Myelin basic protein immunosensor for multiple sclerosis detection based upon label-free electrochemical impedance spectroscopy.

PubMed

Derkus, Burak; Emregul, Emel; Yucesan, Canan; Cebesoy Emregul, Kaan

2013-08-15

A novel highly sensitive impedimetric Myelin Basic Protein (MBP) immunosensor for the determination of a Multiple Sclerosis (MS) autoantibody, Anti-Myelin Basic Protein (Anti-MBP) was developed by immobilization of MBP on Gelatin and Gelatin-Titanium Dioxide (TiO₂) modified platinium electrode. Cyclic voltammetric (CV) and Electrochemical Impedance Spectroscopic (EIS) methods were employed in determination of the electrode responses and applicability. Gelatin-MBP and gelatin-TiO₂-MBP electrodes were prepared by chemical immobilization of the substrates onto the platinium electrodes. The formal potentials of MBP confined on gelatin-MBP and gelatin-TiO₂-MBP surfaces are estimated to be 195 and 205 mV, respectively. Thus, a little more reversible electron transfer reaction occurs on the gelatin-TiO₂-MBP immunosensor surface. The peak separations of MBP (150 mV and 110 mV s(-1) at 100 mV s(-1)) and the asymmetric anodic and cathodic peak currents indicate that the electron transfer between Anti-MBP and gelatin-MBP/gelatin-TiO₂-MBP immunosensor is quasireversible. Control samples containing a nonspecific human immunoglobulin G (hIgG) antibody were also studied, and calibration curves were obtained by subtraction of the responses for specific and nonspecific antibody-based sensors. Gelatin-MBP and gelatin-TiO₂-MBP immunosensors have detection limit of 0.1528 ng ml(-1) and 0.1495 ng ml(-1) respectively. This immunosensor exhibits high sensitivity and low response times (58 s for gelatin-MBP and 46 s for gelatin-TiO₂-MBP immunosensor). The developed label-free impedimetric immunosensors also provide a simple and sensitive detection method for the specific determination of Anti-MBP in human cerebrospinal fluid (CSF) and serum samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Low-picomolar, label-free procalcitonin analytical detection with an electrolyte-gated organic field-effect transistor based electronic immunosensor.

PubMed

Seshadri, Preethi; Manoli, Kyriaki; Schneiderhan-Marra, Nicole; Anthes, Uwe; Wierzchowiec, Piotr; Bonrad, Klaus; Di Franco, Cinzia; Torsi, Luisa

2018-05-01

Herein a label-free immunosensor based on electrolyte-gated organic field-effect transistor (EGOFET) was developed for the detection of procalcitonin (PCT), a sepsis marker. Antibodies specific to PCT were immobilized on the poly-3-hexylthiophene (P3HT) organic semiconductor surface through direct physical adsorption followed by a post-treatment with bovine serum albumin (BSA) which served as the blocking agent to prevent non-specific adsorption. Antibodies together with BSA (forming the whole biorecognition layer) served to selectively capture the procalcitonin target analyte. The entire immunosensor fabrication process was fast, requiring overall 45min to be completed before analyte sensing. The EGOFET immunosensor showed excellent electrical properties, comparable to those of bare P3HT based EGOFET confirming reliable biosensing with bio-functional EGOFET immunosensor. The detection limit of the immunosensor was as low as 2.2pM and within a range of clinical relevance. The relative standard deviation of the individual calibration data points, measured on immunosensors fabricated on different chips (reproducibility error) was below 7%. The developed immunosensor showed high selectivity to the PCT analyte which was evident through control experiments. This report of PCT detection is first of its kind among the electronic sensors based on EGOFETs. The developed sensor is versatile and compatible with low-cost fabrication techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free immunosensor based on gold nanoparticle silver enhancement.

PubMed

Yang, Minghui; Wang, Cunchang

2009-02-01

A label-free immunosensor for the sensitive detection of human immunoglobulin G (IgG) was prepared based on gold nanoparticle-silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection.

Application of a Label-Free Immunosensor for White Spot Syndrome Virus (WSSV) in Shrimp Cultivation Water.

PubMed

Waiyapoka, Thanyaporn; Deachamag, Panchalika; Chotigeat, Wilaiwan; Bunsanong, Nittaya; Kanatharana, Proespichaya; Thavarungkul, Panote; Loyprasert-Thananimit, Suchera

2015-10-01

White spot syndrome virus (WSSV) is a major pathogen affecting the shrimp industry worldwide. In a preliminary study, WSSV binding protein (WBP) was specifically bound to the VP26 protein of WSSV. Therefore, we have developed the label-free affinity immunosensor using the WBP together with anti-GST-VP26 for quantitative detection of WSSV in shrimp pond water. When the biological molecules were immobilized on a gold electrode to form a self-assembled monolayer, it was then used to detect WSSV using a flow injection system with optimized conditions. Binding between the different copies of WSSV and the immobilized biological molecules was detected by an impedance change (ΔZ″) in real time. The sensitivity of the developed immunosensor was in the linear range of 1.6 × 10(1)-1.6 × 10(6) copies/μl. The system was highly sensitive for the analysis of WSSV as shown by the lack of impedance change when using yellow head virus (YHV). The developed immunosensor could be reused up to 37 times (relative standard deviation (RSD), 3.24 %) with a good reproducibility of residual activity (80-110 %). The immunosensor was simple to operate, reliable, reproducible, and could be applied for the detection and quantification of WSSV in water during shrimp cultivation.

Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria.

PubMed

Barreiros dos Santos, M; Azevedo, S; Agusil, J P; Prieto-Simón, B; Sporer, C; Torrents, E; Juárez, A; Teixeira, V; Samitier, J

2015-02-01

Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm(− 2) (0.08 pmol cm(− 2)). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL(− 1)) over a large linear working range (10–10(6) CFU mL(− 1)). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors.

PubMed

Zhang, Sen; Ma, Hongmin; Yan, Liangguo; Cao, Wei; Yan, Tao; Wei, Qin; Du, Bin

2014-09-15

Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.052 pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01 pg/mL to 100 ng/mL and a lower detection limit of 0.0043 pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields. Copyright © 2014 Elsevier B.V. All rights reserved.

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7.

PubMed

Li, Dujuan; Feng, Yangyang; Zhou, Ling; Ye, Zunzhong; Wang, Jianping; Ying, Yibin; Ruan, Chuanmin; Wang, Ronghui; Li, Yanbin

2011-02-14

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10(2)-10(5) cfu mL(-1) and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor. Copyright © 2010 Elsevier B.V. All rights reserved.

A sensitive electrochemical immunosensor based on poly(2-aminobenzylamine) film modified screen-printed carbon electrode for label-free detection of human immunoglobulin G.

PubMed

Putnin, Thitirat; Jumpathong, Watthanachai; Laocharoensuk, Rawiwan; Jakmunee, Jaroon; Ounnunkad, Kontad

2018-08-01

This work focuses on fabricating poly(2-aminobenzylamine)-modified screen-printed carbon electrode as an electrochemical immunosensor for the label-free detection of human immunoglobulin G. To selectively detect immunoglobulin G, the anti-immunoglobulin G antibody with high affinity to immunoglobulin G was covalently linked with the amine group of poly(2-aminobenzylamine) film-deposited screen-printed carbon electrode. The selectivity for immunoglobulin G was subsequently assured by being challenged with redox-active interferences and adventitious adsorption did not significantly interfere the analyte signal. To obviate the use of costly secondary antibody, the [Fe(CN) 6 ] 4-/3- redox probe was instead applied to measure the number of human immunoglobulin G through the immunocomplex formation that is quantitatively related to the level of the differential pulse voltammetric current. The resulting immunosensor exhibited good sensitivity with the detection limit of 0.15 ng mL -1 , limit of quantitation of 0.50 ng mL -1 and the linear range from 1.0 to 50 ng mL -1 . Given those striking analytical performances and the affordability arising from using cheap screen-printed carbon electrode with label-free detection, the immunosensor serves as a promising model for the next-step development of a diagnostic tool.

Ultrasensitive Label-free Electrochemical Immunosensor based on Multifunctionalized Graphene Nanocomposites for the Detection of Alpha Fetoprotein

PubMed Central

Wang, Yaoguang; Zhang, Yong; Wu, Dan; Ma, Hongmin; Pang, Xuehui; Fan, Dawei; Wei, Qin; Du, Bin

2017-01-01

In this work, a novel label-free electrochemical immunosensor was developed for the quantitative detection of alpha fetoprotein (AFP). Multifunctionalized graphene nanocomposites (TB-Au-Fe3O4-rGO) were applied to modify the electrode to achieve the amplification of electrochemical signal. TB-Au-Fe3O4-rGO includes the advantages of graphene, ferroferric oxide nanoparticles (Fe3O4 NPs), gold nanoparticles (Au NPs) and toluidine blue (TB). As a kind of redox probe, TB can produce the electrochemical signal. Graphene owns large specific surface area, high electrical conductivity and good adsorption property to load a large number of TB. Fe3O4 NPs have good electrocatalytic performance towards the redox of TB. Au NPs have good biocompatibility to capture the antibodies. Due to the good electrochemical performance of TB-Au-Fe3O4-rGO, the effective and sensitive detection of AFP was achieved by the designed electrochemical immunosensor. Under optimal conditions, the designed immunosensor exhibited a wide linear range from 1.0 × 10−5 ng/mL to 10.0 ng/mL with a low detection limit of 2.7 fg/mL for AFP. It also displayed good electrochemical performance including good reproducibility, selectivity and stability, which would provide potential applications in the clinical diagnosis of other tumor markers. PMID:28186128

A Label-Free Porous Silicon Immunosensor for Broad Detection of Opiates in a Blind Clinical Study and Result Comparison to Commercial Analytical Chemistry Techniques

PubMed Central

Bonanno, Lisa M.; Kwong, Tai C.; DeLouise, Lisa A.

2010-01-01

In this work we evaluate for the first time the performance of a label-free porous silicon (PSi) immunosensor assay in a blind clinical study designed to screen authentic patient urine specimens for a broad range of opiates. The PSi opiate immunosensor achieved 96% concordance with liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS) results on samples that underwent standard opiate testing (n=50). In addition, successful detection of a commonly abused opiate, oxycodone, resulted in 100% qualitative agreement between the PSi opiate sensor and LC-MS/MS. In contrast, a commercial broad opiate immunoassay technique (CEDIA®) achieved 65% qualitative concordance with LC-MS/MS. Evaluation of important performance attributes including precision, accuracy, and recovery was completed on blank urine specimens spiked with test analytes. Variability of morphine detection as a model opiate target was < 9% both within-run and between-day at and above the cutoff limit of 300 ng ml−1. This study validates the analytical screening capability of label-free PSi opiate immunosensors in authentic patient samples and is the first semi-quantitative demonstration of the technology’s successful clinical use. These results motivate future development of PSi technology to reduce complexity and cost of diagnostic testing particularly in a point-of-care setting. PMID:21062030

A sensitive label-free immunosensor for detection α-Fetoprotein in whole blood based on anticoagulating magnetic nanoparticles.

PubMed

Xu, Tingting; Chi, Bo; Wu, Fan; Ma, Shangshang; Zhan, Shuyue; Yi, Meihui; Xu, Hong; Mao, Chun

2017-09-15

Accurate values of tumor markers in blood play an especially important role in the diagnosis of illness. Here, based on the combination of three techniques include anticoagulant technology, nanotechnology and biosensing technology, a sensitive label-free immunosensor with anti-biofouling electrode for detection α-Fetoprotein (AFP) in whole blood was developed by anticoagulating magnetic nanoparticles. The obtained products of Fe 3 O 4 -ɛ-PL-Hep nanoparticles were characterized by fourier transform infrared (FT-IR) spectra, transmission electron microscopy (TEM), ζ-potential and vibrating sample magnetometry (VSM). Moreover, the blood compatibility of anticoagulating magnetic nanoparticles was characterized by in vitro coagulation tests, hemolysis assay and whole blood adhesion tests. Combining the anticoagulant property of heparin (Hep) and the good magnetism of Fe 3 O 4 , the Fe 3 O 4 -ɛ-PL-Hep nanoparticles could improve not only the anti-biofouling property of the electrode surface when they contact with whole blood, but also the stability and reproducibility of the proposed immunosensor. Thus, the prepared anticoagulating magnetic nanoparticles modified immunosensor for the detection of AFP showed excellent electrochemical properties with a wide concentration range from 0.1 to 100ng/mL and a low detection limit of 0.072ng/mL. Furthermore, five blood samples were assayed using the developed immunosensor. The results showed satisfactory accuracy with low relative errors. It indicated that our developed immunoassay was competitive and could be potentially used for the detection of whole blood samples directly. Copyright © 2017 Elsevier B.V. All rights reserved.

pH responsive label-assisted click chemistry triggered sensitivity amplification for ultrasensitive electrochemical detection of carbohydrate antigen 24-2.

PubMed

Zheng, Yun; Zhao, Lihua; Ma, Zhanfang

2018-05-15

Sensitivity amplification strategy by implementing click chemistry in the construction of biosensing interface can efficiently improve the performance of immunosensor. Herein, we developed a sandwich-type amperometric immunosensor for ultrasensitive detection of carbohydrate antigen 24-2 (CA 242) based on pH responsive label-assisted click chemistry triggered sensitivity amplification strategy. The sensitivity of amperometric immunosensor relies on the current response differences (ΔI) caused by per unit concentration target analyte. The pH responsive Cu 2+ -loaded polydopamine (CuPDA) particles conjugated with detection antibodies were employed as labels, which can release Cu(II) ions by regulating pH. In the presence of ascorbic acid (reductant), Cu(II) ions were reduced to Cu(I) ions. Azide-functionalized double-stranded DNA (dsDNA) as signal enhancer was immobilized on the substrate through Cu + -catalyzed azide/alkyne cycloaddition reaction. With the help of the click reaction, the ΔI caused by target was elevated prominently, resulting in sensitivity amplification of the immunosensor. Under optimal condition, the proposed immunosensor exhibited excellent performance with linear range from 0.0001 to 100 U mL -1 and ultralow detection limit of 20.74 μU mL -1 . This work successfully combines click chemistry with pH-responsive labels in sandwich-type amperometric immunosensor, providing a promising sensitivity amplification strategy to construct immunosensing platform for analysis of other tumor marker. Copyright © 2018 Elsevier B.V. All rights reserved.

Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip†

PubMed Central

Kim, Jong-Hoon; Hiraiwa, Morgan; Lee, Hyun-Boo; Lee, Kyong-Hoon; Cangelosi, Gerard A.; Chung, Jae-Hyun

2013-01-01

Electric detection using a nanocomponent may lead to platforms for rapid and simple biosensing. Sensors composed of nanotips or nanodots have been described for highly sensitive amperometry enabled by confined geometry. However, both fabrication and use of nanostructured sensors remain challenging. This paper describes a dendritic nanotip used as an amperometric biosensor for highly sensitive detection of target bacteria. A dendritic nanotip is structured by Si nanowires coated with single-walled carbon nanotubes (SWCNTs) for generation of a high electric field. For reliable measurement using the dendritic structure, Si nanowires were uniformly fabricated by ultraviolet (UV) lithography and etching. The dendritic structure effectively increased the electric current density near the terminal end of the nanotip according to numerical computation. The electrical characteristics of a dendritic nanotip with additional protein layers was studied by cyclic voltammetry and I–V measurement in deionized (DI) water. When the target bacteria dielectrophoretically captured onto a nanotip were bound with fluorescence antibodies, the electric current through DI water decreased. Measurement results were consistent with fluorescence- and electron microscopy. The sensitivity of the amperometry was 10 cfu/sample volume (103 cfu/mL), which was equivalent to the more laborious fluorescence measurement method. The simple configuration of a dendritic nanotip can potentially offer an electrolyte-free detection platform for sensitive and rapid biosensors. PMID:23585927

Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip.

PubMed

Kim, Jong-Hoon; Hiraiwa, Morgan; Lee, Hyun-Boo; Lee, Kyong-Hoon; Cangelosi, Gerard A; Chung, Jae-Hyun

2013-01-01

Electric detection using a nanocomponent may lead to platforms for rapid and simple biosensing. Sensors composed of nanotips or nanodots have been described for highly sensitive amperometry enabled by confined geometry. However, both fabrication and use of nanostructured sensors remain challenging. This paper describes a dendritic nanotip used as an amperometric biosensor for highly sensitive detection of target bacteria. A dendritic nanotip is structured by Si nanowires coated with single-walled carbon nanotubes (SWCNTs) for generation of a high electric field. For reliable measurement using the dendritic structure, Si nanowires were uniformly fabricated by ultraviolet (UV) lithography and etching. The dendritic structure effectively increased the electric current density near the terminal end of the nanotip according to numerical computation. The electrical characteristics of a dendritic nanotip with additional protein layers was studied by cyclic voltammetry and I-V measurement in deionized (DI) water. When the target bacteria dielectrophoretically captured onto a nanotip were bound with fluorescence antibodies, the electric current through DI water decreased. Measurement results were consistent with fluorescence- and electron microscopy. The sensitivity of the amperometry was 10 cfu/sample volume (10 3 cfu/mL), which was equivalent to the more laborious fluorescence measurement method. The simple configuration of a dendritic nanotip can potentially offer an electrolyte-free detection platform for sensitive and rapid biosensors.

An Amperometric Immunosensor Based on Multi-Walled Carbon Nanotubes-Thionine-Chitosan Nanocomposite Film for Chlorpyrifos Detection

PubMed Central

Sun, Xia; Cao, Yaoyao; Gong, Zhili; Wang, Xiangyou; Zhang, Yan; Gao, Jinmei

2012-01-01

In this work, a novel amperometric immunosensor based on multi-walled carbon nanotubes-thionine-chitosan (MWCNTs-THI-CHIT) nanocomposite film as electrode modified material was developed for the detection of chlorpyrifos residues. The nanocomposite film was dropped onto a glassy carbon electrode (GCE), and then the anti-chlorpyrifos monoclonal antibody was covalently immobilized onto the surface of MWCNTs-THI-CHIT/GCE using the crosslinking agent glutaraldehyde (GA). The modification procedure was characterized by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimized conditions, a linear relationship between the relative change in peak current of different pulse voltammetry (DPV) and the logarithm of chlorpyrifos solution concentration was obtained in the range from 0.1 to 1.0 × 105 ng/mL with a detection limit of 0.046 ng/mL. The proposed chlorpyrifos immunosensor exhibited high reproducibility, stability, and good selectivity and regeneration, making it a potential alternative tool for ultrasensitive detection of chlorpyrifos residues in vegetables and fruits. PMID:23443396

Label-Free Electrical Immunosensor for Highly Sensitive and Specific Detection of Microcystin-LR in Water Samples.

PubMed

Tan, Feng; Saucedo, Nuvia Maria; Ramnani, Pankaj; Mulchandani, Ashok

2015-08-04

Microcystin-LR (MCLR) is one of the most commonly detected and toxic cyclic heptapeptide cyanotoxins released by cyanobacterial blooms in surface waters, for which sensitive and specific detection methods are necessary to carry out its recognition and quantification. Here, we present a single-walled carbon nanotube (SWCNTs)-based label-free chemiresistive immunosensor for highly sensitive and specific detection of MCLR in different source waters. MCLR was initially immobilized on SWCNTs modified interdigitated electrode, followed by incubation with monoclonal anti-MCLR antibody. The competitive binding of MCLR in sample solutions induced departure of the antibody from the antibody-antigen complexes formed on SWCNTs, resulting in change in the conductivity between source and drain of the sensor. The displacement assay greatly improved the sensitivity of the sensor compared with direct immunoassay on the same device. The immunosensor exhibited a wide linear response to log value of MCLR concentration ranging from 1 to 1000 ng/L, with a detection limit of 0.6 ng/L. This method showed good reproducibility, stability and recovery. The proposed method provides a powerful tool for rapid and sensitive monitoring of MCLR in environmental samples.

New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish.

