulation, due to the presence or reaction of biomolecules such as proteins and nucleic acids. In this study, we successfully fabricated and electrically characterized the sensors and demonstrated successful detection of target protein. In this study, we used biotinylated bovine serum albumin as the receptor and streptavidin as the target analyte PMID:23355762

  17. 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.

  18. 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).

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Optical weak measurement system with common path implementation for label-free biomolecule sensing.

    PubMed

    Zhang, Yilong; Li, Dongmei; He, Yonghong; Shen, Zhiyuan; He, Qinghua

    2016-11-15

    A reflection-type phase-sensitive weak measurement for biosensing and chemical label-free sensing is presented. The phase difference between p and s polarizations in total internal reflection caused by biomolecular recognition is measured by weak value amplification. The system with p and s polarizations in a common path is stable and robust. The sensing process occurring on the silicon dioxide surface is achieved with a resolution of 3.6×10-6 refractive index units. The applicability is demonstrated by real-time monitoring biomolecular interaction of IgG and protein A.

  6. Label-free CMOS bio sensor with on-chip noise reduction scheme for real-time quantitative monitoring of biomolecules.

    PubMed

    Seong-Jin Kim; Euisik Yoon

    2012-06-01

    We present a label-free CMOS field-effect transistor sensing array to detect the surface potential change affected by the negative charge in DNA molecules for real-time monitoring and quantification. The proposed CMOS bio sensor includes a new sensing pixel architecture implemented with correlated double sampling for reducing offset fixed pattern noise and 1/f noise of the sensing devices. We incorporated non-surface binding detection which allows real-time continuous monitoring of DNA concentrations without immobilizing them on the sensing surface. Various concentrations of 19-bp oligonucleotides solution can be discriminated using the prototype device fabricated in 1- μm double-poly double-metal standard CMOS process. The detection limit was measured as 1.1 ng/μl with a dynamic range of 40 dB and the transient response time was measured less than 20 seconds.

  7. A label-free gold-nanoparticle-based SERS assay for direct cyanide detection at the parts-per-trillion level.

    PubMed

    Senapati, Dulal; Dasary, Samuel S R; Singh, Anant K; Senapati, Tapas; Yu, Hongtao; Ray, Paresh C

    2011-07-18

    Cyanide is an extremely toxic lethal poison known to humankind. Developing rapid, highly sensitive, and selective detection of cyanide from water samples is extremely essential for human life safety. Driven by the need, here we report a gold-nanoparticle-based label-free surface-enhanced Raman spectroscopy (SERS) system for highly toxic cyanide ion recognition in parts-per-trillion level and to examine gold-nanoparticle-cyanide interaction. We have shown that the SERS assay can be used to probe the gold nanoparticle dissociation process in the presence of cyanide ions. Our experimental data indicates that gold-nanoparticle-based SERS can detect cyanide from a water sample at the 110 ppt level with excellent discrimination against other common anions and cations. The results also show that the SERS probe can be used to detect cyanide from environmental samples.

  8. 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.

  9. Ligand engagement on material surfaces is discriminated by cell mechanosensoring.

    PubMed

    Battista, Edmondo; Causa, Filippo; Lettera, Vincenzo; Panzetta, Valeria; Guarnieri, Daniela; Fusco, Sabato; Gentile, Francesco; Netti, Paolo A

    2015-03-01

    Peptide or protein ligands can be used for molecular decoration to enhance the functionality of synthetic materials. However, some skepticism has arisen about the efficacy of such strategy in practical contexts since serum proteins largely adsorb. To address this issue, it is crucial to ascertain whether a chemically conjugated integrin-binding peptide is fully recognized by a cell even if partially covered by a physisorbed layer of serum protein; in more general terms, if competitive protein fragments physisorbed onto the surface are distinguishable from those chemically anchored to it. Here, we engraft an RGD peptide on poly-ε-caprolactone (PCL) surfaces and follow the dynamics of focal adhesion (FA) and cytoskeleton assembly at different times and culture conditions using a variety of analytical tools. Although the presence of serum protein covers the bioconjugated RGD significantly, after the first adhesion phase cells dig into the physisorbed layer and reach the submerged signal to establish a more stable adhesion structure (mature FAs). Although the spreading area index is not substantially affected by the presence of the RGD peptide, cells attached to chemically bound signals develop a stronger adhesive interaction with the materials and assemble a mechanically stable cytoskeleton. This demonstrates that cells are able to discriminate, via mechanosensoring, between adhesive motives belonging to physisorbed proteins and those firmly anchored on the material surface.

  10. Rapid quantitative detection of Brucella melitensis by a label-free impedance immunosensor based on a gold nanoparticle-modified screen-printed carbon electrode.

    PubMed

    Wu, Haiyun; Zuo, Yueming; Cui, Chuanjin; Yang, Wei; Ma, Haili; Wang, Xiaowen

    2013-07-04

    A rapid and simple method for quantitative monitoring of Brucella melitensis using electrochemical impedance spectroscopy (EIS) is reported for the first time. The label-free immunosensors were fabricated by immobilizing Brucella melitensis antibody on the surface of gold nanoparticle-modified screen-printed carbon electrodes (GNP-SPCEs). Cyclic voltammetry (CV) and EIS were used to characterize the Brucella melitensis antigen interaction on the surface of GNP-SPCEs with antibody. A general electronic equivalent model of an electrochemical cell was introduced for interpretation of the impedance components of the system. The results showed that the change in electron-transfer resistance (Rct) was significantly different due to the binding of Brucella melitensis cells. A linear relationship between the Rct variation and logarithmic value of the cell concentration was found from 4 × 10(4) to 4 × 10(6) CFU/mL in pure culture. The label-free impedance biosensor was able to detect as low as 1 × 10(4) and 4 × 10(5) CFU/mL of Brucella melitensis in pure culture and milk samples, respectively, in less than 1.5 h. Moreover, a good selectivity versus Escherichia coli O157:H7 and Staphylococcus aureus cells was obtained for our developed immunosensor demonstrating its specificity towards only Brucella melitensis.

  11. Label-free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study.

    PubMed

    Cheong, Youjin; Jin Kim, Young; Kang, Heeyoon; Choi, Samjin; Joo Lee, Hee

    2017-02-01

    We introduce a label-free spectroscopic method to classify subtypes of quinolone-nonsusceptible Escherichia coli (E. coli) isolates obtained from human blood cultures. Raman spectroscopy with a 30-nm gold-deposited, surface-enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)-predefined groups including E. coli ATCC25922, E. coli ST131:O75, and E. coli ST1193:O25b. Although there was a coffee-ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001-1004 cm(-1) (CC aromatic ring breathing stretching vibrational mode of phenylalanine), 1447-1448 cm(-1) (CH2 scissoring deformation vibrational mode), and 1667 cm(-1) (amide I α-helix). Although the three MLST-predefined E. coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm-assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone-nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone-nonsusceptible E. coli ST131:O75 and E. coli ST1193:O25b isolates. Therefore, this label-free and nondestructive technique is likely to be useful for clinically diagnosing quinolone-nonsusceptible E. coli isolates with the MLST method.

  12. A Method for Label-Free, Differential Top-Down Proteomics

    PubMed Central

    Ntai, Ioanna; Toby, Timothy K.; LeDuc, Richard D.; Kelleher, Neil L.

    2016-01-01

    Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research. PMID:26867742

  13. A Method for Label-Free, Differential Top-Down Proteomics.

    PubMed

    Ntai, Ioanna; Toby, Timothy K; LeDuc, Richard D; Kelleher, Neil L

    2016-01-01

    Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.

  14. 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.

  15. 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.

  16. Real-time dual-loop electric current measurement for label-free nanofluidic preconcentration chip.

    PubMed

    Chung, Pei-Shan; Fan, Yu-Jui; Sheen, Horn-Jiunn; Tian, Wei-Cheng

    2015-01-07

    An electrokinetic trapping (EKT)-based nanofluidic preconcentration device with the capability of label-free monitoring trapped biomolecules through real-time dual-loop electric current measurement was demonstrated. Universal current-voltage (I-V) curves of EKT-based preconcentration devices, consisting of two microchannels connected by ion-selective channels, are presented for functional validation and optimal operation; universal onset current curves indicating the appearance of the EKT mechanism serve as a confirmation of the concentrating action. The EKT mechanism and the dissimilarity in the current curves related to the volume flow rate (Q), diffusion coefficient (D), and diffusion layer (DL) thickness were explained by a control volume model with a five-stage preconcentration process. Different behaviors of the trapped molecular plug were categorized based on four modes associated with different degrees of electroosmotic instability (EOI). A label-free approach to preconcentrating (bio)molecules and monitoring the multibehavior molecular plug was demonstrated through real-time electric current monitoring, rather than through the use of microscope images.

  17. Label-free in situ imaging of oil body dynamics and chemistry in germination

    PubMed Central

    Waschatko, Gustav; Billecke, Nils; Schwendy, Sascha; Jaurich, Henriette; Bonn, Mischa; Vilgis, Thomas A.

    2016-01-01

    Plant oleosomes are uniquely emulsified lipid reservoirs that serve as the primary energy source during seed germination. These oil bodies undergo significant changes regarding their size, composition and structure during normal seedling development; however, a detailed characterization of these oil body dynamics, which critically affect oil body extractability and nutritional value, has remained challenging because of a limited ability to monitor oil body location and composition during germination in situ. Here, we demonstrate via in situ, label-free imaging that oil bodies are highly dynamic intracellular organelles that are morphologically and biochemically remodelled extensively during germination. Label-free, coherent Raman microscopy (CRM) combined with bulk biochemical measurements revealed the temporal and spatial regulation of oil bodies in native soya bean cotyledons during the first eight days of germination. Oil bodies undergo a cycle of growth and shrinkage that is paralleled by lipid and protein compositional changes. Specifically, the total protein concentration associated with oil bodies increases in the first phase of germination and subsequently decreases. Lipids contained within the oil bodies change in saturation and chain length during germination. Our results show that CRM is a well-suited platform to monitor in situ lipid dynamics and local chemistry and that oil bodies are actively remodelled during germination. This underscores the dynamic role of lipid reservoirs in plant development. PMID:27798279