PubMed

Wu, Haiyun; Ohnuki, Hitoshi; Ota, Shirei; Murata, Masataka; Yoshiura, Yasutoshi; Endo, Hideaki

2017-07-15

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. As a well-known indicator of fish stress, a simple and rapid method for detecting cortisol changes especially sudden increases is desired. In this study, we describe an enzyme-functionalized label-free immunosensor system for detecting fish cortisol levels. Detection of cortisol using amperometry was achieved by immobilizing both anti-cortisol antibody (selective detection of cortisol) and glucose oxidase (signal amplification and non-toxic measurement) on an Au electrode surface with a self-assembled monolayer. This system is based on the maximum glucose oxidation output current change induced by the generation of a non-conductive antigen-antibody complex, which depends on the levels of cortisol in the sample. The immunosensor responded to cortisol levels with a linear decrease in the current in the range of 1.25-200ngml -1 (R=0.964). Since the dynamic range of the sensor can cover the normal range of plasma cortisol in fish, the samples obtained from the fish did not need to be diluted. Further, electrochemical measurement of one sample required only ~30min. The sensor system was applied to determine the cortisol levels in plasma sampled from Nile tilapia (Oreochromis niloticus), which were then compared with levels of the same samples determined using the conventional method (ELISA). Values determined using both methods were well correlated. These findings suggest that the proposed label-free immunosensor could be useful for rapid and convenient analysis of cortisol levels in fish without sample dilution. We also believe that the proposed system could be integrated in a miniaturized potentiostat for point-of-care cortisol detection and useful as a portable diagnostic in fish farms in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

An ultrasensitive label-free electrochemiluminescent immunosensor for measuring Cry1Ab level and genetically modified crops content.

PubMed

Gao, Hongfei; Wen, Luke; Wu, Yuhua; Fu, Zhifeng; Wu, Gang

2017-11-15

The development of genetically modified (GM) insect-resistant crops has aroused great public concern about the risks on the eco-environment resulting from a release of toxic Cry proteins (such as Cry1Ab) to the soil. Therefore, it is of crucial importance to measure the Cry proteins level and the GM crops content. Here, we have tested for the first time a method that uses novel carbon nanospheres (CNPs) label-free electrochemiluminescent (ECL) immunosensor for the ultrasensitive quantification of Cry1Ab and GM crops. In this work, novel CNPs were prepared from printer toner with a very facile approach, and linked with anti-Cry1Ab antibodies to modify a golden working electrode. The immunoreaction between Cry1Ab and its antibody formed an immunocomplex on the bioreceptor region of the sensor, which inhibited electron transfer between the electrode surface and the ECL substance, leading to a decrease of ECL response. Under the optimal conditions, the fabricated label-free ECL immunosensor determined Cry1Ab down to 3.0pgmL -1 within a linear range of 0.010-1.0ngmL -1 , showing significant improvement of sensitivity than that of most previous reports. Meanwhile, the proposed method was successfully applied for GM rice BT63 and GM maize MON810 detections down to 0.010% and 0.020%, respectively. Due to its outstanding advantages such as high sensitivity, ideal selectivity, simple fabrication, rapid detection, and low cost, the developed method can be considered as a powerful and pioneering tool for GM crops detection. Its use can also be extended to other toxin protein sensing in foods. Copyright © 2017. Published by Elsevier B.V.

Label-free Detection of Influenza Viruses using a Reduced Graphene Oxide-based Electrochemical Immunosensor Integrated with a Microfluidic Platform

NASA Astrophysics Data System (ADS)

Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

2017-02-01

Reduced graphene oxide (RGO) has recently gained considerable attention for use in electrochemical biosensing applications due to its outstanding conducting properties and large surface area. This report presents a novel microfluidic chip integrated with an RGO-based electrochemical immunosensor for label-free detection of an influenza virus, H1N1. Three microelectrodes were fabricated on a glass substrate using the photolithographic technique, and the working electrode was functionalized using RGO and monoclonal antibodies specific to the virus. These chips were integrated with polydimethylsiloxane microchannels. Structural and morphological characterizations were performed using X-ray photoelectron spectroscopy and scanning electron microscopy. Electrochemical studies revealed good selectivity and an enhanced detection limit of 0.5 PFU mL-1, where the chronoamperometric current increased linearly with H1N1 virus concentration within the range of 1 to 104 PFU mL-1 (R2 = 0.99). This microfluidic immunosensor can provide a promising platform for effective detection of biomolecules using minute samples.

Amperometric immunosensor based on multiwalled carbon nanotubes/Prussian blue/nanogold-modified electrode for determination of α-fetoprotein.

PubMed

Jiang, Wen; Yuan, Ruo; Chai, Ya-Qin; Yin, Bing

2010-12-01

In this article, a conspicuously simple and highly sensitive amperometric immunosensor based on the sequential electrodeposition of Prussian blue (PB) and gold nanoparticles (GNPs) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrode (GCE) surface is proposed for the detection of α-fetoprotein (AFP). By comparison with PB, the MWCNT/PB composite film had been proven to show much better electrochemical stability and a larger response current. The electrodeposited GNP film can be used not only to immobilize biomolecules but also to avoid the leakage of PB and to prevent shedding of MWCNT/PB composite film from the electrode surface. The performance and factors influencing the performance of the immunosensor were investigated. Under optimal experimental conditions, the proposed immunosensor for AFP was observed with an ultralow limit of detection (LOD) equal to 3 pg/ml (at 3δ), and the linear working range spanned the concentrations of AFP from 0.01 to 300 ng/ml. Moreover, the immunosensor, as well as a commercially available kit, was examined for use in the determination of AFP in real human serum specimens. More significant, the assay mentioned here is simpler than the traditional enzyme-linked immunosorbent assay (ELISA), and an excellent correlation of levels of AFP measured was obtained, indicating that the developed immunoassay could be a promising alternative approach for detection of AFP and other tumor markers in the clinical diagnosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Rapid Quantitative Detection of Brucella melitensis by a Label-Free Impedance Immunosensor Based on a Gold Nanoparticle-Modified Screen-Printed Carbon Electrode

PubMed Central

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

2013-01-01

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 × 104 to 4 × 106 CFU/mL in pure culture. The label-free impedance biosensor was able to detect as low as 1 × 104 and 4 × 105 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. PMID:23881126

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.

A comparative Study of Aptasensor Vs Immunosensor for Label-Free PSA Cancer Detection on GQDs-AuNRs Modified Screen-Printed Electrodes.

PubMed

Srivastava, Monika; Nirala, Narsingh R; Srivastava, S K; Prakash, Rajiv

2018-01-31

Label-free and sensitive detection of PSA (Prostate Specific Antigen) is still a big challenge in the arena of prostate cancer diagnosis in males. We present a comparative study for label-free PSA aptasensor and PSA immunosensor for the PSA-specific monoclonal antibody, based on graphene quantum dots-gold nanorods (GQDs-AuNRs) modified screen-printed electrodes. GQDs-AuNRs composite has been synthesized and used as an electro-active material, which shows fast electron transfer and catalytic property. Aptamer or anti-PSA has immobilized onto the surface of modified screen printed electrodes. Three techniques are used simultaneously, viz. cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedence spectroscopy (EIS) to investigate the analytical performance of both PSA aptasensor and PSA immunosensor with its corresponding PSA antigen. Under optimum conditions, both sensors show comparable results with an almost same limit of detection (LOD) of 0.14 ng mL -1 . The results developed with aptasensor and anti-PSA is also checked through the detection of PSA in real samples with acceptable results. Our study suggests some advantages of aptasensor in terms of better stability, simplicity and cost effectiveness. Further our present work shows enormous potential of our developed sensors for real application using voltammetric and EIS techniques simultaneous to get reliable detection of the disease.

Label-free electrical detection using carbon nanotube-based biosensors.

PubMed

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.

Amperometric immunosensor for α-fetoprotein antigen in human serum based on co-immobilizing dinuclear copper complex and gold nanoparticle doped chitosan film

NASA Astrophysics Data System (ADS)

Gan, Ning; Meng, Ling Hua; Wang, Feng

2009-09-01

A sensitive amperometric immunosensor for α-fetoprotein (AFP), a tumor marker for the diagnosis of hepatocellular carcinoma (HCC), was constructed, The immunosensor is prepared by co-immobilizing [Cu2(phen)2Cl2] (μ-Cl)2 (CuL), nano-Au/Chitosan(Chit) composite, horseradish peroxidase (HRP) and AFP antibody(anti-AFP) on a glassy carbon electrode (GCE). Firstly, CuL was irreversibly absorb on GCE electrode through π-π stacking interaction; then nano-Au/Chit composite was immobilized onto the electrode because of its excellent membrane-forming ability, finally HRP and anti-AFP was adsorbed onto the surface of the gold nanoparticles to construct GCE | CuL/nanoAu-chit/HRP/anti-AFP immunosensor. The preparation procedure of the electrode was characterized by electrochemical and spectroscopy method. The results showed that this immunosensor exhibited an excellent electrocatalytic response to the reduction of hydrogen peroxide (H2O2) without the aid of an electron mediator, offers a high-sensitivity (1710 nA · ng-1 · ml-1) for the detection of AFP and has good correlation for detection of AFP in the range of 0.2 to 120.0 ng/ml with a detection limit of 0.05 ng/ml. The biosensor showed high selectivity as well as good stability and reproductivity.

Label-free as-grown double wall carbon nanotubes bundles for Salmonella typhimurium immunoassay.

PubMed

Punbusayakul, Niramol; Talapatra, Saikat; Ajayan, Pulickel M; Surareungchai, Werasak

2013-01-01

A label-free immunosensor from as-grown double wall carbon nanotubes (DW) bundles was developed for detecting Salmonella typhimurium. The immunosensor was fabricated by using the as-grown DW bundles as an electrode material with an anti-Salmonella impregnated on the surface. The immunosensor was electrochemically characterized by cyclic voltammetry. The working potential (100, 200, 300 and 400 mV vs. Ag/AgCl) and the anti-Salmonella concentration (10, 25, 50, 75, and 100 μg/mL) at the electrode were subsequently optimized. Then, chronoamperometry was used with the optimum potential of 100 mV vs. Ag/AgCl) and the optimum impregnated anti-Salmonella of 10 μg/mL to detect S. typhimurium cells (0-10(9) CFU/mL). The DW immunosensor exhibited a detection range of 10(2) to 10(7) CFU/mL for the bacteria with a limit of detection of 8.9 CFU/mL according to the IUPAC recommendation. The electrode also showed specificity to S. typhimurium but no current response to Escherichia coli. These findings suggest that the use of a label-free DW immunosensor is promising for detecting S. typhimurium.

An electrochemical immunosensor for cardiac Troponin I using electrospun carboxylated multi-walled carbon nanotube-whiskered nanofibres.

PubMed

Rezaei, Babak; Shoushtari, Ahmad Mousavi; Rabiee, Mohammad; Uzun, Lokman; Mak, Wing Cheung; Turner, Anthony P F

2018-05-15

A sandwich-type nanostructured immunosensor based on carboxylated multi-walled carbon nanotube (CMWCNT)-embedded whiskered nanofibres (WNFs) was developed for detection of cardiac Troponin I (cTnI). WNFs were directly fabricated on glassy carbon electrodes (GCE) by removing the sacrificial component (polyethylene glycol, PEG) after electrospinning of polystyrene/CMWCNT/PEG nanocomposite nanofibres, and utilised as a transducer layer for enzyme-labeled amperometric immunoassay of cTnI. The whiskered segments of CMWCNTs were activated and utilised to immobilise anti-cTnT antibodies. It was observed that the anchored CMWCNTs within the nanofibres were suitably stabilised with excellent electrochemical repeatability. A sandwich-type immuno-complex was formed between cTnI and horseradish peroxidase-conjugated anti-cTnI (HRP-anti-cTnI). The amperometric responses of the immunosensor were studied using cyclic voltammetry (CV) through an enzymatic reaction between hydrogen peroxide and HRP conjugated to the secondary antibody. The nanostructured immunosensor delivered a wide detection range for cTnI from the clinical borderline for a normal person (0.5-2ngmL -1 ) to the concentration present in myocardial infarction patients (> 20ngmL -1 ), with a detection limit of ~ 0.04ngmL -1 . It also showed good reproducibility and repeatability for three different cTnI concentration (1, 10 and 25ngmL -1 ) with satisfactory relative standard deviations (RSD). Hence, the proposed nanostructured immunosensor shows potential for point-of-care testing. Copyright © 2018 Elsevier B.V. All rights reserved.

An electrochemical immunosensor for brain natriuretic peptide prepared with screen-printed carbon electrodes nanostructured with gold nanoparticles grafted through aryl diazonium salt chemistry.

PubMed

Serafín, V; Torrente-Rodríguez, R M; González-Cortés, A; García de Frutos, P; Sabaté, M; Campuzano, S; Yáñez-Sedeño, P; Pingarrón, J M

2018-03-01

A sensitive amperometric immunosensor has been prepared by immobilization of capture antibodies onto gold nanoparticles (AuNPs) grafted on a screen-printed carbon electrode (SPCE) through aryl diazonium salt chemistry using 4-aminothiophenol (AuNPs-S-Phe-SPCE). The immunosensor was designed for the accurate determination of clinically relevant levels of B-type natriuretic peptide (BNP) in human serum samples. The nanostructured electrochemical platform resulted in an ordered layer of AuNPs onto SPCEs which combined the advantages of high conductivity and improved stability of immobilized biomolecules. The resulting disposable immunosensor used a sandwich type immunoassay involving a peroxidase-labeled detector antibody. The amperometric transduction was carried out at -0.20V (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H 2 O 2 as the enzyme substrate. The nanostructured immunosensors show a storage stability of at least 25 days, a linear range between 0.014 and 15ngmL -1 , and a LOD of 4pgmL -1 , which is 100 times lower than the established cut-off value for heart failure (HF) diagnosis. The performance of the immunosensor is advantageously compared with that provided with immunosensors prepared by grafting SPCE with p-phenylendiamine (H 2 N-Phe-SPCE) and attaching AuNPs by immersion into an AuNPs suspension or by electrochemical deposition, as well as with immunosensors constructed using commercial AuNPs-modified SPCEs. The developed immunosensor was applied to the successful analysis of human serum from heart failure (HF) patients upon just a 10-times dilution as sample treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

A sensitive label-free electrochemical immunosensor for detection of cytokeratin 19 fragment antigen 21-1 based on 3D graphene with gold nanopaticle modified electrode.

PubMed

Zeng, Yan; Bao, Jing; Zhao, Yanan; Huo, Danqun; Chen, Mei; Yang, Mei; Fa, Huanbao; Hou, Changjun

2018-02-01

Previous studies have confirmed that cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) serves as a powerful biomarker in non-small cell lung cancer (NSCLC). Herein, we report for the first time a label-free electrochemical immunosensor for sensitive and selective detection of tumor marker CYFRA21-1. In this work, three-dimensional graphene @ gold nanoparticles (3D-G@Au) nanocomposite was modified on the glassy carbon electrode (GCE) surface to enhance the conductivity of immunosensor. The anti-CYFRA21-1 captured and fixed on the modified GCE through the cross-linking of chitosan (CS), glutaraldehyde (GA) and anti-CYFRA21-1. The differential pulse voltammetry (DPV) peak current change due to the specific interaction between anti-CYFRA21-1 and CYFRA21-1 on the modified electrode surface was utilized to detect CYFRA21-1. Under optimized conditions, the proposed electrochemical immunosensor was employed to detect CYFRA21-1 and exhibited a wide linear range of 0.25-800ngmL -1 and low detection limit of 100pgmL -1 (S/N = 3). Moreover, the recovery rates of serum samples were in the range from 95.2% to 108.7% and the developed immunosensor also shows a good correlation (less than 6.6%) with enzyme-linked immunosorbent assay (ELISA) in the detection of clinical serum samples. Therefore, it is expected that the proposed immunosensor based on a 3D-G@Au has great potential in clinical medical diagnosis of CYFRA21-1. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of an enrofloxacin immunosensor based on label-free electrochemical impedance spectroscopy.

PubMed

Wu, Ching-Chou; Lin, Chia-Hung; Wang, Way-Shyan

2009-06-30

Enrofloxacin is the most widespread antibiotic in the fluoroquinolone family. As such, the development of a rapid and sensitive method for the determination of trace amounts of enrofloxacin is an important issue in the health field. The interaction of the enrofloxacin antigen to a specific antibody (Ab) immobilized on an 11-mercapto-undecanoic acid-coated gold electrode was quantified by electrochemical impedance spectroscopy. Two equivalent circuits were separately used to interpret the obtained impedance spectra. These circuits included one resistor in series with one parallel circuit comprised of a resistor and a capacitor (1R//C), and one resistor in series with two parallel RC circuits (2R//C). The results indicate that the antigen-antibody reaction analyzed using the 1R//C circuit provided a more sensitive resistance increment against the enrofloxacin concentration than that of the 2R//C circuit. However, the 2R//C circuit provided a better fitting for impedance spectra, and therefore supplies more detailed results of the enrofloxacin-antibody interaction, causing the increase of electron transfer resistance selectively to the modified layer, and not the electrical double layer. The antibody-modified electrode allowed for analysis of the dynamic linear range of 1-1000 ng/ml enrofloxacin with a detection limit of 1 ng/ml. The reagentless and label-free impedimetric immunosensors provide a simple and sensitive detection method for the specific determination of enrofloxacin.

A label-free electrochemical impedance immunosensor based on AuNPs/PAMAM-MWCNT-Chi nanocomposite modified glassy carbon electrode for detection of Salmonella typhimurium in milk.

PubMed

Dong, Jing; Zhao, Han; Xu, Minrong; Ma, Qiang; Ai, Shiyun

2013-12-01

A sensitive and stable label-free electrochemical impedance immunosensor for the detection of Salmonella typhimurium was developed by immobilising anti-Salmonella antibodies onto the gold nanoparticles and poly(amidoamine)-multiwalled carbon nanotubes-chitosan nanocomposite film modified glassy carbon electrode (AuNPs/PAMAM-MWCNT-Chi/GCE). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to verify the stepwise assembly of the immunosensor. Co-addition of MWCNT, PAMAM and AuNPs greatly enhanced the sensitivity of the immunosensor. The immobilisation of antibodies and the binding of Salmonella cells to the modified electrode increased the electron-transfer resistance (Ret), which was directly measured with EIS using [Fe(CN)6](3-/4-) as a redox probe. A linear relationship of Ret and Salmonella concentration was obtained in the Salmonella concentration range of 1.0×10(3) to 1.0×10(7) CFU mL(-1) with a detection limit of 5.0×10(2) CFU mL(-1). Additionally, the proposed method was successfully applied to determine S. typhimurium content in milk samples with satisfactory results. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ultrasensitive electrochemical immunosensors for multiplexed determination using mesoporous platinum nanoparticles as nonenzymatic labels.

PubMed

Cui, Zhentao; Wu, Dan; Zhang, Yong; Ma, Hongmin; Li, He; Du, Bin; Wei, Qin; Ju, Huangxian

2014-01-07

An ultrasensitive multiplexed immunoassay method was developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. M-Pt NPs were prepared by ultrasonic method and employed to label the secondary antibody (Ab2) for signal amplification. The immunosensor array was constructed by covalently immobilizing capture antibody (Ab1) on graphene modified screen printed carbon electrodes (SPECs). After the sandwich-type immunoreactions, the M-Pt-Ab2 was bound to immunosensor surface to catalyze the electro-reduction of H2O2 reaction, which produced detectable signals for readout of analytes. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this method showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002 U mL(-1), 0.001 U mL(-1) and 7.0 pg mL(-1) for CA125, CA153 and CEA, respectively. The disposable immunosensor array possessed excellent clinical value in cancer screening as well as convenient point of care diagnostics. Copyright © 2013 Elsevier B.V. All rights reserved.

A cytokine immunosensor for Multiple Sclerosis detection based upon label-free electrochemical impedance spectroscopy using electroplated printed circuit board electrodes.

PubMed

Bhavsar, Kinjal; Fairchild, Aaron; Alonas, Eric; Bishop, Daniel K; La Belle, Jeffrey T; Sweeney, James; Alford, T L; Joshi, Lokesh

2009-10-15

A biosensor for the serum cytokine, Interleukin-12 (IL-12), based upon a label-free electrochemical impedance spectroscopy (EIS) monitoring approach is described. Overexpression of IL-12 has been correlated to the diagnosis of Multiple Sclerosis (MS). An immunosensor has been fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode and immobilizing anti-IL-12 monoclonal antibodies (MAb) onto the surface of the electrode. This approach yields a robust sensor that facilitates reproducible mass fabrication and easy alteration of the electrode shape. Results indicate that this novel PCB sensor can detect IL-12 at physiological levels, <100 fM with f-values of 0.05 (typically <0.0001) in a label-free and rapid manner. A linear (with respect to log concentration) detectable range was achieved. Detection in a complex biological solution is also explored; however, significant loss of dynamic range is noted in the 100% complex solution. The cost effective approach described here can be used potentially for diagnosis of diseases (like MS) with known biomarkers in body fluids and for monitoring physiological levels of biomolecules with healthcare, food, and environmental relevance.

Interferometer immunosensor based on porous silicon for determining alpha-fetoprotein

NASA Astrophysics Data System (ADS)

Lv, Xiaoyi; Jiang, Jing; Lv, Guodong; Mo, Jiaqing; Jia, Zhenhong

2016-10-01

An increased level of alpha-fetoprotein ( AFP) in the blood may be a sign of liver cancer. Porous silicon based optical microcavities structure is prepared as a label-free immunosensor platform for detecting AFP. After the antigen-antibody reaction, it is monitored that the red shift of the reflection spectrum of the immunosensor increases

A membrane-based immunosensor for the analysis of the herbicide isoproturon.

PubMed

Baskeyfield, Damian E H; Davis, Frank; Magan, Naresh; Tothill, Ibtisam E

2011-08-12

A membrane based heterogeneous competitive enzyme-linked immunosorbent assay (ELISA) was used in this work to develop an immunosensor for the detection of a common herbicide, isoproturon. A screen-printed carbon working electrode with carbon counter and silver-silver chloride pseudo-reference electrode was utilized incorporating a membrane fixed into intimate contact with the working electrode to facilitate signal transduction. The membrane containing an immobilized isoproturon-ovalbumin conjugate was laminated onto the carbon working electrode and horseradish peroxidase (HRP) labeled polyclonal antibody was then applied for the competitive assay. Two different amperometric systems, hydroquinone and o-phenylenediamine (OPD) mediation reduction were utilised and the properties of the resultant sensors were compared. A flow injection apparatus was also developed utilising the immunosensor. Limits of detection for isoproturon (LLD(90)) were found to be as low as 0.84 ng mL(-1). The senor was also validated using spiked extracted soil samples and also isoproturon contaminated samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Disposable electrochemical immunosensor for Brettanomyces bruxellensis based on nanogold-reduced graphene oxide hybrid nanomaterial.

PubMed

Borisova, Boryana; Villalonga, María L; Arévalo-Villena, María; Boujakhrout, Abderrahmane; Sánchez, Alfredo; Parrado, Concepción; Pingarrón, José M; Briones-Pérez, Ana; Villalonga, Reynaldo

2017-09-01

The assembly of a novel disposable amperometric immunosensor for the detection of the red wine spoilage yeast Brettanomyces bruxellensis is reported. The nanostructured sensing interface was prepared by first coating carbon screen printed electrodes with a gold nanoparticles-reduced graphene oxide hybrid nanomaterial, which was then modified with 3-mercaptopropionic acid to further immobilize specific antibodies for B. bruxellensis via a carbodiimide-coupling reaction. The functionalized electrode allowed the amperometric detection of B. bruxellensis in buffered solutions and red wine samples in the range of 10-10 6  CFU/mL and 10 2 -10 6  CFU/mL, with low detection limits of 8 CFU/mL and 56 CFU/mL, respectively. The electrochemical immunosensor also exhibited high reproducibility, selectivity, and storage stability. Graphical abstract A novel disposable electrochemical immunosensor for the detection of the red wine spoilage yeast B. bruxellensis.

Sensitive electrochemical immunosensor for α-fetoprotein based on graphene/SnO2/Au nanocomposite.

PubMed

Liu, Junfeng; Lin, Guanhua; Xiao, Can; Xue, Ying; Yang, Ankang; Ren, Hongxuan; Lu, Wensheng; Zhao, Hong; Li, Xiangjun; Yuan, Zhuobin

2015-09-15

A label-free electrochemical immunosensor for sensitive detection of α-fetoprotein (AFP) was developed based on graphene/SnO2/Au nanocomposite. The graphene/SnO2/Au nanocomposite modified glassy carbon electrode was used to immobilize α-fetoprotein antibody (anti-AFP) and to construct the immunosensor. Results demonstrated that the peak currents of [Ru(NH3)6](3+) decreased due to the interaction between antibody and antigen on the modified electrode. Thus, a label-free immunosensor for the detection of AFP was realized by monitoring the peak current change of [Ru(NH3)6](3+). The factors influencing the performance of the immunosensor were investigated in details. Under optimal conditions, the peak currents obtained by DPV decreased linearly with the increasing AFP concentrations in the range from 0.02 to 50 ng mL(-1) with a linear coefficient of 0.9959. This electrochemical immunoassay has a low detection limit of 0.01 ng mL(-1) (S/N=3) and was successfully applied to the determination of AFP in serum samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Portable amperometric immunosensor for histamine detection using Prussian blue-chitosan-gold nanoparticle nanocomposite films.

PubMed

Dong, Xiu-Xiu; Yang, Jin-Yi; Luo, Lin; Zhang, Yi-Feng; Mao, Chuanbin; Sun, Yuan-Ming; Lei, Hong-Tao; Shen, Yu-Dong; Beier, Ross C; Xu, Zhen-Lin

2017-12-15

Histamine (HA) is a biogenic amine that can accumulate to high concentration levels in food as a result of microbial activity and can cause toxic effects in consumers. In this work, a portable electrochemical immunosensor capable of detecting HA with high sensitivity and selectivity was developed. Prussian blue-chitosan-gold nanoparticle (PB-CS-AuNP) nanocomposite films with excellent biocompatibility were synthesized and characterized by scanning electron microscopy and energy dispersive X-ray analysis. The PB-CS-AuNP were coated onto a screen-printed electrode by one-step electrodeposition and used to conjugate the HA ovalbumin conjugate (HA-Ag). HA was determined by a competition between the coating HA-Ag and the HRP labeled HA antibody (HRP-HA-Ab). After careful optimization of assay conditions and Box-Behnken analysis, the developed immunosensor showed a linear range from 0.01 to 100μg/mL for HA in fish samples. The average recoveries from spiked samples ranged from 97.25% to 105%. The biosensor also showed good specificity, reproducibility, and stability, indicating its potential application in monitoring HA in a simple and low cost manner. Copyright © 2017 Elsevier B.V. All rights reserved.

Recent advances in immunosensor for narcotic drug detection

PubMed Central

Gandhi, Sonu; Suman, Pankaj; Kumar, Ashok; Sharma, Prince; Capalash, Neena; Suri, C. Raman

2015-01-01

Introduction: Immunosensor for illicit drugs have gained immense interest and have found several applications for drug abuse monitoring. This technology has offered a low cost detection of narcotics; thereby, providing a confirmatory platform to compliment the existing analytical methods. Methods: In this minireview, we define the basic concept of transducer for immunosensor development that utilizes antibodies and low molecular mass hapten (opiate) molecules. Results: This article emphasizes on recent advances in immunoanalytical techniques for monitoring of opiate drugs. Our results demonstrate that high quality antibodies can be used for immunosensor development against target analyte with greater sensitivity, specificity and precision than other available analytical methods. Conclusion: In this review we highlight the fundamentals of different transducer technologies and its applications for immunosensor development currently being developed in our laboratory using rapid screening via immunochromatographic kit, label free optical detection via enzyme, fluorescence, gold nanoparticles and carbon nanotubes based immunosensing for sensitive and specific monitoring of opiates. PMID:26929925

Glucose-oxidase label-based redox cycling for an incubation period-free electrochemical immunosensor.

PubMed

Singh, Amardeep; Park, Seonhwa; Yang, Haesik

2013-05-21

Catalytic reactions of enzyme labels in enzyme-linked immunosorbent assays require a long incubation period to obtain high signal amplification. We present herein a simple immunosensing scheme in which the incubation period is minimized without a large increase in the detection limit. This scheme is based on electrochemical-enzymatic (EN) redox cycling using glucose oxidase (GOx) as an enzyme label, Ru(NH3)6(3+) as a redox mediator, and glucose as an enzyme substrate. Fast electron mediation of Ru(NH3)6(3+) between the electrode and the GOx label attached to the electrode allows high signal amplification. The acquisition of chronocoulometric charges at a potential in the mass transfer-controlled region excludes the influence of the kinetics of Ru(NH3)6(2+) electrooxidation and also facilitates high signal-to-background ratios. The reaction between reduced GOx and Ru(NH3)6(3+) is rapid even in air-saturated Tris buffer, where the faster competitive reaction between reduced GOx and dissolved oxygen also occurs. The direct electrooxidation of glucose at the electrode and the direct electron transfer between glucose and Ru(NH3)6(3+) that undesirably increase background levels occur relatively slowly. The detection limit for the EN redox cycling-based detection of cancer antigen 125 (CA-125) in human serum is slightly higher than 0.1 U/mL for the incubation period of 0 min, and the detection limits for the incubation periods of 5 and 10 min are slightly lower than 0.1 U/mL, indicating that the detection limits are almost similar irrespective of the incubation period and that the immunosensor is highly sensitive.

A novel electrochemical immunosensor based on Au nanoparticles and horseradish peroxidase signal amplification for ultrasensitive detection of α-fetoprotein.

PubMed

Lu, Dingqiang; Xu, Qiuda; Pang, Guangchang; Lu, Fuping

2018-06-05

An electrochemical double-layer Au nanoparticle membrane immunosensor was developed using an electrochemical biosensing signal amplification system with Au nanoparticles, thionine, chitosan, and horseradish peroxidase, which was fabricated using double self-adsorption of Au nanoparticle sol followed by anti-α-fetoprotein Balb/c mouse monoclonal antibody adsorption. The AuNPs sol was characterized by spectrum scanning and transmission electron microscopy. The immunosensor was characterized by atomic force microscopy, cyclic voltammetry, and alternating-current impedance during each stage of adsorption and assembly. The amperometric I-t curve method was used to measure α-fetoprotein (AFP) diluted in phosphate buffered saline. The result indicated a wide linear range, and the change rate of steady-current before and after immune response had linear correlation within the range 0.1-10 4  pg/mL AFP. The current change rate equation was △I = 5.82334 lgC + 37.01195 (R 2  = 0.9922). The lowest limit of detection was 0.03 pg/mL (S/N = 3), and the reproducibility of the sensor was good. Additionally, the sensor could be stably stored above phosphate buffered saline at 4 °C for more than 24 days. More importantly, the sensor is label-free, reagentless and low fouling, making it capable of assaying AFP in real serum samples without suffering from significant interference or biofouling.

Amperometric immunoassay for the obesity biomarker amylin using a screen printed carbon electrode functionalized with an electropolymerized carboxylated polypyrrole.

PubMed

Martínez-García, Gonzalo; Sánchez-Tirado, Esther; González-Cortés, Araceli; Yáñez-Sedeño, Paloma; Pingarrón, José M

2018-06-09

Amylin (the islet amyloid polypeptide) is a hormone related to adiposity, hunger and satiety. It is co-secreted with insulin from pancreatic B-cells. An amperometric immunosensor is presented here for the determination of amylin. It is making use of a screen printed carbon electrode (SPCE) functionalized with electropolymerized poly(pyrrole propionic acid) (pPPA) with abundant carboxyl groups that facilitate covalent binding of antibody against amylin. A competitive immunoassay was implemented using biotinylated amylin and streptavidin labeled with horse radish peroxidase (HRP-Strept) as the enzymatic tracer. The amperometric detection of H 2 O 2 mediated by hydroquinone was employed as an electrochemical probe to monitor the affinity reaction. The variables involved in the preparation and function of the immunosensor were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The calibration graph for amylin, obtained by amperometry at -200 mV vs Ag pseudo-reference electrode, showed a range of linearity extending from 1.0 fg∙mL -1 to 50 pg∙mL -1 , with a detection limit of 0.92 fg∙mL -1 . This is approximately 7000 times lower than the minimum detectable concentration reported for the ELISA immunoassays available for amylin. The assay has excellent reproducibility and good selectivity over potential interferents. Graphical abstract Schematic of an amperometric competitive immunoassay for the obesity biomarker amylin using a poly(pyrrole propionic acid)-modified screen-printed electrode. The detection limit is 0.92 fg∙mL-1 amylin. The method provides excellent reproducibility for the measurements, good selectivity and successful applicability to human urine and serum samples.

Solid-state voltammetry-based electrochemical immunosensor for Escherichia coli using graphene oxide-Ag nanoparticle composites as labels.

PubMed

Jiang, Xiaochun; Chen, Kun; Wang, Jing; Shao, Kang; Fu, Tao; Shao, Feng; Lu, Donglian; Liang, Jiangong; Foda, M Frahat; Han, Heyou

2013-06-21

A new electrochemical immunosensor based on solid-state voltammetry was fabricated for the detection of Escherichia coli (E. coli) by using graphene oxide-Ag nanoparticle composites (P-GO-Ag) as labels. To construct the platform, Au nanoparticles (AuNPs) were first self-assembled on an Au electrode surface through cysteamine and served as an effective matrix for antibody (Ab) attachment. Under a sandwich-type immunoassay format, the analyte and the probe (P-GO-Ag-Ab) were successively captured onto the immunosensor. Finally, the bonded AgNPs were detected through a solid-state redox process in 0.2 M of KCl solution. Combining the advantages of the high-loading capability of graphene oxide with promoted electron-transfer rate of AuNPs, this immunosensor produced a 26.92-fold signal enhancement compared with the unamplified protocol. Under the optimal conditions, the immunosensor exhibited a wide linear dependence on the logarithm of the concentration of E. coli ranging from 50 to 1.0 × 10(6) cfu mL(-1) with a detection limit of 10 cfu mL(-1). Moreover, as a practical application, the proposed immunosensor was used to monitor E. coli in lake water with satisfactory results.

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

2015-05-01

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

[Fabrication and evaluation of the enzyme immunosensor for rapid detection of Vibrio parahaemolyticus based on chitosan-SiO2 hybrid membrane].

PubMed

Zhao, Guang-Ying; Ma, Chao; Li, Jian-Rong

2010-01-01

To improve the key technology of immunesensors in immobilizing bio-sensitive element and keeping its bioactivity, an enzyme immunosensor based on chitosan-SiO(2) (CS-Sio(2)) hybrid membrane was fabricated. To estimate the new immunosensor Vibrio parahaemolyticus which was the main pathogens of aquatic products. A CS-SiO(2) hybrid membrane was prepared using sol-gel method. The enzyme immunosensor was fabricated by coating the membrane and horseradish peroxidase labeled Vibrio parahaemolyticus antibody (HRP-anti-VP) on the surface of four-channel screen-printed carbon electrode. The immunosensor was characterized by cyclic voltammetry. Vibrio parahaemolyticus could be detected according to the decrease percentage (DP) of peak current before and after immune response, while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction. Seven kinds of bacteria, like Vibrio alginolyticus, were selected for specific experiments. By studying the infrared spectrum of three kinds of films, the CS-SiO(2) hybrid membrane was prepared and HRP-anti-VP was fixed in the hybrid membrane. Under the optimum conditions of immunoreaction and electrochemical detection, the DP of peak current before and after immune response showed a linear relation with lgC in the range of 10(4) - 10(9) cfu/ml, while the linear regression equation was: DP = 6.5 lgC-3.319, the correlation coefficient was 0.9958 and the detection limit was 6.9 x 10(3) cfu/ml (S/N = 3). The immunosensor possessed acceptable specificity, reproducibility (RSD < 6%), stability (the amperometric response was 95% of the initial response after a week) and accuracy (96.7% of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789.7-2003). The enzyme immunosensor based on CS-SiO(2) hybrid membrane gave a good performance in rapid detection of Vibrio parahaemolyticus.

Simple and suitable immunosensor for β-lactam antibiotics analysis in real matrixes: milk, serum, urine.

PubMed

Merola, Giovanni; Martini, Elisabetta; Tomassetti, Mauro; Campanella, Luigi

2015-03-15

The anti-penicillin G was conjugated to avidin-peroxidase and biotin to obtain immunogen and competitor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other β-lactam antibiotics, with Kaff values of the order of 10(8) M(-1). The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of "in situ" analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumental methods reported in the literature and used for the analysis of β-lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amperometric electrode for hydrogen peroxide as transducer, the BSA-penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10(-11)M. Lastly, the new immunosensor displayed low selectivity versus the entire class of β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics. Copyright © 2014 Elsevier B.V. All rights reserved.

Carbon nanotubes-based chemiresistive immunosensor for small molecules: detection of nitroaromatic explosives.

PubMed

Park, Miso; Cella, Lakshmi N; Chen, Wilfred; Myung, Nosang V; Mulchandani, Ashok

2010-12-15

In recent years, there has been a growing focus on use of one-dimensional (1-D) nanostructures, such as carbon nanotubes and nanowires, as transducer elements for label-free chemiresistive/field-effect transistor biosensors as they provide label-free and high sensitivity detection. While research to-date has elucidated the power of carbon nanotubes- and other 1-D nanostructure-based field effect transistors immunosensors for large charged macromolecules such as proteins and viruses, their application to small uncharged or charged molecules has not been demonstrated. In this paper we report a single-walled carbon nanotubes (SWNTs)-based chemiresistive immunosensor for label-free, rapid, sensitive and selective detection of 2,4,6-trinitrotoluene (TNT), a small molecule. The newly developed immunosensor employed a displacement mode/format in which SWNTs network forming conduction channel of the sensor was first modified with trinitrophenyl (TNP), an analog of TNT, and then ligated with the anti-TNP single chain antibody. Upon exposure to TNT or its derivatives the bound antibodies were displaced producing a large change, several folds higher than the noise, in the resistance/conductance of SWNTs giving excellent limit of detection, sensitivity and selectivity. The sensor detected between 0.5 ppb and 5000 ppb TNT with good selectivity to other nitroaromatic explosives and demonstrated good accuracy for monitoring TNT in untreated environmental water matrix. We believe this new displacement format can be easily generalized to other one-dimensional nanostructure-based chemiresistive immuno/affinity-sensors for detecting small and/or uncharged molecules of interest in environmental monitoring and health care. Copyright © 2010 Elsevier B.V. All rights reserved.

Fabrication of SrTiO3 Layer on Pt Electrode for Label-Free Capacitive Biosensors

PubMed Central

Carapella, Giovanni; Pilloton, Roberto; Di Matteo, Marisa

2018-01-01

Due to their interesting ferroelectric, conductive and dielectric properties, in recent years, perovskite-structured materials have begun to attract increasing interest in the biosensing field. In this study, a strontium titanate perovskite layer (SrTiO3) has been synthesized on a platinum electrode and exploited for the development of an impedimetric label-free immunosensor for Escherichia coli O157:H7 detection. The electrochemical characterization of the perovskite-modified electrode during the construction of the immunosensor, as well as after the interaction with different E. coli O157:H7 concentrations, showed a reproducible decrease of the total capacitance of the system that was used for the analytical characterization of the immunosensor. Under optimized conditions, the capacitive immunosensor showed a linear relationship from to 1 to 7 log cfu/mL with a low detection limit of 1 log cfu/mL. Moreover, the atomic force microscopy (AFM) technique underlined the increase in roughness of the SrTiO3-modified electrode surface after antibody immobilization, as well as the effective presence of cells with the typical size of E. coli. PMID:29547521

Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples.