  18. Design low crosstalk ring-slot array structure for label-free multiplexed sensing.

    PubMed

    Huang, Lijun; Tian, Huiping; Zhou, Jian; Ji, Yuefeng

    2014-08-25

    We theoretically demonstrate a low crosstalk ring-slot array structure used for label-free multiplexed sensing. The proposed sensors array is based on an array of three ring-slot and input/output line defect coupling waveguides. Each ring-slot cavity has slightly different cavity spacing and different resonant frequency. Results obtained using two dimensional finite-difference time-domain (2D-FDTD) simulation indicate that the resonant frequencies of each sensor unit in response to the refractive index variations are independent. The refractive index sensitivity is 134 ~ 145.5 nm/RIU (refractive index unit) and the Q factors more than 10(4) can be achieved. The calculated detect limit lower than 1.13 × 10(-4) RIU is obtained. In addition, an extremely small crosstalk lower than -25.8 dB is achieved among the array of three ring-slot cavities. The results demonstrate that this multiplexed sensor array is a promising platform for integrated optical devices and enables highly parallel label-free detection.

  19. Capillary Microfluidics-Assembled Virus-like Particle Bionanoreceptor Interfaces for Label-Free Biosensing.

    PubMed

    Zang, Faheng; Gerasopoulos, Konstantinos; Brown, Adam D; Culver, James N; Ghodssi, Reza

    2017-03-15

    A capillary microfluidics-integrated sensor system is developed for rapid assembly of bionanoreceptor interfaces on-chip and label-free biosensing. Genetically engineered Tobacco mosaic virus (TMV) virus-like particles (VLPs), displaying thousands copies of identical receptor peptides FLAG-tags, are utilized as nanoceptors for antibody sensing. Controlled and accelerated assembly of VLP receptor layer on impedance sensor has been achieved using capillary action and surface evaporation from an open-channel capillary microfluidic system. VLPs create a dense and localized receptor monolayer on the impedance sensor using only 5 μL of VLP sample solution (0.2 mg/mL) in only 6 min at room temperature. The VLP-functionalized impedance sensor is capable of label-free detection of target antibodies down to 55 pM concentration within 5 min. These results highlight the significant potentials of an integrated microsystem for rapid and controlled receptor-transducer interface creation and the nanoscale VLP-based sensors for fast, accurate, and decentralized pathogen detection.

  20. Optical Microfibre Based Photonic Components and Their Applications in Label-Free Biosensing

    PubMed Central

    Wang, Pengfei; Bo, Lin; Semenova, Yuliya; Farrell, Gerald; Brambilla, Gilberto

    2015-01-01

    Optical microfibre photonic components offer a variety of enabling properties, including large evanescent fields, flexibility, configurability, high confinement, robustness and compactness. These unique features have been exploited in a range of applications such as telecommunication, sensing, optical manipulation and high Q resonators. Optical microfibre biosensors, as a class of fibre optic biosensors which rely on small geometries to expose the evanescent field to interact with samples, have been widely investigated. Due to their unique properties, such as fast response, functionalization, strong confinement, configurability, flexibility, compact size, low cost, robustness, ease of miniaturization, large evanescent field and label-free operation, optical microfibres based biosensors seem a promising alternative to traditional immunological methods for biomolecule measurements. Unlabeled DNA and protein targets can be detected by monitoring the changes of various optical transduction mechanisms, such as refractive index, absorption and surface plasmon resonance, since a target molecule is capable of binding to an immobilized optical microfibre. In this review, we critically summarize accomplishments of past optical microfibre label-free biosensors, identify areas for future research and provide a detailed account of the studies conducted to date for biomolecules detection using optical microfibres. PMID:26287252

  1. Spiked proteomic standard dataset for testing label-free quantitative software and statistical methods.

    PubMed

    Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Dorssaeler, Alain Van; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

    2016-03-01

    This data article describes a controlled, spiked proteomic dataset for which the "ground truth" of variant proteins is known. It is based on the LC-MS analysis of samples composed of a fixed background of yeast lysate and different spiked amounts of the UPS1 mixture of 48 recombinant proteins. It can be used to objectively evaluate bioinformatic pipelines for label-free quantitative analysis, and their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. More specifically, it can be useful for tuning software tools parameters, but also testing new algorithms for label-free quantitative analysis, or for evaluation of downstream statistical methods. The raw MS files can be downloaded from ProteomeXchange with identifier PXD001819. Starting from some raw files of this dataset, we also provide here some processed data obtained through various bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in different workflows, to exemplify the use of such data in the context of software benchmarking, as discussed in details in the accompanying manuscript [1]. The experimental design used here for data processing takes advantage of the different spike levels introduced in the samples composing the dataset, and processed data are merged in a single file to facilitate the evaluation and illustration of software tools results for the detection of variant proteins with different absolute expression levels and fold change values.