PubMed

Prieto-Simón, Beatriz; Campàs, Mònica; Marty, Jean-Louis; Noguer, Thierry

2008-02-28

The increasing concern about ochratoxin A (OTA) contamination of different food and feedstuffs demands high-performing detection techniques for quality assessment. Two indirect competitive enzyme-linked immunosorbent assay (ELISA) strategies were investigated for the development of OTA electrochemical immunosensors based on different OTA immobilisation procedures. Immunosensors based on avidin/biotin-OTA showed enhanced performance characteristics compared to those based on the adsorption of bovine serum albumin (BSA)-OTA conjugate. Performance of polyclonal (PAb) and monoclonal (MAb) antibodies against OTA was compared, showing at least one-order of magnitude lower IC(50) values when working with MAb. Alkaline phosphatase (ALP)- and horseradish peroxidase (HRP)-labelled secondary antibodies were evaluated. Both conjugates led to similar results when working with OTA standard solutions in buffer. However, whereas electroactive interferences present in spiked wine samples did not affect HRP-labelled immunosensors (4% slope deviation), they were likely oxidised at 0.225 V versus Ag/AgCl, the working potential for ALP-labelled immunosensors (25% slope deviation). Considering 80% of antibody binding as the limit of detection, values of 0.7 and 0.3 ng/mL for HRP- and ALP-labelled immunosensors respectively, validate these immunosensors as useful screening tools to assess OTA levels in wine.

Electrochemical immunosensor for ethinylestradiol using diazonium salt grafting onto silver nanoparticles-silica-graphene oxide hybrids.

PubMed

Cincotto, Fernando H; Martínez-García, Gonzalo; Yáñez-Sedeño, Paloma; Canevari, Thiago C; Machado, S A S; Pingarrón, José M

2016-01-15

This work describes the preparation of an electrochemical immunosensor for ethinylestradiol (EE2) based on grafting of diazonium salt of 4-aminobenzoic acid onto a glassy carbon electrode modified with silver nanoparticles/SiO2/graphene oxide hybrid followed by covalent binding of anti-ethinylestradiol (anti-EE2) to activated carboxyl groups. A competitive immunoassay was developed for the determination of the hormone using peroxidase-labeled ethinylestradiol (HRP-EE2) and measurement of the amperometric response at -200mV in the presence of hydroquinone (HQ) as redox mediator. The calibration curve for EE2 exhibited a linear range between 0.1 and 50ng/mL (r(2)=0.996), with a detection limit of 65pg/mL. Interference studies with other hormones related with EE2 revealed the practical specificity of the developed method for the analyte. A good reproducibility, with RSD=4.5% (n=10) was also observed. The operating stability of a single bioelectrode modified with anti-EE2 was maintained at least for 15 days when it was stored at 4°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of spiked urine with good results. Copyright © 2015 Elsevier B.V. All rights reserved.

Visible-light driven photoelectrochemical immunosensor for insulin detection based on MWCNTs@SnS2@CdS nanocomposites.

PubMed

Liu, Yixin; Zhang, Yifeng; Wu, Dan; Fan, Dawei; Pang, Xuehui; Zhang, Yong; Ma, Hongmin; Sun, Xu; Wei, Qin

2016-12-15

In this work, a label-free photoelectrochemical (PEC) immunosensor was developed for ultrasensitive detection of insulin based on MWCNTs@SnS2@CdS nanocomposites. As graphene-like 2D nanomaterial, SnS2 nanosheets loaded on the conducting framework of multi-walled carbon nanotubes (MWCNTs) were adopted for the construction of immunosensor for the first time, providing a favorable substrate for in-situ growth of CdS nanocrystal that had suitable band structure matching well with SnS2. The well-matched band structure of these two metal sulfides effectively inhibited the recombination of photogenerated electron-hole pairs, thus improving the photo-to-current conversion efficiency. Besides, the introduction of MWCNTs facilitated electron transfer across the surface of electrodes, leading to a further increment of photocurrent. The as constructed label-free PEC immunosensor based on MWCNTs@SnS2@CdS nanocomposites exhibited excellent PEC performance for the detection of insulin. The concentrations of insulin could be directly detected based on the decrement of photocurrent that was brought by the increased steric hindrances due to the formation of antigen-antibody immunocomplexes. Under the optimal conditions, the PEC immunosensor had a sensitive response to insulin in a linear range of 0.1pgmL(-1) to 5ngmL(-1) with a detection limit of 0.03pgmL(-1). Meanwhile, good stability and selectivity were achieved as well. The design and fabrication of this PEC immunosensor based on MWCNTs@SnS2@CdS nanocomposites not only provided an ideal platform for the detection of insulin, but also opened up a new avenue for the development of immunosensor for some other biomarkers analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitive electrochemical immunosensor for α-synuclein based on dual signal amplification using PAMAM dendrimer-encapsulated Au and enhanced gold nanoparticle labels.

PubMed

An, Yarui; Jiang, Xiaoli; Bi, Wenji; Chen, Hua; Jin, Litong; Zhang, Shengping; Wang, Chuangui; Zhang, Wen

2012-02-15

A novel electrochemical immunosensor for sensitive detection of α-synuclein (α-SYN), a very important neuronal protein, has been developed based on dual signal amplification strategy. Herein, G4-polyamidoamine dendrimer-encapsulated Au nanoparticles (PAMAM-Au nanocomposites) were covalently bound on the poly-o-aminobenzoic acid (poly-o-ABA), which was initially electropolymerized on the electrode surface to perform abundant carboxyl groups. The formed immunosensor platform, PAMAM-Au, was proved to provide numerous amino groups to allow highly dense immobilization of antigen, and facilitate the improvement of electrochemical responses as well. Subsequently, the enhanced gold nanoparticle labels ({HRP-Ab(2)-GNPs}) were fabricated by immobilizing horseradish peroxidase-secondary antibody (HRP-Ab(2)) on the surface of gold nanoparticles (GNPs). After an immunoassay process, the {HRP-Ab(2)-GNPs} labels were introduced onto the electrode surface, and produced an electrocatalytic response by reduction of hydrogen peroxide (H(2)O(2)) in the presence of enzymatically oxidized thionine. On the basis of the dual signal amplification of PAMAM-Au and {HRP-Ab(2)-GNPs} labels, the designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This developed strategy was successfully proved as a simple, cost-effective method, and could be easily extended to other protein analysis schemes. Copyright © 2011 Elsevier B.V. All rights reserved.

An electrochemical immunosensor for quantitative detection of ficolin-3

NASA Astrophysics Data System (ADS)

San, Lili; Zeng, Dongdong; Song, Shiping; Zuo, Xiaolei; Zhang, Huan; Wang, Chenguang; Wu, Jiarui; Mi, Xianqiang

2016-06-01

Diabetes mellitus (DM) is one of the most common metabolic disorders in the world, of which more than 90% is type-2 diabetes mellitus (T2DM). There is a rather urgent need for reliable, sensitive and quick detection techniques in clinical application of T2DM. Ficolin-3 is a potential biomarker of T2DM, because serum ficolin-3 levels are associated with insulin resistance and predict the incidence of T2DM. Herein, a sandwich-type electrochemical immunosensor was developed for the detection of ficolin-3 in human serum. Cyclic voltammetry and the amperometric current versus time were used to characterize the performance of the immunosensor. Under optimal conditions, the detection limitation of ficolin-3 was 100 ng ml-1 and the linear dynamic range was between 2 and 50 μg ml-1. The method has ideal accuracy, excellent stability and selectivity and has wide application prospects in clinical research.

Nanoparticle-based immunosensor with apoferritin templated metallic phosphate label for quantification of phosphorylated acetylcholinesterase

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

Du, Dan; Chen, Aiqiong; Xie, Yunying

2011-05-15

A new sandwich-like electrochemical immunosensor has been developed for quantification of organophosphorylated acetylcholinesterase (OP-AChE), an exposure biomarker of organophosphate pesticides and nerve agents. Zirconia nanoparticles (ZrO2 NPs) were anchored on a screen printed electrode (SPE) to preferably capture OP-AChE adducts by metal chelation with phospho-moieties, which was selectively recognized by lead phosphate-apoferritin labeled anti-AChE antibody (LPA-anti-AChE). The sandwich-like immunoreactions were performed among ZrO2 NPs, OP-AChE and LPA-anti-AChE to form ZrO2/OP-AChE/LPA-anti-AChE complex and the released lead ions were detected on a disposable SPE. The binding affinity was investigated by both square wave voltammetry (SWV) and quartz crystal microbalance (QCM) measurements. Themore » proposed immunosensor yielded a linear response current over a broad OP-AChE concentrations range from 0.05 nM to 10 nM, with detection limit of 0.02 nM, which has enough sensitivity for monitoring of low-dose exposure to OPs. This method avoids the drawback of unavailability of commercial OP-specific antibody as well as amplifies detection signal by using apoferritin encoded metallic phosphate nanoparticle tags. This nanoparticle-based immunosensor offers a new method for rapid, sensitive, selective and inexpensive quantification of phosphorylated adducts for monitoring of OP pesticides and nerve agents exposures.« less

Electrochemical detection of fluoroquinolone antibiotics in milk using a magneto immunosensor.

PubMed

Pinacho, Daniel G; Sánchez-Baeza, Francisco; Pividori, María-Isabel; Marco, María-Pilar

2014-08-28

An amperometric magneto-immunosensor (AMIS) for the detection of residues of fluoroquinolone antibiotics in milk samples is described for the first time. The immunosensor presented combines magnetic beads biomodified with an antibody with a broad recognition profile of fluoroquinolones, a haptenized enzyme and a magnetic graphite-epoxy composite (m-GEC) electrode. After the immunochemical reaction with specific enzyme tracer, the antibody biomodified magnetic beads are easily captured by an electrode made of graphite-epoxy composite containing a magnet, which also acts as transducer for the electrochemical detection. In spite of the complexity of milk, the use of magnetic beads allows elimination of potential interferences caused by the matrix components; hence the AMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. The immunosensor is able to detect up to seven different fluoroquinolones far below the MRLs defined by the UE for milk; for example ciprofloxacin is detected directly in milk with an IC50 of 0.74 µg/L and a LOD of 0.009 µg/L. This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis fluoroquinolones in complex samples.

Electrochemical Detection of Fluoroquinolone Antibiotics in Milk Using a Magneto Immunosensor

PubMed Central

Pinacho, Daniel G.; Sánchez-Baeza, Francisco; Pividori, María-Isabel; Marco, María-Pilar

2014-01-01

An amperometric magneto-immunosensor (AMIS) for the detection of residues of fluoroquinolone antibiotics in milk samples is described for the first time. The immunosensor presented combines magnetic beads biomodified with an antibody with a broad recognition profile of fluoroquinolones, a haptenized enzyme and a magnetic graphite–epoxy composite (m-GEC) electrode. After the immunochemical reaction with specific enzyme tracer, the antibody biomodified magnetic beads are easily captured by an electrode made of graphite-epoxy composite containing a magnet, which also acts as transducer for the electrochemical detection. In spite of the complexity of milk, the use of magnetic beads allows elimination of potential interferences caused by the matrix components; hence the AMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. The immunosensor is able to detect up to seven different fluoroquinolones far below the MRLs defined by the UE for milk; for example ciprofloxacin is detected directly in milk with an IC50 of 0.74 μg/L and a LOD of 0.009 μg/L. This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis fluoroquinolones in complex samples. PMID:25171120

Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin.

PubMed

Gobi, K Vengatajalabathy; Iwasaka, Hiroyuki; Miura, Norio

2007-02-15

A simple and rapid continuous-flow immunosensor based on surface plasmon resonance (SPR) has been developed for detection of insulin as low as 1 ng ml-1 (ppb) with a response time of less than 5 min. At first, a heterobifunctional oligo(ethyleneglycol)-dithiocarboxylic acid derivative (OEG-DCA) containing dithiol and carboxyl end groups was used to functionalize the thin Au-film of SPR chip. Insulin was covalently bound to the Au-thiolate monolayer of OEG-DCA for activating the sensor surface to immunoaffinity interactions. An on-line competitive immunosensing principle is examined for detection of insulin, in which the direct affinity binding of anti-insulin antibody to the insulin on sensor surface is examined in the presence and absence of various concentrations of insulin. Immunoreaction of anti-insulin antibody with the sensor surface was optimized with reference to antibody concentration, sample analysis time and flow-rate to provide the desired detection limit and determination range. With the immunosensor developed, the lowest detectable concentration of insulin is 1 ng ml-1 and the determination range covers a wide concentration of 1-300 ng ml-1. The developed OEG-monolayer based sensor chip exhibited high resistance to non-specific adsorption of proteins, and an uninterrupted highly sensitive detection of insulin from insulin-impregnated serum samples has been demonstrated. After an immunoreaction cycle, active sensor surface was regenerated simply by a brief flow of an acidic buffer (glycine.HCl; pH 2.0) for less than 1 min. A same sensor chip was found reusable for more than 25 cycles without an appreciable change in the original sensor activity.

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode.

PubMed

Kong, Fen-Ying; Xu, Mao-Tian; Xu, Jing-Juan; Chen, Hong-Yuan

2011-10-15

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels. Copyright © 2011 Elsevier B.V. All rights reserved.

Towards an Electrochemical Immunosensor System with Temperature Control for Cytokine Detection.

PubMed

Metzner, Julia; Luckert, Katrin; Lemuth, Karin; Hämmerle, Martin; Moos, Ralf

2018-04-24

The cytokine interleukin-13 (IL-13) plays a major role in airway inflammation and is a target of new anti-asthmatic drugs. Hence, IL-13 determination could be interesting in assessing therapy success. Thus, in this work an electrochemical immunosensor for IL-13 was developed and integrated into a fluidic system with temperature control for read-out. Therefore, two sets of results are presented. First, the sensor was set up in sandwich format on single-walled carbon nanotube electrodes and was read out by applying the hydrogen peroxide⁻hydroquinone⁻horseradish peroxidase (HRP) system. Second, a fluidic system was built up with an integrated heating function realized by Peltier elements that allowed a temperature-controlled read-out of the immunosensor in order to study the influence of temperature on the amperometric read-out. The sensor was characterized at the temperature optimum of HRP at 30 °C and at 12 °C as a reference for lower performance. These results were compared to a measurement without temperature control. At the optimum operation temperature of 30 °C, the highest sensitivity (slope) was obtained compared to lower temperatures and a limit of detection of 5.4 ng/mL of IL-13 was calculated. Taken together, this approach is a first step towards an automated electrochemical immunosensor platform and shows the potential of a temperature-controlled read-out.

Enzyme-less electrochemical displacement heterogeneous immunosensor for diclofenac detection.

PubMed

Nguyen, T T K; Vu, T T; Anquetin, G; Tran, H V; Reisberg, S; Noël, V; Mattana, G; Nguyen, Q V; Dai Lam, Tran; Pham, M C; Piro, B

2017-11-15

We describe an electrochemical immunosensor based on functionalization of a working electrode by electrografting two functional diazonium salts. The first one is a molecular probe, diclofenac, coupled with an arylamine onto which a specific antibody is immobilized by affinity interactions; the second is a redox probe (a quinone) also coupled with an arylamine, able to transduce the hapten-antibody association into a change in electroactivity. The steric hindrance induced by the antibody leads to a current decrease upon binding of the antibody on the grafted molecular probe; conversely, when diclofenac is present in solution, a displacement equilibrium occurs between the target diffusing into the solution and the grafted probe. This leads to dissociation of the antibody from the electrode surface, event which is transduced into a current increase ("signal-on" detection). The detection limit is ca. 20 fM, corresponding to 6pgL -1 diclofenac, which is competitive compared to other label-free immunosensors. We demonstrate that the sensor is selective and is able to quantify diclofenac in tap water. Copyright © 2017 Elsevier B.V. All rights reserved.

Optical waveguide lightmode spectroscopy technique-based immunosensor development for aflatoxin B1 determination in spice paprika samples.

PubMed

Majer-Baranyi, Krisztina; Zalán, Zsolt; Mörtl, Mária; Juracsek, Judit; Szendrő, István; Székács, András; Adányi, Nóra

2016-11-15

Optical waveguide lightmode spectroscopy (OWLS) technique has been applied to label-free detection of aflatoxin B1 in a competitive immunoassay format, with the aim to compare the analytical goodness of the developed OWLS immunosenor with HPLC and enzyme-linked immunosorbent assay (ELISA) methods for the detection of aflatoxin in spice paprika matrix. We have also assessed applicability of the QuEChERS method prior to ELISA measurements, and the results were compared to those obtained by traditional solvent extraction followed by immunoaffinity clean-up. The AFB1 content of sixty commercial spice paprika samples from different countries were measured with the developed and optimized OWLS immunosensor. Comparing the results from the indirect immunosensor to that obtained by HPLC or ELISA provided excellent correlation (with regression coefficients above 0.94) indicating that the competitive OWLS immunosensor has a potential for quick determination of aflatoxin B1 in paprika samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Less label, more free: approaches in label-free quantitative mass spectrometry.

PubMed

Neilson, Karlie A; Ali, Naveid A; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Gariné; Lee, Albert; van Sluyter, Steven C; Haynes, Paul A

2011-02-01

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineered biomarkers for leprosy diagnosis using labeled and label-free analysis.

PubMed

de Santana, Juliana F; da Silva, Mariângela R B; Picheth, Guilherme F; Yamanaka, Isabel B; Fogaça, Rafaela L; Thomaz-Soccol, Vanete; Machado-de-Avila, Ricardo A; Chávez-Olórtegui, Carlos; Sierakowski, Maria Rita; de Freitas, Rilton Alves; Alvarenga, Larissa M; de Moura, Juliana

2018-09-01

The biotechnological evolution towards the development of antigens to detect leprosy has been progressing. However, the identification of leprosy in paucibacillary patients, based solely on the antigen-antibody interaction still remains a challenge. The complexity of clinical manifestations requires innovative approaches to improve the sensitivity of assays to detect leprosy before the onset of symptoms, thus avoiding disabilities and contributing, indirectly, to reduce transmission. In this study, the strategies employed for early leprosy diagnosis were: i. using a phage-displayed mimotope (APDDPAWQNIFNLRR) which mimics an immunodominant sequence (PPNDPAWQRNDPILQ) of an antigen of Mycobacterium leprae known as Ag85B; ii. engineering the mimotope by adding a C-terminal flexible spacer (SGSG-C); iii. conjugating the mimotope to a carrier protein to provide better exposure to antibodies; iv. amplifying the signal using biotin-streptavidin detection system in an ELISA; and v. coating the optimized mimotope on a quartz crystal microbalance (QCM) sensor for label-free biosensing. The ELISA sensitivity increased up to 91.7% irrespective of the immunological profile of the 132 patients assayed. By using comparative modeling, the M. tuberculosis Ag85B was employed as a template to ascertain which features make the mimotope a good antigen in terms of its specificity. For the first time, a sensitive QCM-based immunosensor to detect anti M. leprae antibodies in human serum was used. M. leprae antibodies could also be detected in the sera of paucibacillary patients; thus, the use of a mimotope-derived synthetic peptide as bait for antibodies in a novel analytical label-free immunoassay for leprosy diagnosis exhibits great potential. Copyright © 2018 Elsevier B.V. All rights reserved.

A sensitive electrochemical immunosensor for label-free detection of Zika-virus protein.

PubMed

Kaushik, Ajeet; Yndart, Adriana; Kumar, Sanjeev; Jayant, Rahul Dev; Vashist, Arti; Brown, Ashley N; Li, Chen-Zhong; Nair, Madhavan

2018-06-26

This work, as a proof of principle, presents a sensitive and selective electrochemical immunosensor for Zika-virus (ZIKV)-protein detection using a functionalized interdigitated micro-electrode of gold (IDE-Au) array. A miniaturized IDE-Au immunosensing chip was prepared via immobilization of ZIKV specific envelop protein antibody (Zev-Abs) onto dithiobis(succinimidyl propionate) i.e., (DTSP) functionalized IDE-Au (electrode gap/width of 10 µm). Electrochemical impedance spectroscopy (EIS) was performed to measure the electrical response of developed sensing chip as a function of ZIKV-protein concentrations. The results of EIS studies confirmed that sensing chip detected ZIKV-protein selectively and exhibited a detection range from 10 pM to 1 nM and a detection limit of 10 pM along with a high sensitivity of 12 kΩM -1 . Such developed ZIKV immune-sensing chip can be integrated with a miniaturized potentiostat (MP)-interfaced with a smartphone for rapid ZIKV-infection detection required for early stage diagnostics at point-of-care application.

An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies

PubMed Central

Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan

2016-01-01

Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)32+-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)32+-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)32+-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility. PMID:26927130

A novel electrochemical immunosensor using β-cyclodextrins functionalized silver supported adamantine-modified glucose oxidase as labels for ultrasensitive detection of alpha-fetoprotein.

PubMed

Gao, Jian; Ma, Hongmin; Lv, Xiaohui; Yan, Tao; Li, Na; Cao, Wei; Wei, Qin

2015-09-17

In this work, a novel sandwich-type electrochemical immunosensor based on host-guest interaction was fabricated for the detection of alpha-fetoprotein (AFP). Due to the large specific surface area of multiwalled carbon nanotubes and the unique supramolecular recognition ability of β-cyclodextrins, ferrocenecarboxylic acid (Fc) was incorporated into this sensor platform by host-guest interaction to generate an electrochemical signal. And β-cyclodextrins functionalized silver supported adamantine-modified glucose oxidase (GOD-CD-Ag), was used as a label to improve the analytical performance of the immunosensor by the dual amplification strategy. The obtained GOD-CD-Ag conjugates could convert glucose into gluconic acid with the formation of hydrogen peroxide (H2O2). And then silver nanoparticles could in situ catalyze the reduction of the generated H2O2, dramatically improving the oxidation reaction of Fc. The developed immunosensor shows a wide linear calibration range from 0.001 to 5.0 ng/mL with a low detection limit (0.2 pg/mL) for the detection of AFP. The method, with ideal reproducibility and selectivity, has a wide application prospect in clinical research. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food.

PubMed

Morlay, Alexandra; Roux, Agnès; Templier, Vincent; Piat, Félix; Roupioz, Yoann

2017-01-01

Foodborne diseases are a major concern for both food industry and health organizations due to the economic costs and potential threats for human lives. For these reasons, specific regulations impose the research of pathogenic bacteria in food products. Nevertheless, current methods, references and alternatives, take up to several days and require many handling steps. In order to improve pathogen detection in food, we developed an immune-sensor, based on Surface Plasmon Resonance imaging (SPRi) and bacterial growth which allows the detection of a very low number of Listeria monocytogenes in food sample in one day. Adequate sensitivity is achieved by the deposition of several antibodies in a micro-array format allowing real-time detection. This label-free method thus reduces handling and time to result compared with current methods.

A sandwich HIV p24 amperometric immunosensor based on a direct gold electroplating-modified electrode.

PubMed

Zheng, Lei; Jia, Liyong; Li, Bo; Situ, Bo; Liu, Qinlan; Wang, Qian; Gan, Ning

2012-05-18

Acquired immune deficiency syndrome (AIDS) is a severe communicable immune deficiency disease caused by the human immune deficiency virus (HIV). The analysis laboratory diagnosis of HIV infection is a crucial aspect of controlling AIDS. The p24 antigen, the HIV-1 capsid protein, is of considerable diagnostic interest because it is detectable several days earlier than host-generated HIV antibodies following HIV exposure. We present herein a new sandwich HIV p24 immunosensor based on directly electroplating an electrode surface with gold nanoparticles using chronoamperometry, which greatly increased the conductivity and reversibility of the electrode. Under optimum conditions, the electrochemical signal showed a linear relationship with the concentration of p24, ranging from 0.01 ng/mL to 100 ng/mL (R > 0.99), and the detection limit was 0.008 ng/mL. Compared with ELISA, this method increased the sensitivity by more than two orders of magnitude (the sensitivity of ELISA for p24 is about 1 ng/mL). This immunosensor may be broadly applied to clinical samples, being distinguished by its ease of use, mild reaction conditions, guaranteed reproducibility, and good anti-interference ability.

Ultrasensitive Immunosensor for Cancer Biomarker Proteins using Gold Nanoparticle Film Electrodes and Multienzyme-Particle Amplification

PubMed Central

Mani, Vigneshwaran; Chikkaveeraiah, Bhaskara V.; Patel, Vyomesh; Gutkind, J. Silvio; Rusling, James F.

2009-01-01

A densely packed gold nanoparticle platform combined with a multiple-enzyme labeled detection antibody-magnetic bead bioconjugate was used as the basis for an ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum. Sensitivity was greatly amplified by synthesizing magnetic bioconjugates particles containing 7500 horseradish peroxidase (HRP) labels along with detection antibodies (Ab2) attached to activated carboxyl groups on 1 µm diameter magnetic beads. These sensors had sensitivity of 31.5 µA mL ng−1 and detection limit (DL) of 0.5 pg mL−1 for prostate specific antigen (PSA) in 10 µL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, eight fold better than a previously reported carbon nanotube (CNT) forest immunosensor featuring multiple labels on carbon nanotubes, and near or below the normal serum levels of most cancer biomarkers. Measurements of PSA in cell lysates and human serum of cancer patients gave excellent correlations with standard ELISA assays. These easily fabricated AuNP immunosensors show excellent promise for future fabrication of bioelectronic arrays. PMID:19216571

A photoelectrochemical immunosensor for detection of α-fetoprotein based on Au-ZnO flower-rod heterostructures

NASA Astrophysics Data System (ADS)

Han, Zhizhong; Luo, Min; Chen, Li; Chen, Jinghua; Li, Chunyan

2017-04-01

In this work, a novel label free photoelectrochemical (PEC) immunosensor has been developed for the detection of α-fetoprotein (AFP). The immunosensor was based on Au-ZnO flower-rods (FRs) heterostructure, where Au nanoparticles (NPs) were firstly electrodeposited by cyclic voltammetry methods. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Mott-Schottky plot (MS), UV-vis diffuse reflectance spectrum and fluorescence emission spectrum were used for the characterizations of Au-ZnO FRs. The results demonstrated that Au NPs not only obviously enhanced the visible light absorption of ZnO FRs due to surface plasmon resonance (SPR) but also improved the separation of photo-generated electron-hole pairs. Therefore, the photocurrent of Au-ZnO FRs was increased under simulated sunlight. The photocurrent was reduced after the specific antibody-antigen immune reaction. And the photocurrent decrement was linear with the logarithm of AFP antigen concentration in the range from 0.005 ng mL-1 to 50 ng mL-1 with a low detection limit of 0.56 pg mL-1 (S/N = 3). The PEC immunosensor also exhibited high anti-interference property and acceptable stability. This work would provide a promising photoelectrochemical strategy for the detection of other proteins in clinical diagnosis.

Controlled carbon nanotube layers for impedimetric immunosensors: High performance label free detection and quantification of anti-cholera toxin antibody.

PubMed

Palomar, Quentin; Gondran, Chantal; Holzinger, Michael; Marks, Robert; Cosnier, Serge

2017-11-15

An original impedimetric immunosensor was developed based on carbon nanotube (CNT) deposits with controlled thicknesses for enhanced electroactive surface areas leading to improved sensor performances. Cholera monitoring was chosen as the model immune system for this setup. These CNT deposits were characterized using confocal laser microscopy and electrochemical methods. To form the sensor device, the CNT deposits were functionalized via electrocoating of polypyrrole-nitrilotriacetic acid (poly(pyrrole-NTA)) followed by the formation of a Cu (II) complex with the NTA functions. The bioreceptor unit, cholera toxin B Subunit, modified with biotin, was then immobilized via coordination of the biotin groups with the NTA-Cu(II) complex. Each step of the formation of the immunosensor and the subsequent binding of the analyte antibody anti-cholera toxin were investigated with cyclic voltammetry and Electrochemical Impedance Spectroscopy. After optimization, the resulting impedimetric cholera sensor shows excellent reproducibility, increased sensitivities, a very satisfying detection limit of 10 -13 gmL -1 and an exceptional linear range for anti-cholera detection of 8 orders of magnitude (10 -13 -10 -5 gmL -1 ) and a sensitivity of 24.7 ± 0.4Ω per order of magnitude. Copyright © 2017 Elsevier B.V. All rights reserved.

Amperometric biosensor based on a single antibody of dual function for rapid detection of Streptococcus agalactiae.

PubMed

Vásquez, Gersson; Rey, Alba; Rivera, Camilo; Iregui, Carlos; Orozco, Jahir

2017-01-15

Pathogenic bacteria are responsible for several diseases in humans and in a variety of hosts. Detection of pathogenic bacteria is imperative to avoid and/or fight their potential harmful effects. This work reports on the first amperometric biosensor for the rapid detection of Streptococcus agalactiae (S. agalactiae). The biosensor relies on a single biotinylated antibody that immobilizes the bacteria on a screen-printed carbon electrode while is further linked to a streptavidin-conjugated HRP reporter. The biotinylated antibody provides selectivity to the biosensor whereas serves as an anchoring point to the reporter for further amplification of the electrochemical signal. The resultant immunosensor is simple, responds rapidly, and allows for the selective and highly sensitive quantification of S. agalactiae cells in a concentration range of 10 1 -10 7 CFUml -1 , with a detection limit of 10CFUml -1 . The approach not only enables a rapid detection and quantification of S. agalactiae in environmental samples but also opens up new opportunities for the simple fabrication of electrochemical immunosensors for different target pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

An electrochemical magneto immunosensor (EMIS) for the determination of paraquat residues in potato samples.

PubMed

Garcia-Febrero, Raul; Valera, Enrique; Muriano, Alejandro; Pividori, M-Isabel; Sanchez-Baeza, Francisco; Marco, M-Pilar

2013-09-01

An electrochemical magneto immunosensor for the detection of low concentrations of paraquat (PQ) in food samples has been developed and its performance evaluated in a complex sample such as potato extracts. The immunosensor presented uses immunoreagents specifically developed for the recognition of paraquat, a magnetic graphite-epoxy composite (m-GEC) electrode and biofunctionalized magnetic micro-particles (PQ1-BSAMP) that allow reduction of the potential interferences caused by the matrix components. The amperometric signal is provided by an enzymatic probe prepared by covalently linking an enzyme to the specific antibodies (Ab198-cc-HRP). The use of hydroquinone, as mediator, allows recording of the signal at a low potential, which also contributes to reducing the background noise potentially caused by the sample matrix. The immunocomplexes formed on top of the modified MP are easily captured by the m-GEC, which acts simultaneously as transducer. PQ can be detected at concentrations as low as 0.18 ± 0.09 μg L(-1). Combined with an efficient extraction procedure, PQ residues can be directly detected and accurately quantified in potato extracts without additional clean-up or purification steps, with a limit of detection (90% of the maximum signal) of 2.18 ± 2.08 μg kg(-1), far below the maximum residue level (20 μg kg(-1)) established by the EC. The immunosensor presented here is suitable for on-site analysis. Combined with the use of magnetic racks, multiple samples can be run simultaneously in a reasonable time.

Amplification-free point of care immunosensor for detecting type V collagen at a concentration level of ng/ml

NASA Astrophysics Data System (ADS)

Chung, Pei-Yu; Bracho-Sanchez, Evelyn R.; Jiang, Peng; Seagrave, JeanClare; Duncan, Matthew R.; Grotendorst, Gary R.; Schultz, Gregory; Batich, Christopher

2011-06-01

Point-of-care testing (POCT) is applicable in the immediate vicinity of the patient, where timely diagnosis or prognostic information could help doctors decide the following treatment. Among types of developed POCT, gold nanoparticle based lateral flow strip technology provides advantages such as simple operation, cost-effectiveness, and a user-friendly platform. Therefore, this type of POCT is most likely to be used in battlefields and developing countries. However, conventional lateral flow strips suffer from low detection limits. Although enzyme-linked amplification was demonstrated to improve the detection limit and sensitivity by stronger visible lines or by permitting electrochemical analytical instrumentation, the enzyme labels have potential to cause interference with other enzymes in our body fluids. To eliminate this limitation, we developed an amplification-free gold nanoparticle-based immunosensor applied for detecting collagen type V, which is produced or released abnormally during rejection of lung transplants and sulfur mustard exposure. By using suitable blocking protein to stabilize gold nanoparticles as the reporter probe, a low detection limit of ng/ml was achieved. This strategy is a promising platform for clinical POCT, with potential applications in military or disaster response.

Extended-gate-type IGZO electric-double-layer TFT immunosensor with high sensitivity and low operation voltage

NASA Astrophysics Data System (ADS)

Liang, Lingyan; Zhang, Shengnan; Wu, Weihua; Zhu, Liqiang; Xiao, Hui; Liu, Yanghui; Zhang, Hongliang; Javaid, Kashif; Cao, Hongtao

2016-10-01

An immunosensor is proposed based on the indium-gallium-zinc-oxide (IGZO) electric-double-layer thin-film transistor (EDL TFT) with a separating extended gate. The IGZO EDL TFT has a field-effect mobility of 24.5 cm2 V-1 s-1 and an operation voltage less than 1.5 V. The sensors exhibit the linear current response to label-free target immune molecule in the concentrations ranging from 1.6 to 368 × 10-15 g/ml with a detection limit of 1.6 × 10-15 g/ml (0.01 fM) under an ultralow operation voltage of 0.5 V. The IGZO TFT component demonstrates a consecutive assay stability and recyclability due to the unique structure with the separating extended gate. With the excellent electrical properties and the potential for plug-in-card-type multifunctional sensing, extended-gate-type IGZO EDL TFTs can be promising candidates for the development of a label-free biosensor for public health applications.

Screening of boldenone and methylboldenone in bovine urine using disposable electrochemical immunosensors.

PubMed

Lu, Huihui; Conneely, Gráinne; Pravda, Miloslav; Guilbault, George G

2006-09-01

Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone-bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG-horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3',5,5'-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9+/-4.3 pg ml(-1) for boldenone and 120.2+/-8.2 pg ml(-1) for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC(50)=1.0+/-0.3 ng ml(-1)(n=3, N=3), R(2)=0.969, R.S.D.=9.6% for boldenone and 1.5+/-0.3 ng ml(-1), 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of alpha-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.

Multiplexed enzyme-free electrochemical immunosensor based on ZnO nanorods modified reduced graphene oxide-paper electrode and silver deposition-induced signal amplification strategy.

PubMed

Sun, Guoqiang; Zhang, Lina; Zhang, Yan; Yang, Hongmei; Ma, Chao; Ge, Shenguang; Yan, Mei; Yu, Jinghua; Song, Xianrang

2015-09-15

Herein, an origami multiplexed enzyme-free electrochemical (EC) immunodevice is developed for the first time. Typically, ZnO nanorods (ZNRs) modified reduced graphene oxide (rGO)-paper electrode is used as a sensor platform, in which rGO improves the electronic transmission rate and ZNRs provide abundant sites for capture probes binding. Furthermore, by combining the large surface area of rGO and high catalytic activity of bovine serum protein (BSA)-stabilized silver nanoparticles (Ag@BSA) toward H2O2 reduction, rGO/Ag@BSA composites can be used as an excellent signal labels. The current signal is generated from the reduction of H2O2 and further amplified by a subsequent signal labels-promoted deposition of silver. Under optimal conditions, the proposed immunoassays exhibit excellent precision, high sensitivity and a wide linear range of 0.002-120 mIU mL(-1) for human chorionic gonadotropin, 0.001-110 ng mL(-1) for prostate-specific antigen, and 0.001-100 ng mL(-1) for carcinoembryonic antigen. The results for real sample analysis demonstrate that the newly constructed immunosensor arrays provide a simple and cost-effective method for clinical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection.

PubMed

Cheng, Yinfeng; Yuan, Ruo; Chai, Yaqin; Niu, Huan; Cao, Yaling; Liu, Huijing; Bai, Lijuan; Yuan, Yali

2012-10-01

In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl(4) and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol-H(2)O(2) system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL(-1) to 80 ng mL(-1) and with a detection limit of 3.3 pg mL(-1) (SN(-1)=3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers. Copyright © 2012 Elsevier B.V. All rights reserved.

Ultrasensitive environmental assessment of xeno-estrogens in water samples using label-free graphene immunosensors.

PubMed

Barton, Huw; Berbel-Filho, Waldir M; Consuegra, Sofia; Francis, Lewis; Tizaoui, Chedly; Conlan, R Steven; Teixeira, Sofia Rodrigues

2018-05-01

There is a growing interest in the possible environmental health impact posed by endocrine-disrupting chemicals (EDCs). A challenge to the field of endocrine disruption is that these substances are diverse and may not appear to share any structural similarity other than usually being low molecular mass (<1000 Da) compounds. Here we demonstrate the effectiveness of sensor device for the detection of low molecular weight, poorly water soluble, estrogenic compounds E1, E2 and EE2, fabricated by electropolymerization over graphene screen printed electrode (SPE). The PANI/Gr-SPE-devices displayed linear responses to estrogenic substances, in EIS assays, from 0.0975 ng/L to 200 ng/L in water samples, with a detection limit of 0.043 pg/L for E1, 0.19 ng/L for E2 and 0.070 pg/L for EE2 which is lower than other current biosensing techniques. This portable, disposable immunosensor offers a solution for immediate measurement at sample collection sites, due to its excellent sensitivity and selectivity when testing water samples obtained directly from rivers and waste water treatment facilities. The simple screen printing production method will enable the low cost, high volume production required for this type of environmental analysis. Copyright © 2018 Elsevier Inc. All rights reserved.

An impedimetric immunosensor for highly sensitive detection of IL-8 in human serum and saliva samples: A new surface modification method by 6-phosphonohexanoic acid for biosensing applications.

PubMed

Aydın, Elif Burcu; Sezgintürk, Mustafa Kemal

2018-08-01

In this study, we fabricated a sensitive and label-free impedimetric immunosensor based on 6-phosphonohexanoic acid (PHA) modified ITO electrode for detection of interleukin-8 (IL-8) in human serum and saliva. PHA was first employed to cancer biomarker sensing platform. Anti-IL-8 antibody was used as a biorecognition element and the detection principle of this immunosensor was based on monitoring specific interaction between anti-IL-8 antibody and IL-8 antigen. The morphological characterization of each electrode modification step was analyzed by scanning electron microscopy (SEM), SEM-energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) while electrochemical characterization was performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and single frequency impedance (SFI) techniques. Moreover, the antibody immobilization on the electrode surface was proved Fourier-transform infrared spectroscopy (FTIR) and Raman Spectroscopy. This proposed impedimetric immunosensor exhibited good performances with a wide linear in the range from 0.02 pg/mL to 3 pg/mL as well as a relative low detection limit of 6 fg/mL. The impedimetric immunosensor had a good specificity, stability and reproducibility. This study proved that PHA was a suitable interface material to fabricate an electrochemical biosensor. Copyright © 2018 Elsevier Inc. All rights reserved.

Interferometric nanoimmunosensor for label-free and real-time monitoring of Irgarol 1051 in seawater.

PubMed

Chocarro-Ruiz, Blanca; Herranz, Sonia; Fernández Gavela, Adrián; Sanchís, Josep; Farré, Marinella; Marco, M Pilar; Lechuga, Laura M

2018-05-26

An interferometric nanobiosensor for the specific and label-free detection of the pollutant Irgarol 1051 directly in seawater has been settled. Due to the low molecular weight of Irgarol pollutant and its expected low concentration in seawater, the sensor is based on a competitive inhibition immunoassay. Parameters as surface biofunctionalization, concentration of the selective antibody and regeneration conditions have been carefully evaluated. The optimized immunosensor shows a limit of detection of only 3 ng/L, well below the 16 ng/L set by the EU as the maximum allowable concentration in seawater. It can properly operate during 30 assay-regeneration cycles using the same sensor biosurface and with a time-to-result of only 20 min for each cycle. Moreover, the interferometric nanosensor is able to directly detect low concentrations of Irgarol 1051 in seawater without requiring sample pre-treatments and without showing any background signal due to sea matrix effect. Copyright © 2018 Elsevier B.V. All rights reserved.