  2. A Multiplex Label-Free Approach to Avian Influenza Surveillance and Serology

    PubMed Central

    Bucukovski, Joseph; Latorre-Margalef, Neus; Stallknecht, David E.; Miller, Benjamin L.

    2015-01-01

    Influenza serology has traditionally relied on techniques such as hemagglutination inhibition, microneutralization, and ELISA. These assays are complex, challenging to implement in a format allowing detection of several types of antibody-analyte interactions at once (multiplex), and troublesome to implement in the field. As an alternative, we have developed a hemagglutinin microarray on the Arrayed Imaging Reflectometry (AIR) platform. AIR provides sensitive, rapid, and label-free multiplex detection of targets in complex analyte samples such as serum. In preliminary work, we demonstrated the application of this array to the testing of human samples from a vaccine trial. Here, we report the application of an expanded label-free hemagglutinin microarray to the analysis of avian serum samples. Samples from influenza virus challenge experiments in mallards yielded strong, selective detection of antibodies to the challenge antigen in most cases. Samples acquired in the field from mallards were also analyzed, and compared with viral hemagglutinin inhibition and microneutralization assays. We find that the AIR hemagglutinin microarray can provide a simple and robust alternative to standard methods, offering substantially greater information density from a simple workflow. PMID:26241048

  3. Optical waveguide sensor based on silica nanotube arrays for label-free biosensing.

    PubMed

    Fan, Yong; Ding, Yu; Ma, Hui; Teramae, Norio; Sun, Shuqing; He, Yonghong

    2015-05-15

    Label-free biosensing based on optical waveguide spectroscopy of silica nanotube (SNT) arrays is realized with high sensitivity. The SNT arrays fabricated using a porous anodic alumina (PAA) template assisted by surface sol-gel (SSG) method showed a high value of 552 reciprocal refractive index unit as the sensing figure of merit by exchanging the sensing environment with water and ethanol. A standard biotin-streptavidin affinity model was tested using the SNT arrays which support a TM1 mode and the fundamental response of the system was investigated. Results show that the response of the SNT arrays for adsorption of streptavidin is higher than the one using substrate without removing the PAA template due to the larger surface area and the stronger electromagnetic field. The limit of detection (LOD) of the SNT arrays for detection of streptavidin was estimated as 93 pM, with the detection time of 40 min. Additionally, the Fresnel calculations suggested higher potential sensitivity of the current system compared to that of the conventional SPR sensors. Thus, the SNT arrays may be used as a versatile platform for high-sensitive label-free optical biosensing due to the high performance and the large potential of the surface functionality.

  4. Hyperspectral stray light imaging of chromosomes: a novel concept for label-free karyotyping

    NASA Astrophysics Data System (ADS)

    Luckow, Sabrina; Rebner, Karsten; Oelkrug, Dieter; Kessler, Rudolf W.

    2012-03-01

    We describe the design and performance of a Hyperspectral Imaging System (HSI) for label-free characterization of human metaphase chromosomes. Chromosomes consist of a DNA-protein complex that is organized in sub-structures and can be described by an array of "particles" with different size and refractive indices. This locally resolved stray light pattern can be used to visualize and characterize unstained chromosomes. The paper describes an imaging system where stray light spectra of chromosomes are detected using a Pushbroom Imaging device attached to a standard microscope in combination with multivariate data analysis. To proof the concept, single particle spectra as well as particle array spectra are analyzed and explained by means of Mie scattering theory and the results are confirmed with FDTD (Finite Difference Time Domain) simulations. This label free signature is due to the superposition of the interference pattern of the different layer thicknesses, the spectral interference of the band pattern, changes in refractive indices along the chromosome axis as well as the absorption of chromophores in different spectral regions of the chromatin condensation. This complex spectral signature can be analyzed by means of a principal component analysis (PCA) and classified in a multidimensional PCA space.

  5. Ultrasensitive Sensing Material Based on Opal Photonic Crystal for Label-Free Monitoring of Transferrin.

    PubMed

    Wu, Enqi; Peng, Yuan; Zhang, Xihao; Bai, Jialei; Song, Yanqiu; He, Houluo; Fan, Longxing; Qu, Xiaochen; Gao, Zhixian; Liu, Ying; Ning, Baoan

    2017-02-22

    A new opal photonic crystal (PC) sensing material, allowing label-free detection of transferrin (TRF), is proposed in the current study. This photonic crystal was prepared via a vertical convective self-assembly method with monodisperse microspheres polymerized by methyl methacrylate (MMA) and 3-acrylamidophenylboronic acid (AAPBA). FTIR, TG, and DLS were used to characterize the components and particle size of the monodisperse microspheres. SEM was used to observe the morphology of the PC. The diffraction peak intensity decreases as the TRF concentration increase. This was due to the combination of TRF to the boronic acid group of the photonic crystal. After condition optimization, a standard curve was obtained and the linear range of TRF concentration was from 2 × 10(-3) ng/mL to 200 ng/mL. Measurement of TRF concentration in simulated urine sample was also investigated using the sensing material. The results indicated that the PC provided a cheap, label-free, and easy-to-use alternative for TRF determination in clinical diagnostics.