A sandwich-type electrochemical immunosensor based on in situ silver deposition for determination of serum level of HER2 in breast cancer patients.

PubMed

Shamsipur, Mojtaba; Emami, Mahdi; Farzin, Leila; Saber, Reza

2018-04-30

The sensitive quantification of Human Epidermal growth factor Receptor 2 (HER2), as a key prognostic tumor marker, plays a critical role in screening, early diagnosis and management of breast cancer. This paper describes a sandwich-type immunoassay with silver signal enhancement strategy for highly sensitive detection of HER2. For this purpose, the target capturing step was designed by functionalization of 3-aminopropyltrimethoxysilane coated magnetite nanoparticles with antibody (antiHER2/APTMS-Fe 3 O 4 ), as a platform bioconjugate (PB), and immobilized at a bare GCE. Then, in the presence of label-free immunosensor, the PB was covered by magnetic gold nanoparticles self-assembled with thiolated antibodies (antiHER2/Hyd@AuNPs-APTMS-Fe 3 O 4 ) containing chemically reduced silver ions, as a label bioconjugate (LB). Under optimum conditions, a linear relationship between the differential pulse voltammetric (DPV) stripping signal of silver and the logarithm of HER2 concentrations was obtained in the range of 5.0 × 10 -4 -50.0ngmL -1 (R 2 = 0.9906) with a detection limit of 2.0 × 10 -5 ngmL -1 . The effectiveness of this protocol was evaluated experimentally through employing of designed immunosensor for detection of the serum level of tumor marker. The good consistency of the results with those obtained by the enzyme-linked immunosorbent assay (ELISA) conventional method (p-value of < 0.05) showed that this immunosensor can be applied for the testing of HER2 in clinical samples of breast cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy using highly loaded palladium nanoparticles/carbon decorated magnetic microspheres as signal labels.

PubMed

Ji, Lei; Guo, Zhankui; Yan, Tao; Ma, Hongmin; Du, Bin; Li, Yueyun; Wei, Qin

2015-06-15

An ultrasensitive sandwich-type electrochemical immunosensor for quantitative detection of alpha fetoprotein (AFP) was proposed based on a novel signal amplification strategy in this work. Carbon decorated Fe3O4 magnetic microspheres (Fe3O4@C) with large specific surface area and good adsorption property were used as labels to anchor palladium nanoparticles (Pd NPs) and the secondary antibodies (Ab2). Pd NPs were loaded on Fe3O4@C to obtain Fe3O4@C@Pd with core-shell structure by electrostatic attraction, which were further used to immobilize Ab2 due to the bonding of Pd-NH2. A signal amplification strategy was the noble metal nanoparticles, such as Pd NPs, exhibiting high electrocatalytic activities toward hydrogen peroxide (H2O2) reduction. This signal amplification was novel not only because of the great capacity, but also the ease of magnetic separation from the sample solution based on their magnetic property. Moreover, carboxyl-functionalized multi-walled carbon nanotubes (MWCNTs-COOH) were used for the immobilization of primary antibodies (Ab1). Therefore, high sensitivity could be realized by the designed immunosensor based on this novel signal amplification strategy. Under optimal conditions, the immunosensor exhibited a wide linear range of 0.5 pg/mL to 10 ng/mL toward AFP with a detection limit of 0.16 pg/mL (S/N=3). Moreover, it revealed good selectivity, acceptable reproducibility and stability, indicating a potential application in clinical monitoring of tumor biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative determination of two probiotics by QCM and OWLS-based immunosensors.

PubMed

Szalontai, Helga; Adányi, Nóra; Kiss, Attila

2014-09-25

The regular consumption of foods containing probiotic bacteria has beneficial physiological effects on the health and the digestion system. There is a need for novel analytical approaches for the determination of these bacteria that are faster than the classical plate counting method. For this purpose, two label-free biosensors were investigated and presented in this paper: Quartz Crystal Microbalance (QCM) and Optical Waveguide Lightmode Spectroscopy (OWLS) based direct immunosensors were developed for real-time direct detection of probiotic bacteria in fermented dairy products. Bifidobacterium bifidum O1356 and Lactobacillus acidophilus O1132 were detected by polyclonal anti-B. bifidum IgG and anti-L. acidophilus IgG immobilized on the sensors' surface. Sulfo-LC-SPDP cross linking agent was used to bind antibodies to the gold surface of the QCM's AT-cut quartz wafer. Concerning OWLS, antibodies were covalently bound to the amino groups of the silanized surface of the waveguide by glutaraldehyde. The dynamic measuring range was found between 1.0E+3 and 5.0E+5CFUmL(-1) in 100 fold diluted fermented milk products by QCM and with OWLS. Considering the current legislation of the probiotic content in probiotic products, the two developed immunosensors can be applied for rapid quantification of L. acidophilus and B. bifidum in fermented milk. These examinations offer effective alternatives to the microbiological plate counting method. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrochemiluminescence immunosensor for ultrasensitive detection of biomarker using Ru(bpy)(3)(2+)-encapsulated silica nanosphere labels.

PubMed

Qian, Jing; Zhou, Zhenxian; Cao, Xiaodong; Liu, Songqin

2010-04-14

Here, we describe a new approach for electrochemiluminescence (ECL) assay with Ru(bpy)(3)(2+)-encapsulated silica nanoparticle (SiO(2)@Ru) as labels. A water-in-oil (W/O) microemulsion method was employed for one-pot synthesis of SiO(2)@Ru nanoparticles. The as-synthesized SiO(2)@Ru nanoparticles have a narrow size distribution, which allows reproducible loading of Ru(bpy)(3)(2+) inside the silica shell and of alpha-fetoprotein antibody (anti-AFP), a model antibody, on the silica surface with glutaraldehyde as linkage. The silica shell effectively prevents leakage of Ru(bpy)(3)(2+) into the aqueous solution due to strong electrostatic interaction between the positively charged Ru(bpy)(3)(2+) and the negatively charged surface of silica. The porous structure of silica shell allowed the ion to move easily through the pore to exchange energy/electrons with the entrapped Ru(bpy)(3)(2+). The as-synthesized SiO(2)@Ru can be used as a label for ultrasensitive detection of biomarkers through a sandwiched immunoassay process. The calibration range of AFP concentration was 0.05-30 ng mL(-1) with linear relation from 0.05 to 20 ng mL(-1) and a detection limit of 0.035 ng mL(-1) at 3sigma. The resulting immunosensors possess high sensitivity and good analytical performance. Copyright 2010 Elsevier B.V. All rights reserved.

Exploration of two-dimensional bio-functionalized phosphorene nanosheets (black phosphorous) for label free haptoglobin electro-immunosensing applications

NASA Astrophysics Data System (ADS)

Tuteja, Satish K.; Neethirajan, Suresh

2018-04-01

We report on the development of an antibody-functionalized interface based on electrochemically active liquid-exfoliated two-dimensional phosphorene (Ph) nanosheets—also known as black phosphorous nanosheets—for the label-free electrochemical immunosensing of a haptoglobin (Hp) biomarker, a clinical marker of severe inflammation. The electrodeposition has been achieved over the screen-printed electrode (SPE) using liquid-assisted ultrasonically exfoliated black phosphorus nanosheets. Subsequently, Ph-SPEs bioconjugated with Hp antibodies (Ab), using electrostatic interactions via a poly-L-lysine linker for biointerface development. Electrochemical analysis demonstrates that the Ab-modified Ph-SPEs (Ab@Ph-SPE) exhibit enhanced electroconducting behavior as compared to the pristine electrodes. This Ab-functionalized phosphorene-based electrochemical immunosensor platform has demonstrated remarkable sensitivity and specificity, having a dynamic linear response range from 0.01-10 mg ml-1 for Hp in standard and serum samples with a low detection limit (˜0.011 mg ml-1) using the label-free electrochemical technique. The sensor electrodes were also studied with other closely relative interferents to investigate cross reactivity and specificity. This strategy opens up avenues to POC (point-of-care) and on-farm livestock disease monitoring technologies for multiplexed diagnosis in complex biological samples such as serum. The technique is simple in fabrication and provides an analytical response in less than 60 s.

Ultrasensitive electrochemical immunosensor for alpha fetoprotein detection based on platinum nanoparticles anchored on cobalt oxide/graphene nanosheets for signal amplification.

PubMed

Liu, Li; Tian, Lihui; Zhao, Guanhui; Huang, Yuzhen; Wei, Qin; Cao, Wei

2017-09-15

An ultrasensitive sandwich-type electrochemical immunosensor was developed for quantitative monitoring of Alpha fetoprotein (AFP). To achieve this objective, an incorporated signal amplification strategy of platinum nanoparticles anchored on cobalt oxide/graphene nanosheets (Pt NPs/Co 3 O 4 /graphene) was proposed by acting as the label of secondary antibodies. The prepared label not only empowered by advantages of each component but exhibited better electrochemical performance than single Pt NPs, Co 3 O 4 and graphene, which has shown large specific surface area and good catalytic activity towards the reduction of H 2 O 2 . Meanwhile, the nanocomposite of gold nanoparticles adhered on 3-mercaptopropyltriethoxysilane functionalized graphene sheets (Au@MPTES-GS) was used as matrix to accelerate electron transfer and immobilize primary antibodies in this system. The signal amplification mechanism of the matrix and the label were explored successfully. Under optimal conditions, the electrochemical immunosensor exhibited a wide linear range from 0.1 pg mL -1 to 60 ng mL -1 with a low detection limit of 0.029 pg mL -1 for AFP. The proposed immunosensor may have promising application in the clinical diagnosis of AFP and other tumor markers. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis and characterization of a stable, label-free optical biosensor from TiO2-coated porous silicon.

PubMed

Li, Jianlin; Sailor, Michael J

2014-05-15

A nanoscale layer of TiO2 is coated on the inner pore walls of a porous silicon (PSi) film by room-temperature infiltration of a TiO2 sol-gel precursor and firing at 500 °C. The PSi:TiO2 composite films are characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), energy dispersive X-ray spectral analysis (EDS), scanning electron microscopy (SEM) and reflective interferometric Fourier transform spectroscopy (RIFTS). The analysis indicates that TiO2 conformally coats the inner pore surfaces of the PSi film. The film displays greater aqueous stability in the pH range 2-12 relative to a PSi:SiO2 surface. A label-free optical interference immunosensor based on the TiO2-coated PSi film is demonstrated by real-time monitoring of the physical adsorption of protein A, followed by the specific binding of rabbit anti-sheep immunoglobulin (IgG) and then specific capture of sheep IgG. The time to achieve equilibrium for the physical adsorption of protein A on the surface of TiO2-coated PSi film is significantly greater than that of PSi film. The specificity of the protein A and rabbit anti-sheep IgG construct on the sensor is confirmed by tests with non-binding chicken IgG. The sensitivity of the immunosensor is shown to be 8210 ± 170 nm/refractive index unit (RIU). Copyright © 2013 Elsevier B.V. All rights reserved.

An Ultrasensitive Electrochemical Immunosensor for Alpha-Fetoprotein Using an Envision Complex-Antibody Copolymer as a Sensitive Label

PubMed Central

Xiong, Ping; Gan, Ning; Cao, Yuting; Hu, Futao; Li, Tianhua; Zheng, Lei

2012-01-01

A novel strategy is presented for sensitive detection of alfa-fetoprotein (AFP), using a horseradish peroxidase (HRP)-functionalized Envision antibody complex (EVC) as the label. The Envision-AFP signal antibody copolymer (EVC-AFP Ab2) was composed of a dextran amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of secondary antibody, and acted as a signal tag in the immunosensor. The sensor was constructed using the following steps: First, gold electrode (GE) was modified with nano-gold (AuNPs) by electro-deposition in HAuCl4 solution. The high affinity of the AuNPs surface facilitates direct formation of a self-assembled thiolated protein G layer. Next, the coated GE was incubated in a solution of AFP capture antibody (AFP Ab1); these antibodies attach to the thiolated protein G layer through their non-antigenic regions, leaving the antigen binding sites for binding of target analyte. Following a sandwich immunoreaction, an EVC-AFP Ab2-AFP-AFP Ab1 immunocomplex was formed on the electrode surface, allowing large amounts of HRP on the complex to produce an amplified electrocatalytic current of hydroquinone (HQ) in the presence of hydrogen peroxide (H2O2). Highly amplified detection was achieved, with a detection limit of 2 pg/mL and a linear range of 0.005–0.2 ng/mL for AFP in 10 μL undiluted serum; this is near or below the normal levels of most cancer biomarker proteins in human serum. Measurements of AFP in the serum of cancer patients correlated strongly with standard enzyme-linked immunosorbent assays. These easily fabricated EVC-modified immunosensors show excellent promise for future fabrication of bioelectronic arrays. By varying the target biomolecules, this technique may be easily extended for use with other immunoassays, and thus represents a versatile design route.

Dual-responsive immunosensor that combines colorimetric recognition and electrochemical response for ultrasensitive detection of cancer biomarkers.

PubMed

Hong, Wooyoung; Lee, Sooyeon; Cho, Youngnam

2016-12-15

We developed a nanoroughened, biotin-doped polypyrrole immunosensor for the detection of tumor markers through dual-signal (electrochemical and colorimetric) channels, electrochemical and colorimetric, that demonstrates remarkable analytical performance. A rapid, one-step electric field-mediated method was employed to fabricate the immunosensor with nanoscale roughness by simply modulating the applied electrical potential. We demonstrated the successful detection of three tumor markers (CA125, CEA, and PSA) via the double enzymatic signal amplifications in the presence of a target antigen, ultimately leading to desired diagnostic accuracy and reliability. The addition of multiple horseradish peroxidase (HRP)- and antibody-labeled nanoparticles greatly amplified the signal and simplified the measurement of cancer biomarker proteins by sequentially magnifying electrochemical and colorimetric signals in a single platform. The two parallel assays performed using the proposed immunosensor have yielded highly consistent and reproducible results. Additionally, for the analysis of plasma samples in a clinical setting, the values obtained with our immunosensor were validated by correlating the results with those of a standard radioimmunoassay (RIA), which obtained very similar clinically valid responses. Copyright © 2016 Elsevier B.V. All rights reserved.

Smartphone-based immunosensor for CA125 detection.

PubMed

Hosu, Oana; Ravalli, Andrea; Lo Piccolo, Giuseppe Mattia; Cristea, Cecilia; Sandulescu, Robert; Marrazza, Giovanna

2017-05-01

In this work, we report the design, the development and the characterization of the analytical performances of a colorimetric smartphone-based immunosensor for the detection of cancer antigen 125 (CA125). The immunosensor was based on a sandwich strategy in which the primary antibody was immobilized by spotting onto the 3D nitrocellulose membrane. The immunospots were subsequently incubated with CA125 solutions, followed by the affinity reaction with a secondary antibody labeled with gold nanoparticles (AuNPs). The antibody-AuNPs captured onto immunospots induced the silver deposition from a silver enhancer solution leading to the formation of gold-silver nanoparticles of different grey color spots depending on CA125 concentration. The 8 megapixels smartphone camera was integrated in a home-made dark box and used as transducer of color image acquisition and data handling. The pixel intensity of the captured images was determined by an image processing algorithm. The experimental parameters involved in each step of the immunosensor design were studied and optimized, obtaining a limit of detection of 30U/mL CA125. The selectivity of the immunoassay was proven against different concentration solutions of Vascular Endothelial Growth Factor (VEGF) antigen as an unspecific protein when a blank signal was obtained for all tested solutions. Finally, preliminary experiments in human serum samples spiked with CA125 protein were also performed. Therefore, the proposed system could represent a powerful point-of-care tool for the next generation technology for detecting and monitoring cancer biomarkers at early stages by taking advantage of nowadays gadgets with enhanced features such as smartphones. Copyright © 2017 Elsevier B.V. All rights reserved.

Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element.

PubMed

Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

2016-08-11

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

PubMed Central

Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

2016-01-01

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1. PMID:27529243

Electrochemical magneto immunosensor for the detection of anti-TG2 antibody in celiac disease.

PubMed

Kergaravat, Silvina V; Beltramino, Luis; Garnero, Nidia; Trotta, Liliana; Wagener, Marta; Isabel Pividori, Maria; Hernandez, Silvia R

2013-10-15

An electrochemical magneto immunosensor for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The immunological reaction is performed on magnetic beads (MBs) as a solid support in which the transglutaminase enzyme (TG2) is covalently immobilized (TG2-MB) and then ATG2 were revealed by an antibody labeled with peroxidase. The electrochemical response of the enzymatic reaction with o-phenilendiamine and H₂O₂ as substrates by square wave voltammetry was correlated with the ATG2. Graphite-epoxi composite cylindrical electrodes and screen printed electrodes were used as transducers in the immunosensor. A total number of 29 sera from clinically confirmed cases of celiac disease and 19 negative control sera were tested by the electrochemical magneto immunosensor. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 16.95 units was the most effective cut-off value (COV) to discriminate correctly between celiac and non-celiac patients. Using this point for prediction, sensitivity was found to be 100%, while specificity was 84%. Copyright © 2013 Elsevier B.V. All rights reserved.

Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly.

PubMed

Parfenov, Vladislav A; Koudan, Elizaveta V; Bulanova, Elena A; Karalkin, Pavel A; Pereira, Frederico DAS; Norkin, Nikita E; Knyazeva, Alisa D; Gryadunova, Anna A; Petrov, Oleg F; Vasiliev, M M; Myasnikov, Maxim; Chernikov, Valery P; Kasyanov, Vladimir A; Marchenkov, Artem Yu; Brakke, Kenneth A; Khesuani, Yusef D; Demirci, Utkan; Mironov, Vladimir A

2018-05-31

Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first-time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd3+) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd3+. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering. . © 2018 IOP Publishing Ltd.

Development of a Plastic-Based Microfluidic Immunosensor Chip for Detection of H1N1 Influenza

PubMed Central

Lee, Kyoung G.; Lee, Tae Jae; Jeong, Soon Woo; Choi, Ho Woon; Heo, Nam Su; Park, Jung Youn; Park, Tae Jung; Lee, Seok Jae

2012-01-01

Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device. PMID:23112630

Microfluidic immunosensor for rapid and highly-sensitive salivary cortisol quantification.

PubMed

Pinto, V; Sousa, P; Catarino, S O; Correia-Neves, M; Minas, G

2017-04-15

This paper presents a novel poly(dimethylsiloxane) (PDMS) microfluidic immunosensor that integrates a complementary metal-oxide-semiconductor (CMOS) optical detection system for a rapid and highly-sensitive quantification of salivary cortisol. The simple and non-invasive method of saliva sampling provides an interesting alternative to the blood, allowing a fast sampling at short intervals, relevant for many clinical diagnostic applications. The developed approach is based on the covalent immobilization of a coating antibody (Ab), a polyclonal anti-IgG, onto a treated PDMS surface. The coating Ab binds the capture Ab, an IgG specific for cortisol, allowing its correct orientation. Horseradish peroxidase (HRP)-labelled cortisol is added to compete with the cortisol in the sample, for the capture Ab binding sites. The HRP-labelled cortisol, bonded to the capture Ab, is measured through the HRP enzyme and the tetramethylbenzidine (TMB) substrate reaction. The cortisol quantification is performed by colorimetric detection of HRP-labelled cortisol, through optical absorption at 450nm, using a CMOS silicon photodiode as the photodetector. Under the developed optimized conditions presented here, e.g., microfluidic channels geometry, immobilization method and immunoassay conditions, the immunosensor shows a linear range of detection between 0.01-20ng/mL, a limit of detection (LOD) of 18pg/mL and an analysis time of 35min, featuring a great potential for point-of-care applications requiring continuous monitoring of the salivary cortisol levels during a circadian cycle. Copyright © 2016 Elsevier B.V. All rights reserved.

78 FR 47154 - Food Labeling; Gluten-Free Labeling of Foods

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-05

...The Food and Drug Administration (FDA or we) is issuing a final rule to define the term ``gluten-free'' for voluntary use in the labeling of foods. The final rule defines the term ``gluten-free'' to mean that the food bearing the claim does not contain an ingredient that is a gluten-containing grain (e.g., spelt wheat); an ingredient that is derived from a gluten-containing grain and that has not been processed to remove gluten (e.g., wheat flour); or an ingredient that is derived from a gluten-containing grain and that has been processed to remove gluten (e.g., wheat starch), if the use of that ingredient results in the presence of 20 parts per million (ppm) or more gluten in the food (i.e., 20 milligrams (mg) or more gluten per kilogram (kg) of food); or inherently does not contain gluten; and that any unavoidable presence of gluten in the food is below 20 ppm gluten (i.e., below 20 mg gluten per kg of food). A food that bears the claim ``no gluten,'' ``free of gluten,'' or ``without gluten'' in its labeling and fails to meet the requirements for a ``gluten-free'' claim will be deemed to be misbranded. In addition, a food whose labeling includes the term ``wheat'' in the ingredient list or in a separate ``Contains wheat'' statement as required by a section of the Federal Food, Drug, and Cosmetic Act (the FD&C Act) and also bears the claim ``gluten-free'' will be deemed to be misbranded unless its labeling also bears additional language clarifying that the wheat has been processed to allow the food to meet FDA requirements for a ``gluten-free'' claim. Establishing a definition of the term ``gluten-free'' and uniform conditions for its use in food labeling will help ensure that individuals with celiac disease are not misled and are provided with truthful and accurate information with respect to foods so labeled. We are issuing the final rule under the Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA).

An impedimetric immunosensor based on diamond nanowires decorated with nickel nanoparticles.

PubMed

Subramanian, Palaniappan; Motorina, Anastasiia; Yeap, Weng Siang; Haenen, Ken; Coffinier, Yannick; Zaitsev, Vladimir; Niedziolka-Jonsson, Joanna; Boukherroub, Rabah; Szunerits, Sabine

2014-04-07

Nanostructured boron-doped diamond has been investigated as a sensitive impedimetric electrode for the detection of immunoglobulin G (IgG). The immunosensor was constructed in a three-step process: (i) reactive ion etching of flat boron-doped diamond (BDD) interfaces to synthesize BDD nanowires (BDD NWs), (ii) electrochemical deposition of nickel nanoparticles (Ni NPs) on the BDD NWs, and (iii) immobilization of biotin-tagged anti-IgG onto the Ni NPs. Electrochemical impedance spectroscopy (EIS) was used to follow the binding of IgG at different concentrations without the use of any additional label. A detection limit of 0.3 ng mL(-1) (2 nM) with a dynamic range up to 300 ng mL(-1) (2 μM) was obtained with the interface. Moreover, the study demonstrated that this immunosensor exhibits good stability over time and allows regeneration by incubation in ethylenediaminetetraacetic acid (EDTA) aqueous solution.

A novel electrochemical immunosensor based on PG for early screening of depression markers-heat shock protein 70.

PubMed

Sun, Bolu; Cai, Jinying; Li, Wuyan; Gou, Xiaodan; Gou, Yuqiang; Li, Dai; Hu, Fangdi

2018-07-15

In this study, a novel electrochemical immunosensor for early screening of depression markers-heat shock protein 70 (HSP70) was successfully developed based on the porous graphene (PG) with huge specific surface area and excellent structure. Benefiting from the strong adsorption and good bioactivity of PG which was initially prepared via a simple pyrolysis process, a variety of heat shock protein70 (HSP70) can be firmly loaded on the PG to construct the basic electrode (HSP70/PG/GCE)，which was characterized by the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. Due to the HSP70 fixed on the surface of basic electrode and the HSP70 in the samples can competitively combine with the horseradish peroxidase labeled human HSP 70 antibody (HRP-Strept-Biotin-Ab). As a result, it presented a negative correlation between the concentration of HSP70 in samples and the detection signal of the proposed electrochemical immunosensor (HRP-Strept-Biotin-Ab-HSP70/PG/GCE) in the test liquid. The application of PG with excellent electrical conductivity in construction of immunosensor remarkably improved the sensitivity of the immunosensor for detection of HSP70. The proposed immunosensor demonstrated a wide linear range of 0.0448 ~ 100 ng/mL with a low detection limit of 0.02 ng/mL at 3σ. Moreover, the proposed immunosensor could be applied for the sensitive and efficient detection of HSP70 in real samples with good precision, acceptable stability, reproducibility and satisfactory results. Therefore, the HSP70 immunosensor provides a novel and convenient method for early clinical screening of depression markers-heat shock protein 70. Copyright © 2018 Elsevier B.V. All rights reserved.

Magneto immunosensor for gliadin detection in gluten-free foodstuff: towards food safety for celiac patients.

PubMed

Laube, T; Kergaravat, S V; Fabiano, S N; Hernández, S R; Alegret, S; Pividori, M I

2011-09-15

Gliadin is a constituent of the cereal protein gluten, responsible for the intolerance generated in celiac disease. Its detection is of high interest for food safety of celiac patients, since the only treatment known until now is a lifelong avoidance of this protein in the diet. Therefore, it is essential to have an easy and reliable method of analysis to control the contents in gluten-free foods. An electrochemical magneto immunosensor for the quantification of gliadin or small gliadin fragments in natural or pretreated food samples is described for the first time and compared to a novel magneto-ELISA system based on optical detection. The immunological reaction was performed on magnetic beads as solid support by the oriented covalent immobilization, of the protein gliadin on tosyl-activated beads. Direct, as well as indirect competitive immunoassays were optimized, achieving the best analytical performance with the direct competitive format. Excellent detection limits (in the order of μg L(-1)) were achieved, according to the legislation for gluten-free products. The matrix effect, as well as the performance of the assays was successfully evaluated using spiked gluten-free foodstuffs (skimmed milk and beer), obtaining excellent recovery values in the results. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of electrochemical based sandwich enzyme linked immunosensor for Cryptosporidium parvum detection in drinking water.

PubMed

Thiruppathiraja, Chinnasamy; Saroja, Veerappan; Kamatchiammal, Senthilkumar; Adaikkappan, Periyakaruppan; Alagar, Muthukaruppan

2011-10-01

Cryptosporidium parvum is one of the most important biological contaminants in drinking water and generates significant risks to public health. Due to low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industry analysis. This present study describes a simple, sensitive, enzyme amplified sandwich form of an electrochemical immunosensor using dual labeled gold nanoparticles (alkaline phosphatase and anti-oocysts monoclonal antibody) in indium tin oxide (ITO) as an electrode to detect C. parvum. The biosensor was fabricated by immobilizing the anti-oocysts McAb on a gold nanoparticle functionalized ITO electrode, followed by the corresponding capture of analytes and dual labeled gold nanoparticle probe to detect the C. parvum target. The outcome shows the sensitivity of electrochemical immune sensor enhanced by gold nanoparticles with a limit of detection of 3 oocysts/mL in a minimal processing period. Our results demonstrated the sensitivity of the new approach compared to the customary method and the immunosensors showed acceptable precision, reproducibility, stability, and could be readily applied to multi analyte determination for environmental monitoring.

Porous silicon based photoluminescence immunosensor for rapid and highly-sensitive detection of Ochratoxin A.

PubMed

Myndrul, Valerii; Viter, Roman; Savchuk, Maryna; Shpyrka, Nelya; Erts, Donats; Jevdokimovs, Daniels; Silamiķelis, Viesturs; Smyntyna, Valentyn; Ramanavicius, Arunas; Iatsunskyi, Igor

2018-04-15

A rapid and low cost photoluminescence (PL) immunosensor for the determination of low concentrations of Ochratoxin A (OTA) has been developed. This immunosensor was based on porous silicon (PSi) and modified by antibodies against OTA (anti-OTA). PSi layer was fabricated by metal-assisted chemical etching (MACE) procedure. Main structural parameters (pore size, layer thickness, morphology and nanograins size) and composition of PSi were investigated by means of X-Ray diffraction (XRD), scanning electron microscopy (SEM) and Raman spectroscopy. PL-spectroscopy of PSi was performed at room temperature and showed a wide emission band centered at 680 ± 20nm. Protein A was covalently immobilized on the surface of PSi, which in next steps was modified by anti-OTA and BSA in this way a anti-OTA/Protein-A/PSi structure sensitive towards OTA was designed. The anti-OTA/Protein-A/PSi-based immunosensors were tested in a wide range of OTA concentrations from 0.001 upto 100ng/ml. Interaction of OTA with anti-OTA/Protein-A/PSi surface resulted in the quenching of photoluminescence in comparison to bare PSi. The limit of detection (LOD) and the sensitivity range of anti-OTA/Protein-A/PSi immunosensors were estimated. Association constant and Gibbs free energy for the interaction of anti-OTA/Protein-A/PSi with OTA were calculated and analyzed using the interaction isotherms. Response time of the anti-OTA/Protein-A/PSi-based immunosensor toward OTA was in the range of 500-700s. These findings are very promising for the development of highly sensitive, and potentially portable immunosensors suitable for fast determination of OTA in food and beverages. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrochemical immunosensor for interferon-γ based on disposable ITO detector and HRP-antibody-conjugated nano gold as signal tag.

PubMed

Zhang, Yaru; Zhang, Bin; Ye, Xiaoli; Yan, Yuqi; Huang, Langhuan; Jiang, Zhenyou; Tan, Shaozao; Cai, Xiang

2016-02-01

Tuberculosis is the most frequent cause of infection-related death worldwide. A new disposable electrochemical immunosensor with low cost and simple fabrication was proposed to detect interferon-γ (IFN-γ). Diallyldimethylammonium chloride (PDDA) and Au nanoparticle (AuNP) composite were used to provide an efficient biointerface, horseradish peroxidase (HRP)-labeled antibody-conjugated AuNP (HRP-Ab2-AuNP) bioconjugates were used as a novel signal tag. The large amounts of HRP on the signal tag can catalyze the oxidation of Hydroquinone (HQ) by H2O2, which can induce an amplified reductive current. The catalytic reduction current was related to the amount of HRP immobilized on the surface, which itself was related to the concentration of IFN-γ. Under optimized conditions, the proposed immunosensor showed a high sensitivity and a linear range of 0.1-10,000pg/mL with a detection limit of 0.048pg/mL. The assay results of clinical serum samples obtained by the immunosensor were in acceptable agreement with the reference values. Therefore, the immunosensor possessed excellent clinical value in early diagnosis and control of tuberculosis. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Dithiobis(succinimidyl propionate) modified gold microarray electrode based electrochemical immunosensor for ultrasensitive detection of cortisol.

PubMed

Arya, Sunil K; Chornokur, Ganna; Venugopal, Manju; Bhansali, Shekhar

2010-06-15

Gold microelectrode arrays functionalized with dithiobis(succinimidyl propionate) self-assembled monolayer (SAM) have been used to fabricate an ultrasensitive, disposable, electrochemical cortisol immunosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized on the surface of gold microelectrode array and the sensors were exposed to solutions with different cortisol concentration. After C-Mab binding, unreacted active groups of DTSP were blocked using ethanol amine (EA) and label-free electrochemical impedance (EIS) technique was used to determine cortisol concentration. EIS results confirmed that EA/C-Mab/DTSP/Au based biosensor can accurately detect cortisol in the range of 1pM-100nM. The biosensor was successfully used for the measurement of cortisol in interstitial fluid in vitro. This research establishes the feasibility of using impedance based biosensor architecture for disposable, wearable cortisol detector. Copyright 2010 Elsevier B.V. All rights reserved.

The sandwich-type electrochemiluminescence immunosensor for α-fetoprotein based on enrichment by Fe3O4-Au magnetic nano probes and signal amplification by CdS-Au composite nanoparticles labeled anti-AFP.

PubMed

Zhou, Hankun; Gan, Ning; Li, Tianhua; Cao, Yuting; Zeng, Saolin; Zheng, Lei; Guo, Zhiyong

2012-10-09

A novel and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor was fabricated on a glassy carbon electrode (GCE) for ultra trace levels of α-fetoprotein (AFP) based on sandwich immunoreaction strategy by enrichment using magnetic capture probes and quantum dots coated with Au shell (CdS-Au) as the signal tag. The capture probe was prepared by immobilizing the primary antibody of AFP (Ab1) on the core/shell Fe(3)O(4)-Au nanoparticles, which was first employed to capture AFP antigens to form Fe(3)O(4)-Au/Ab1/AFP complex from the serum after incubation. The product can be separated from the background solution through the magnetic separation. Then the CdS-Au labeled secondary antibody (Ab2) as signal tag (CdS-Au/Ab2) was conjugated successfully with Fe(3)O(4)-Au/Ab1/AFP complex to form a sandwich-type immunocomplex (Fe(3)O(4)-Au/Ab1/AFP/Ab2/CdS-Au), which can be further separated by an external magnetic field and produce ECL signals at a fixed voltage. The signal was proportional to a certain concentration range of AFP for quantification. Thus, an easy-to-use immunosensor with magnetic probes and a quantum dots signal tag was obtained. The immunosensor performed at a level of high sensitivity and a broad concentration range for AFP between 0.0005 and 5.0 ng mL(-1) with a detection limit of 0.2 pg mL(-1). The use of magnetic probes was combined with pre-concentration and separation for trace levels of tumor markers in the serum. Due to the amplification of the signal tag, the immunosensor is highly sensitive, which can offer great promise for rapid, simple, selective and cost-effective detection of effective biomonitoring for clinical application. Copyright © 2012 Elsevier B.V. All rights reserved.

Interpretation of interference signals in label free integrated interferometric biosensors

NASA Astrophysics Data System (ADS)

Heikkinen, Hanna; Wang, Meng; Okkonen, Matti; Hast, Jukka; Myllylä, Risto

2006-02-01

In the future fast, simple and reliable biosensors will be needed to detect various analytes from different biosamples. This is due to fact that the needs of traditional health care are changing. In the future homecare of patients and peoples' responsibility for their own health will increase. Also, different wellness applications need new parameters to be analysed, reducing costs of traditional health care, which are increasing rapidly. One fascinating and promising sensor type for these applications is an integrated optical interferometric immunosensor, which is manufactured using organic materials. The use of organic materials opens up enormous possibilities to develop different biochemical functions. In label free biosensors the measurement is based on detecting changes in refractive index, which typically are in the range of 10 -6-10 -8 [1]. In this research, theoretically generated interferograms are used to compare various signal processing methods. The goal is to develop an efficient method to analyse the interferogram. Different time domain signal processing methods are studied to determine the measuring resolution and efficiency of these methods. A low cost CCD -element is used in detecting the interferogram dynamics. It was found that in most of the signal processing methods the measuring resolution was mainly limited by pixel size. With calculation of Pearson's correlation coefficient, subpixel resolution was achieved which means that nanometer range optical path differences can be measured. This results in the refractive index resolution of the order of 10 -7.

Faraday cage-type electrochemiluminescence immunosensor for ultrasensitive detection of Vibrio vulnificus based on multi-functionalized graphene oxide.

PubMed

Guo, Zhiyong; Sha, Yuhong; Hu, Yufang; Yu, Zhongqing; Tao, Yingying; Wu, Yanjie; Zeng, Min; Wang, Sui; Li, Xing; Zhou, Jun; Su, Xiurong

2016-10-01

A novel Faraday cage-type electrochemiluminescence (ECL) immunosensor devoted to the detection of Vibrio vulnificus (VV) was fabricated. The sensing strategy was presented by a unique Faraday cage-type immunocomplex based on immunomagnetic beads (IMBs) and multi-functionalized graphene oxide (GO) labeled with (2,2'-bipyridine)(5-aminophenanthroline)ruthenium (Ru-NH2). The multi-functionalized GO could sit on the electrode surface directly due to the large surface area, abundant functional groups, and good electronic transport property. It ensures that more Ru-NH2 is entirely caged and become "effective," thus improving sensitivity significantly, which resembles extending the outer Helmholtz plane (OHP) of the electrode. Under optimal conditions, the developed immunosensor achieves a limit of detection as low as 1 CFU/mL. Additionally, the proposed immunosensor with high sensitivity and selectivity can be used for the detection of real samples. The novel Faraday cage-type method has shown potential application for the diagnosis of VV and opens up a new avenue in ECL immunoassay. Graphical abstract Faraday cage-type immunoassay mode for ultrasensitive detection by extending OHP.

Label-free detection of protein-ligand interactions by the quartz crystal microbalance.

PubMed

Janshoff, Andreas; Steinem, Claudia

2005-01-01

In recent years the quartz crystal microbalance (QCM) has been accepted as a powerful technique to monitor adsorption processes at interfaces in different chemical and biological research areas. In the last decade, the investigation of adsorption of biomolecules on functionalized surfaces turned out to be one of the paramount applications of the QCM comprising the interaction of nucleic acids, specific molecular recognition of protein-receptor couples, and antigen-antibody reactions realized in immunosensors. The advantage of the QCM technique is that it allows for a label free detection of molecules. This is a result of the fact that the frequency response of the quartz resonator is proportional to the increase in thickness of the adsorbed layer. However, in recent years it became more and more evident that quartz resonators used in fluids are more than mere mass or thickness sensors. The sensor response is also influenced by viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules and surface roughness. These phenomena have been used to get new insights in the adhesion process of living cells and to understand their response to pharmacological substances by determining morphological changes of the cells. In this chapter we describe a protocol to explore the kinetics and thermodynamics of specific interactions of different proteins such as lectins and annexins with their ligands using receptor bearing solid supported lipid bilayers.

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.

A Sensitive Electrochemical Immunosensor Based on PAMAM Dendrimer-Encapsulated Au for Detection of Norfloxacin in Animal-Derived Foods.

PubMed

Liu, Bing; Li, Min; Zhao, Yaoshuai; Pan, Mingfei; Gu, Ying; Sheng, Wei; Fang, Guozhen; Wang, Shuo

2018-06-15

In this work, a sensitive electrochemical immunosensor has been reported for the determination of norfloxacin in animal-derived foods. The poly (amidoamine) dendrimer encapsulated gold nanoparticles (PAMAM-Au) played dual roles in the proposed sensing platform which not only accelerated the electron transfer process of sensing, but also increased the efficiency of the immobilized antibody. The HRP-labeled antigen, as the signal labels in the immunosensor, was introduced to catalyze the following reaction of the substrate hydroquinone with the aid of H₂O₂ in the competitive reaction. On the basis of the signal amplification of PAMAM-Au, the signal intensity was linearly related to the concentration of norfloxacin in the range of 1 μg·L −1 ⁻10 mg·L −1 . All the results showed that the proposed strategy with low LOD (0.3837 μg·L −1 ) and favorable recovery (91.6⁻106.1%) in the practical sample, and it could provide a suitable protocol for norfloxacin detection in animal-derived foods with high sensitivity, good accuracy, and stability.

Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients.

PubMed

Maldonado, Jesús; González-Guerrero, Ana Belén; Domínguez, Carlos; Lechuga, Laura M

2016-11-15

Spontaneous bacterial peritonitis is an acute bacterial infection of ascitic fluid; it has a high incidence in cirrhotic patients and it is associated with high mortality. In such a situation, early diagnosis and treatment is crucial for the survival of the patient. However, bacterial analysis in ascitic fluid is currently based on culture methods, which are time-consuming and laborious. We report here the application of a photonic interferometer biosensor based on a bimodal waveguide (BiMW) for the rapid and label-free detection of bacteria directly in ascitic fluid. The device consists of a straight waveguide in which two modes of the same polarization interfere while interacting with the external medium through their evanescent fields. A bimolecular event occurring on the sensor area of the device (e.g. capturing bacteria) will differently affect each light mode, inducing a variation in the phase of the light exiting at the output of the waveguide. In this work, we demonstrate the quantitative detection of Bacillus cereus in buffer medium and Escherichia coli in undiluted ascitic fluid from cirrhotic patients. In the case of Bacillus cereus detection, the device was able to specifically detect bacteria at relevant concentrations in 12.5min and in the case of Escherichia coli detection, the analysis time was 25min. Extrapolation of the data demonstrated that the detection limits of the biosensor could reach few bacteria per milliliter. Based on the results obtained, we consider that the BiMW biosensor is positioned as a promising new clinical tool for user-friendly, cost-effective and real-time microbiological analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative differentiation of multiple virus in blood using nanoporous silicon oxide immunosensor and artificial neural network.