  6. Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins.

    PubMed

    Liebel, Matz; Hugall, James T; van Hulst, Niek F

    2017-02-08

    Label-free detection, analysis, and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biological interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here we present amplified interferometric scattering microscopy (a-iSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equivalent to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. We showcase the performance of our approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple experimental realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.

  7. Optical Microfibre Based Photonic Components and Their Applications in Label-Free Biosensing.

    PubMed

    Wang, Pengfei; Bo, Lin; Semenova, Yuliya; Farrell, Gerald; Brambilla, Gilberto

    2015-07-22

    Optical microfibre photonic components offer a variety of enabling properties, including large evanescent fields, flexibility, configurability, high confinement, robustness and compactness. These unique features have been exploited in a range of applications such as telecommunication, sensing, optical manipulation and high Q resonators. Optical microfibre biosensors, as a class of fibre optic biosensors which rely on small geometries to expose the evanescent field to interact with samples, have been widely investigated. Due to their unique properties, such as fast response, functionalization, strong confinement, configurability, flexibility, compact size, low cost, robustness, ease of miniaturization, large evanescent field and label-free operation, optical microfibres based biosensors seem a promising alternative to traditional immunological methods for biomolecule measurements. Unlabeled DNA and protein targets can be detected by monitoring the changes of various optical transduction mechanisms, such as refractive index, absorption and surface plasmon resonance, since a target molecule is capable of binding to an immobilized optical microfibre. In this review, we critically summarize accomplishments of past optical microfibre label-free biosensors, identify areas for future research and provide a detailed account of the studies conducted to date for biomolecules detection using optical microfibres.

  8. Whispering gallery mode resonators for rapid label-free biosensing in small volume droplets.

    PubMed

    Wildgen, Sarah M; Dunn, Robert C

    2015-03-23

    Rapid biosensing requires fast mass transport of the analyte to the surface of the sensing element. To optimize analysis times, both mass transport in solution and the geometry and size of the sensing element need to be considered. Small dielectric spheres, tens of microns in diameter, can act as label-free biosensors using whispering gallery mode (WGM) resonances. WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators. The spherical geometry and tens of microns diameter of these resonators provides an efficient target for sensing while their compact size enables detection in limited volumes. Here, we explore conditions leading to rapid analyte detection using WGM resonators as label-free sensors in 10 μL sample droplets. Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed. We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.

  9. Nanostructured biochip for label-free and real-time optical detection of polymerase chain reaction.

    PubMed

    Hiep, Ha Minh; Kerman, Kagan; Endo, Tatsuro; Saito, Masato; Tamiya, Eiichi

    2010-02-19

    In this report, Au-coated nanostructured biochip with functionalized thiolated primers on its surface is developed for label-free and real-time optical detection of polymerase chain reaction (PCR). A PCR chamber of 150 microm in thickness containing Au-coated nanostructured substrate in the bottom layer was bordered with SU-8 100 walls. After immobilization of 5'-thiolated primers on the surface, simultaneous DNA amplification and detection were performed without any labeled molecules via the relative reflected intensity (RRI) of Au-coated nanostructured substrate. When human genomic DNA at several concentrations of 0.2, 0.5 and 1 ng microL(-1) was included in the initial DNA samples, the increases in the RRI peak values were clearly observed with the increasing PCR cycle numbers. We found that the starting point of the optical signal, which was divergent from the background in our PCR biochip, was around 3-4 cycles, much lower than that of the fluorescent real-time PCR analysis (around 23-25 cycles). Our proposed PCR device using Au-coated nanostructured substrate holds noteworthy promise for rapid, label-free and real-time DNA detection for point-of-care testing (POCT) applications.

  10. Zwitterionic polymer-modified silicon microring resonators for label-free biosensing in undiluted human plasma.

    PubMed

    Kirk, James T; Brault, Norman D; Baehr-Jones, Tom; Hochberg, Michael; Jiang, Shaoyi; Ratner, Daniel M

    2013-04-15

    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.

  11. Molecularly imprinted photonic hydrogels as colorimetric sensors for rapid and label-free detection of vanillin.

    PubMed

    Peng, Hailong; Wang, Shenqi; Zhang, Zhong; Xiong, Hua; Li, Jinhua; Chen, Lingxin; Li, Yanbin

    2012-02-29

    A novel colorimetric sensor for the rapid and label-free detection of vanillin, based on the combination of photonic crystal and molecular imprinting technique, was developed. The sensing platform of molecularly imprinted photonic hydrogel (MIPH) was prepared by a noncovalent and self-assembly approach using vanillin as a template molecule. Morphology characterization by scanning electron microscope (SEM) showed that the MIPH possessed a highly ordered three-dimensional (3D) macroporous structure with nanocavities. The vanillin recognition events of the created nonocavities could be directly transferred into readable optical signals through a change in Bragg diffraction of the ordered macropores array of MIPH. The Bragg diffraction peak shifted from 451 to 486 nm when the concentration of the vanillin was increased from 10⁻¹² to 10⁻³ mol L⁻¹ within 60 s, whereas there were no obvious peak shifts for methyl and ethyl vanillin, indicating that the MIPH had high selectivity and rapid response for vanillin. The adsorption results showed that the hierarchical porous structure and homogeneous layers were formed in the MIPH with higher adsorption capacity. The application of such a label-free sensor with high selectivity, high sensitivity, high stability, and easy operation might offer a potential method for rapid real-time detection of trace vanillin.

  12. Label-free high-throughput assays to screen and characterize novel lactate dehydrogenase inhibitors.

    PubMed

    Vanderporten, Erica; Frick, Lauren; Turincio, Rebecca; Thana, Peter; Lamarr, William; Liu, Yichin

    2013-10-15

    Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD⁺) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD⁺. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Z' scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction.

  13. A Label-Free Biosensing Platform Using a PLL Circuit and Biotin-Streptavidin Binding System.

    PubMed

    Yunseog Hong; Hee-Jo Lee; Sang-Gyu Kim; Byung-Hyun Kim; Gi-Ho Yun; Jong-Gwan Yook

    2015-06-01

    This paper proposes a novel RF biosensor that utilizes a frequency synthesizer associated with a microstrip open-loop resonator for label-free biomolecular detection. The RF biosensor consists mainly of a resonance-assisted transducer and a phase locked loop (PLL) circuit. In this work, the performance of the RF biosensor is validated using the well-known biotin-streptavidin binding system. When biotin is bound to streptavidin, the input impedance of the resonator is varied, resulting in a change in the oscillation frequency. The concentration of the streptavidin is ultimately detected by a voltage signal of the PLL's loop filter with simple measurement equipment. According to the experimental results, the RF biosensor has revealed excellent sensitivity ( ~ 61 kHz/ngml(-1)) and a low detection limit ( ~ 1 ng/ml), as well as a rapid response. These results demonstrate that the RF biosensor can be an effective sensing platform for label-free detection in a biomolecular binding system.

  14. Label-free methods of reporting biomolecular interactions by optical biosensors.

    PubMed

    Citartan, Marimuthu; Gopinath, Subash C B; Tominaga, Junji; Tang, Thean-Hock

    2013-07-07

    Reporting biomolecular interactions has become part and parcel of many applications of science towards an in-depth understanding of disease and gene regulation. Apart from that, in diagnostic applications where biomolecules (antibodies and aptamers) are vastly applied, meticulous monitoring of biomolecular interaction is vital for clear-cut diagnosis. Several currently available methods of analyzing the interaction of the ligands with the appropriate analytes are aided by labeling using fluorescence or luminescence techniques. However, labeling is cumbersome and can occupy important binding sites of interactive molecules to be labeled, which may interfere with the conformational changes of the molecules and increase non-specificity. Optical-based sensing can provide an alternative way as a label-free procedure for monitoring biomolecular interactions. Optical sensors affiliated with different operating principles, including surface plasmon changes, scattering and interferometry, can impart a huge impact for in-house and point-of-care applications. This optical-based biosensing permits real-time monitoring, obviating the use of hazardous labeling molecules such as radioactive tags. Herein, label-free ways of reporting biomolecular interactions by various optical biosensors were gleaned.

  15. Automated selected reaction monitoring software for accurate label-free protein quantification.

    PubMed

    Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

    2012-07-06

    Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

  16. Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating

    NASA Astrophysics Data System (ADS)

    Yasui, Takao; Ogawa, Kensuke; Kaji, Noritada; Nilsson, Mats; Ajiri, Taiga; Tokeshi, Manabu; Horiike, Yasuhiro; Baba, Yoshinobu

    2016-08-01

    Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.

  17. 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.

  18. Surface plasmon resonance label-free monitoring of antibody antigen interactions in real time

    SciTech Connect

    Kausaite, A.; van Dijk, M.; Castrop, J.; Ramanaviciene, A.; Baltrus, J.P.; Acaite, J.; Ramanavicius, A.

    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 labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label-free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibody rabbit anti-cow albumin IgG1 (anti-BSA). The formation of a self-assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non-covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology.

  19. Label-free fluorescent aptasensor for potassium ion using structure-switching aptamers and berberine

    NASA Astrophysics Data System (ADS)

    Guo, Yanqing; Chen, Yanxia; Wei, Yanli; Li, Huanhuan; Dong, Chuan

    2015-02-01

    A simple, rapid and label-free fluorescent aptasensor was fabricated for the detection of potassium ion (K+ ion) in aqueous solution using K+ ion-stabilized single stranded DNA (ssDNA) with G-rich sequence as the recognition element and a fluorescent dye, berberine, as the fluorescence probe. In the presence of K+ ion, the G-rich ssDNA is promoted to form the aptamer-target complex with a G-quadruplex conformation, and berberine binding to the G-quadruplex structure results in the enhancement of its fluorescence. The fluorescence intensity of the sensing system displayed a calibration response for K+ ion in the range of 0-1600 μM with a detection limit of 31 nM (S/N = 3) and a relative standard deviation (RSD) of 0.45%. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K+ ion in blood serum samples with the recovery range of 81.7-105.3%. The assay for detection of potassium ion is easy, economical, robust, and stable in rough conditions.

  20. Multispot, label-free biodetection at a phantom plastic-water interface.

    PubMed

    Giavazzi, Fabio; Salina, Matteo; Cerbino, Roberto; Bassi, Mattia; Prosperi, Davide; Ceccarello, Erica; Damin, Francesco; Sola, Laura; Rusnati, Marco; Chiari, Marcella; Chini, Bice; Bellini, Tommaso; Buscaglia, Marco

    2013-06-04

    Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, low-cost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.

  1. Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring.

    PubMed

    Tang, Hsin-Yao; Beer, Lynn A; Barnhart, Kurt T; Speicher, David W

    2011-09-02

    Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.

  2. Label-free detection of cardiac troponin I with a photonic crystal biosensor.

    PubMed

    Zhang, Bailin; Morales, Andres W; Peterson, Ralph; Tang, Liang; Ye, Jing Yong

    2014-08-15

    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 0.1ngmL(-1) has been achieved.

  3. Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating

    PubMed Central

    Yasui, Takao; Ogawa, Kensuke; Kaji, Noritada; Nilsson, Mats; Ajiri, Taiga; Tokeshi, Manabu; Horiike, Yasuhiro; Baba, Yoshinobu

    2016-01-01

    Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications. PMID:27531471

  4. Label-free detection of myocardial ischaemia in the perfused rat heart by spontaneous Raman spectroscopy

    PubMed Central

    Ohira, Suguru; Tanaka, Hideo; Harada, Yoshinori; Minamikawa, Takeo; Kumamoto, Yasuaki; Matoba, Satoaki; Yaku, Hitoshi; Takamatsu, Tetsuro

    2017-01-01

    Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the “stopped-flow,” global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm−1, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm−1 enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions. PMID:28186163

  5. A Quantitative Proteomic Workflow for Characterization of Frozen Clinical Biopsies: Laser Capture Microdissection Coupled with Label-Free Mass Spectrometry

    PubMed Central

    Shapiro, John P.; Biswas, Sabyasachi; Merchant, Anand S.; Satoskar, Anjali; Taslim, Cenny; Lin, Shili; Rovin, Brad H.; Sen, Chandan K.; Roy, Sashwati; Freitas, Michael A.

    2013-01-01

    This paper describes a simple, highly efficient and robust proteomic workflow for routine liquid-chromatography tandem mass spectrometry analysis of Laser Microdissection Pressure Catapulting (LMPC) isolates. Highly efficient protein recovery was achieved by optimization of a “one-pot” protein extraction and digestion allowing for reproducible proteomic analysis on as few as 500 LMPC isolated cells. The method was combined with label-free spectral count quantitation to characterize proteomic differences from 3,000–10,000 LMPC isolated cells. Significance analysis of spectral count data was accomplished using the edgeR tag-count R package combined with hierarchical cluster analysis. To illustrate the capability of this robust workflow, two examples are presented: 1) analysis of keratinocytes from human punch biopsies of normal skin and a chronic diabetic wound and 2) comparison of glomeruli from needle biopsies of patients with kidney disease. Differentially expressed proteins were validated by use of immunohistochemistry. These examples illustrate that tissue proteomics carried out on limited clinical material can obtain informative proteomic signatures for disease pathogenesis and demonstrate the suitability of this approach for biomarker discovery. PMID:23022584

  6. A quantitative proteomic workflow for characterization of frozen clinical biopsies: laser capture microdissection coupled with label-free mass spectrometry.

    PubMed

    Shapiro, John P; Biswas, Sabyasachi; Merchant, Anand S; Satoskar, Anjali; Taslim, Cenny; Lin, Shili; Rovin, Brad H; Sen, Chandan K; Roy, Sashwati; Freitas, Michael A

    2012-12-21

    This paper describes a simple, highly efficient and robust proteomic workflow for routine liquid-chromatography tandem mass spectrometry analysis of Laser Microdissection Pressure Catapulting (LMPC) isolates. Highly efficient protein recovery was achieved by optimization of a "one-pot" protein extraction and digestion allowing for reproducible proteomic analysis on as few as 500 LMPC isolated cells. The method was combined with label-free spectral count quantitation to characterize proteomic differences from 3000-10,000 LMPC isolated cells. Significance analysis of spectral count data was accomplished using the edgeR tag-count R package combined with hierarchical cluster analysis. To illustrate the capability of this robust workflow, two examples are presented: 1) analysis of keratinocytes from human punch biopsies of normal skin and a chronic diabetic wound and 2) comparison of glomeruli from needle biopsies of patients with kidney disease. Differentially expressed proteins were validated by use of immunohistochemistry. These examples illustrate that tissue proteomics carried out on limited clinical material can obtain informative proteomic signatures for disease pathogenesis and demonstrate the suitability of this approach for biomarker discovery.

  7. 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

  8. 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.

  9. 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

  10. Label-free quantitative proteomic analysis of pre-flowering PMeV-infected Carica papaya L.

    PubMed

    Soares, Eduardo de A; Werth, Emily G; Madroñero, Leidy J; Ventura, José A; Rodrigues, Silas P; Hicks, Leslie M; Fernandes, Patricia M B

    2017-01-16

    Papaya meleira virus (PMeV) infects papaya (Carica papaya L.) and leads to Papaya Sticky Disease (PSD) or "Meleira", characterized by a spontaneous exudation of latex from fruits and leaves only in the post-flowering developmental stage. The latex oxidizes in contact with air and accumulates as a sticky substance on the plant organs, impairing papaya fruit's marketing and exportation. To understand pre-flowering C. papaya resistance to PMeV, an LC-MS/MS-based label-free proteomics approach was used to assess the differential proteome of PMeV-infected pre-flowering C. papaya vs. uninfected (control) plants. In this study, 1333 proteins were identified, of which 111 proteins showed a significant abundance change (57 increased and 54 decreased) and supports the hypothesis of increased photosynthesis and reduction of 26S-proteassoma activity and cell-wall remodeling. All of these results suggest that increased photosynthetic activity has a positive effect on the induction of plant immunity, whereas the reduction of caspase-like activity and the observed changes in the cell-wall associated proteins impairs the full activation of defense response based on hypersensitive response and viral movement obstruction in pre-flowering C. papaya plants.

  11. A microelectrode-based sensor for label-free in vitro detection of ischemic effects on cardiomyocytes.

    PubMed

    Krinke, Dana; Jahnke, Heinz-Georg; Pänke, Oliver; Robitzki, Andrea A

    2009-05-15

    Heart diseases represent the most common cause of death in industrialised countries. For this reason target identification and development of novel anti-target drugs are in the focus of pharmaceutical industry. Especially cardiac infarct is a topical field of research. A bottleneck in today's long-duration and high-cost drug development is the lack of fast, label-free and cell-based high throughput/high content screening (HTS/HCS) assays for bridging the gap between cell-free screening and animal experiments. Here, we report for the first time on an in vitro cardiac ischemic model, where pathological consequences of simulated cardiac infarct can be detected quantitatively by microelectrode array-based impedance spectroscopy. Using the contractile HL-1 cell line and defined ischemic conditions we were able to develop a standardised and reproducible pathologic model. We characterised and verified the HL-1 based ischemic model by apoptosis and proliferation assays as well as immunochemical analysis of cell-cell junctions. We showed that the observed cell and biomolecular effects correspond with results obtained by impedance spectroscopy. Functionality of the impedimetric assay was demonstrated by real-time detection of reduced pathological effects due to application of the selective Rac1 inhibitor NCS23766. Numerical analysis by means of an equivalent circuit allowed the quantification of changes in resistance and capacitance of the adherent cell layer after ischemic treatment and application of NSC23766 as drug model. Our findings provide a novel cell-based real-time screening system for testing drug candidates against cardiac infarct and its implications.

  12. Motility imaging via optical coherence phase microscopy enables label-free monitoring of tissue growth and viability in 3D tissue-engineering scaffolds.

    PubMed

    Holmes, Christina; Tabrizian, Maryam; Bagnaninchi, Pierre O

    2015-05-01

    As the field of tissue engineering continues to progress, there is a deep need for non-invasive, label-free imaging technologies that can monitor tissue growth and health within thick three-dimensional (3D) constructs. Amongst the many imaging modalities under investigation, optical coherence tomography (OCT) has emerged as a promising tool, enabling non-destructive in situ characterization of scaffolds and engineered tissues. However, the lack of optical contrast between cells and scaffold materials using this technique remains a challenge. In this communication, we show that mapping the optical phase fluctuations resulting from cellular viability and motility allows for the distinction of live cells from their surrounding scaffold environment. Motility imaging was performed via a common-path optical coherence phase microscope (OCPM), an OCT modality that has been shown to be sensitive to nanometer-level fluctuations. More specifically, we examined the development of human adipose-derived stem cells and/or murine pre-osteoblasts within two distinct scaffold systems, commercially available alginate sponges and custom-microfabricated poly(d, l-lactic-co-glycolic acid) fibrous scaffolds. Cellular motility is demonstrated as an endogenous source of contrast for OCPM, enabling real-time, label-free monitoring of 3D engineered tissue development.

  13. Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

    PubMed Central

    Beretov, Julia; Wasinger, Valerie C.; Millar, Ewan K. A.; Schwartz, Peter; Graham, Peter H.; Li, Yong

    2015-01-01

    Introduction Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression. Method We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20). Results Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were reveal