PubMed

Chakraborty, W; Ray, R; Samanta, N; RoyChaudhuri, C

2017-12-15

In spite of the rapid developments in various nanosensor technologies, it still remains challenging to realize a reliable ultrasensitive electrical biosensing platform which will be able to detect multiple viruses in blood simultaneously with a fairly high reproducibility without using secondary labels. In this paper, we have reported quantitative differentiation of Hep-B and Hep-C viruses in blood using nanoporous silicon oxide immunosensor array and artificial neural network (ANN). The peak frequency output (f p ) from the steady state sensitivity characteristics and the first cut off frequency (f c ) from the transient characteristics have been considered as inputs to the multilayer ANN. Implementation of several classifier blocks in the ANN architecture and coupling them with both the sensor chips, functionalized with Hep-B and Hep-C antibodies have enabled the quantification of the viruses with an accuracy of around 95% in the range of 0.04fM-1pM and with an accuracy of around 90% beyond 1pM and within 25nM in blood serum. This is the most sensitive report on multiple virus quantification using label free method. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

NASA Astrophysics Data System (ADS)

Tran, Quang Huy; Hanh Nguyen, Thi Hong; Mai, Anh Tuan; Thuy Nguyen, Thi; Khue Vu, Quang; Nga Phan, Thi

2012-03-01

This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml-1 to 1 μg ml-1, and the limit of detection was about 10 ng ml-1. This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks.

Immunosensor based on electrodeposition of gold-nanoparticles and ionic liquid composite for detection of Salmonella pullorum.

PubMed

Wang, Dan; Dou, Wenchao; Zhao, Guangying; Chen, Yan

2014-11-01

In order to increase the reproducibility and stability of electrochemical immunosensor, which is a key issue for its application and popularization, an accurate and stable immunosensor for rapid detection of Salmonella pullorum (S. pullorum) was proposed in this study. The immunosensor was fabricated by modifying Screen-printed Carbon Electrode (SPCE) with electrodeposited gold nanoparticles (AuNPs), HRP-labeled anti-S. pullorum and ionic liquids (ILs) (AuNP/HRP/IL). AuNPs are electrodeposited on the working electrode surface to increase the amount of antibodies that bind to the electrode and then modified with ILs to protect the antibodies from being inactivated in the test environment and maintain their biological activity and the stability of the detection electrode. The electrochemical characteristics of the stepwise modified electrodes and the detection of S. pullorum were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). As shown in the results of the experiments, AuNPs with unique electrochemical properties as well as biocompatibility characteristics have been proven to be able to strengthen the antibody combination effectively and to increase the electrochemical response signal. In addition, a crucial assessment regarding implementation of stability and reproducibility analysis of a range of immunosensors is provided. We found that application of AuNPs/ILs in the immune modified electrodes showed obvious improvement when compared with other groups. Given their high levels of reproducibility, stability, target specificity and sensitivity, AuNPs and ILs were considered to be excellent elements for electrode modification. Copyright © 2014 Elsevier B.V. All rights reserved.

Carbon nanohorn sensitized electrochemical immunosensor for rapid detection of microcystin-LR.

PubMed

Zhang, Jing; Lei, Jianping; Xu, Chuanlai; Ding, Lin; Ju, Huangxian

2010-02-01

A sensitive electrochemical immunosensor was proposed by functionalizing single-walled carbon nanohorns (SWNHs) with analyte for microcystin-LR (MC-LR) detection. The functionalization of SWNHs was performed by covalently binding MC-LR to the abundant carboxylic groups on the cone-shaped tips of SWNHs in the presence of linkage reagents and characterized with Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, and a transmission electron micrograph. Compared with single-walled carbon nanotubes, SWNHs as immobilization matrixes showed a better sensitizing effect. Using home-prepared horseradish peroxidase-labeled MC-LR antibody for the competitive immunoassay, under optimal conditions, the immunosensor exhibited a wide linear response to MC-LR ranging from 0.05 to 20 microg/L with a detection limit of 0.03 microg/L at a signal-to-noise of 3. This method showed good accuracy, acceptable precision, and reproducibility. The assay results of MC-LR in polluted water were in a good agreement with the reference values. The proposed strategy provided a biocompatible immobilization and sensitized recognition platform for analytes as small antigens and possessed promising application in food and environmental monitoring.

Development of optical immunosensors for detection of proteins in serum.

PubMed

Kyprianou, Dimitris; Chianella, Iva; Guerreiro, Antonio; Piletska, Elena V; Piletsky, Sergey A

2013-01-15

The detection of proteins in biological samples such as blood, serum or plasma by biosensors is very challenging due to the complex nature of the matrix, which contains a high level of many interfering compounds. Here we show the application of a novel polymeric immobilisation matrix that helps in the detection of specific protein analytes in biological samples by surface plasmon resonance (SPR) immunosensors. This polymer matrix contains thioacetal functional groups included in the network, and these groups do not require any further activation in order to react with proteins, making it attractive for sensor fabrication. The protein prostate specific antigen (PSA) was selected as a model target analyte. A sandwich format with two primary antibodies recognising different parts (epitopes) of the analyte was used for the detection of PSA in serum. The efficiency of the reduction of non-specific binding achieved with novel polymer was compared with those of other techniques such as coating of sensor surface with polyethylene glycol (PEG), use of charged hydrophilic aspartic acid and surfactants such as Tween20. The detection limit of the polymer based immunosensor was 0.1 ng ml(-1) for free form PSA (f-PSA) in buffer and 5 ng ml(-1) in 20% serum. This is an improvement compared with similar devices reported on literature, indicating the potential of the immunosensor developed here for the analysis of real samples. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel piezoelectric quartz micro-array immunosensor for detection of immunoglobulinE.

PubMed

Yao, Chunyan; Chen, Qinghai; Chen, Ming; Zhang, Bo; Luo, Yang; Huang, Qing; Huang, Junfu; Fu, Weiling

2006-12-01

A novel multi-channel 2 x 5 model of piezoelectric (PZ) micro-array immunosensor has been developed for quantitative detection of human immunoglobulinE (IgE) in serum. Every crystal unit of the fabricated piezoelectric IgE micro-array immunosensor can oscillate without interfering each other. A multi-channel 2 x 5 model micro-array immunosensor as compared with the traditional one-channel immunosensor can provide eight times higher detection speeds for IgE assay. The anti-IgE antibody is deposited on the gold electrode's surface of 10 MHz AT-cut quartz crystals by SPA (staphylococcal protein A), and serves as an antibody recognizing layer. The highly ordered antibody monolayers ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The uniform amount of antibody monolayer coated by the SPA is good, and non-specific reaction caused by other immunoglobulin in sample is found. The fabricated PZ immunosensor can be used for human IgE determination in the range of 5-300 IU/ml with high precision (CV is 4%). 50 human serum samples were detected by the micro-array immunosensor, and the results agreed well with those given by the commercially ELISA test kits. The correlation coefficient is 0.94 between ELISA and PZ immunosensor. After regeneration with NaOH the coated immunosensor can be reused 6 times without appreciable loss of activity.

Electrochemical characterization of an immunosensor for Salmonella spp. detection

USDA-ARS?s Scientific Manuscript database

Immunosensors represent a rapid alternative method for diagnosing Salmonella contamination. The objective of this study was to develop and evaluate the performance of an electrochemical immunosensor for the detection of Salmonella spp., the most common foodborne pathogen worldwide. In the immunosens...

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.

Optical immunosensors for detection of Listeria monocytogenes and Salmonella enteritidis from food

NASA Astrophysics Data System (ADS)

Bhunia, Arun K.; Geng, Tao; Lathrop, Amanda; Valadez, Angela; Morgan, Mark T.

2004-03-01

Listeria monocytogenes and Salmonella are two major foodborne pathogens of significant concern. Two optical evanescent wave immunosensors were evaluated for detection: Antibody-coupled fiber-optic biosensor and a surface plasmon resonant (SPR) immunosensor. In the fiber-optic sensor, polyclonal antibodies for the test organisms were immobilized on polystyrene fiber wave -guides using streptavidin - biotin chemistry. Cyanine 5 -labeled monoclonal antibodies C11E9 (for L. monocytogenes) and SF-11 (for Salmonella Enteritidis) were used to generate a specific fluorescent signal. Signal acquisition was performed by launching a laser-light (635 nm) from an Analyte-2000. This immunosensor was able to detect 103 - 109 cfu/ml of L. monocytogenes or 106-109 cfu/ml of Salmonella Enteritidis and the assays were conducted at near real-time with results obtained within one hour of sampling. The assays were specific and showed signal even in the presence of other microorganisms such as E. coli, Enterococcus faecalis or Salmonella Typhimurium. In the SPR system, IAsys instrument (resonant mirror sensor) was used. Monoclonal antibody-C11E9 was directly immobilized onto a carboxylate cuvette. Whole Listeria cells at various concentrations did not yield any signal while surface protein extracts did. Crude protein extracts from L. monocytogenes and L. innocua had average binding responses of around 150 arc sec (0.25 ng/mm2), which was significantly different from L. grayi, L. ivanovii, or L. welshimeri with average responses of <48 arc sec. Both fiber-optic and SPR sensors show promise in near real-time detection of foodborne L. monocytogenes and Salmonella Enteritidis.

Label-Free Sensors Based on Graphene Field-Effect Transistors for the Detection of Human Chorionic Gonadotropin Cancer Risk Biomarker

PubMed Central

Haslam, Carrie; Damiati, Samar; Whitley, Toby; Ifeachor, Emmanuel

2018-01-01

We report on the development of label-free chemical vapour deposition (CVD) graphene field effect transistor (GFET) immunosensors for the sensitive detection of Human Chorionic Gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFET sensors were fabricated on Si/SiO2 substrate using photolithography with evaporated chromium and sputtered gold contacts. GFET channels were functionalised with a linker molecule to an immobile anti-hCG antibody on the surface of graphene. The binding reaction of the antibody with varying concentration levels of hCG antigen demonstrated the limit of detection of the GFET sensors to be below 1 pg/mL using four-probe electrical measurements. We also show that annealing can significantly improve the carrier transport properties of GFETs and shift the Dirac point (Fermi level) with reduced p-doping in back-gated measurements. The developed GFET biosensors are generic and could find applications in a broad range of medical diagnostics in addition to cancer, such as neurodegenerative (Alzheimer’s and Parkinson’s) and cardiovascular disorders. PMID:29316718

Human heart failure biomarker immunosensor based on excessively tilted fiber gratings.

PubMed

Luo, Binbin; Wu, Shengxi; Zhang, Zhonghao; Zou, Wengen; Shi, Shenghui; Zhao, Mingfu; Zhong, Nianbing; Liu, Yong; Zou, Xue; Wang, Lingling; Chai, Weina; Hu, Chuanmin; Zhang, Lin

2017-01-01

A label-free immunosensor platform based on excessively tilted fiber gratings (Ex-TFGs) was developed for highly specific and fast detection of human N-terminal pro-B-type natriuretic peptide (NT-proBNP), which is considered a powerful biomarker for prognosis and risk stratification of heart failure (HF). High-purity anti-NT-proBNP monoclonal antibodies (MAbs) prepared in our laboratory were immobilized on fiber surface through the staphylococcal protein A (SPA) method for subsequent specific binding of the targeted NT-proBNP. Utilizing fiber optic grating demodulation system (FOGDS), immunoassays were carried out in vitro by monitoring the resonance wavelength shift of Ex-TFG biosensor with immobilized anti-NT-proBNP MAbs. Lowest detectable concentration of ~0.5ng/mL for NT-proBNP was obtained, and average sensitivity for NT-proBNP at a concentration range of 0~1.0 ng/mL was approximately 45.967 pm/(ng/mL). Several human serum samples were assessed by the proposed Ex-TFG biomarker sensor, with high specificity for NT-proBNP, indicating potential application in early diagnosing patients with acute HF symptoms.

Ultrasensitive competitive method-based electrochemiluminescence immunosensor for diethylstilbestrol detection based on Ru(bpy)32+ as luminophor encapsulated in metal-organic frameworks UiO-67.

PubMed

Dong, Xue; Zhao, Guanhui; Liu, Li; Li, Xuan; Wei, Qin; Cao, Wei

2018-07-01

In this work, Ru(bpy) 3 2+ encapsulated in metal-organic frameworks (MOFs) UiO-67 (Ru(bpy) 3 2+ /UiO-67) as luminophor was easily prepared and firstly applied in constructing an electrochemiluminescence (ECL) immunosensor to efficiently estimate diethylstilbestrol (DES). The competitive method-based ECL immunosensor platform was fabricated by amino-silicon dioxide which possesses large surface area. The poriness of UiO-67 was splendid so that Ru(bpy) 3 2+ could be easily encapsulated. Ru(bpy) 3 2+ /UiO-67 with excellent ECL luminescence signal existed large specific surface area for easily labeled with antibodies. DES competed with bovine serum albumin-diethylstilbestrol (BSA-DES) for binding to antibody-specific sites in the constructed immunosensor. However DES was micromolecule, which was easier to bond to antibodies than BSA-DES. The ECL signal was gradually decreases with the increase of the concentration of DES. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.01 pg mL -1 to 50 ng mL -1 with a low detetion limit of 3.27 fg mL -1 (S/N = 3). The novel fabricated immunosensor with interference immunity and high stability may cause an attractive approach for the other targets determination. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel fiber optic immunosensor instrument

NASA Astrophysics Data System (ADS)

Wang, Zhiyu; Huang, Wenling; Tang, Lei; Zhou, Bo; Li, Yugi; He, Jun

1996-09-01

It has developed and performed a novel fiberoptic immunosensor instrument with operating wavelength 400 - 760 nm and repeatability cv equals 0.27%. The instrument has many excellent features such as simplified operation, faster testing time, higher sensitivity and economic cost. It has completely eliminated recovery period which traditional immunosensor owned due to use separative sensor structure. It can widely apply to test for bacteria, virus, hormone, parasite and cancer protein in clinical examination. The instrument has operated in laboratory and relevant medicine units and successfully tested monoclonal rat-anti-human of 413 cases in clinic and prepared with existing ELISA method, the coincidence probability reached 94 to 100%.

On-chip surface-enhanced Raman spectroscopy (SERS)-linked immuno-sensor assay (SLISA) for rapid environmental-surveillance of chemical toxins

NASA Astrophysics Data System (ADS)

Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J.

2005-05-01

The increasing threat of an intentional (attack) or accidental release of toxins, in particular chemical toxins, including chemical warfare agents (CWAs) and toxic industrial chemicals (TICs) has increased public fear. The major problem in such attacks/accidents is to detect toxins present in very low levels. Indeed, several detection techniques are currently being used for the same. However, none of them meet the most critical requirements of a RISE (Rapid, Inexpensive, Simple and Effective) detect-to-protect class of biosensors. To address this critical demand our group has developed a prototype lab-on-a-chip (LOC) using a colloidal silver-based, surface-enhanced Raman spectroscopy (SERS)-linked immuno-sensor assay (SLISA). The LOC-SLISA was tested for the measurement of RAD54, a stress-marker protein expressed by yeast in response to hydrogen peroxide (H2O2), a toxin in the EPA priority list of chemical toxins. We found SLISA has good correlation in accuracy with the traditional ELISA technique and outperforms the latter by being rapid and easy-to-use. SLISA is more sensitive, provides qualitative information on immuno-sensor's chemical characterization and antigen-antibody binding, and allows direct detection with minimal or no chance of uncertainty, which is a stringent limitation of all label-based biosensor technologies including ELISA. For translational significance of our work, we correlated our results to U.S. EPA (environmental protection agency) defined risk exposure guideline levels of H2O2 to validate the commercial potential of our on-chip SLISA. The label-free, cell-based and RISE detection offered by SERS can allow development of biomedical and environmental sensor technology (BEST) needed for direct, rapid and continuous monitoring of human health and environment

A regenerating ultrasensitive electrochemical impedance immunosensor for the detection of adenovirus.

PubMed

Lin, Donghai; Tang, Thompson; Jed Harrison, D; Lee, William E; Jemere, Abebaw B

2015-06-15

We report on the development of a regenerable sensitive immunosensor based on electrochemical impedance spectroscopy for the detection of type 5 adenovirus. The multi-layered immunosensor fabrication involved successive modification steps on gold electrodes: (i) modification with self-assembled layer of 1,6-hexanedithiol to which gold nanoparticles were attached via the distal thiol groups, (ii) formation of self-assembled monolayer of 11-mercaptoundecanoic acid onto the gold nanoparticles, (iii) covalent immobilization of monoclonal anti-adenovirus 5 antibody, with EDC/NHS coupling reaction on the nanoparticles, completing the immunosensor. The immunosensor displayed a very good detection limit of 30 virus particles/ml and a wide linear dynamic range of 10(5). An electrochemical reductive desorption technique was employed to completely desorb the components of the immunosensor surface, then re-assemble the sensing layer and reuse the sensor. On a single electrode, the multi-layered immunosensor could be assembled and disassembled at least 30 times with 87% of the original signal intact. The changes of electrode behavior after each assembly and desorption processes were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy techniques. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Gluten-Free Labeling of Foods

MedlinePlus

... Vaccines, Blood & Biologics Animal & Veterinary Cosmetics Tobacco Products Food Home Food Guidance & Regulation Guidance Documents & Regulatory Information by Topic Allergens Gluten-Free Labeling of Foods Share Tweet Linkedin Pin it More sharing options ...

Current Trends in Nanomaterial-Based Amperometric Biosensors

PubMed Central

Hayat, Akhtar; Catanante, Gaëlle; Marty, Jean Louis

2014-01-01

The last decade has witnessed an intensive research effort in the field of electrochemical sensors, with a particular focus on the design of amperometric biosensors for diverse analytical applications. In this context, nanomaterial integration in the construction of amperometric biosensors may constitute one of the most exciting approaches. The attractive properties of nanomaterials have paved the way for the design of a wide variety of biosensors based on various electrochemical detection methods to enhance the analytical characteristics. However, most of these nanostructured materials are not explored in the design of amperometric biosensors. This review aims to provide insight into the diverse properties of nanomaterials that can be possibly explored in the construction of amperometric biosensors. PMID:25494347

Multiple signal amplification strategies for ultrasensitive label-free electrochemical immunoassay for carbohydrate antigen 24-2 based on redox hydrogel.

PubMed

Tang, Zhongxue; Fu, Yuanyuan; Ma, Zhanfang

2017-05-15

In this work, multiple signal amplification strategies for ultrasensitive label-free electrochemical immunoassay for carbohydrate antigen 24-2 (CA242) were developed using redox sodium alginate-Pb 2+ -graphene oxide (SA-Pb 2+ -GO) hydrogel. The SA-Pb 2+ -GO hydrogel was synthesised by simply mixing SA, GO, and Pb 2+ and then implemented as a novel redox species with a strong current signal at -0.46V (vs. Ag/AgCl). After the three-dimensional and porous SA-Pb 2+ -GO hydrogel was in situ generated on a glassy carbon electrode (GCE), chitosan was adsorbed on the obtained electrode to further enrich Pb 2+ . When chitosan-Pb 2+ /SA-Pb 2+ -GO/GCE was incubated with anti-CA242 using glutaraldehyde and blocked by bovine serum albumin, the immunoassay platform for CA242 was obtained. Owing to the addition of GO, the obtained conductive SA-GO/GCE was beneficial for signal amplification. After incubating SA-GO/GCE with excessive amounts of Pb 2+ , the resistance of SA-Pb 2+ -GO/GCE further decreased and a strong redox signal was obtained. The chitosan fixed by electrostatic adsorption resulted in further adsorption of Pb 2+ , behaving as further amplifying the signal and improving conductivity. In this case, multiple signal amplification strategies were involved in the proposed immunosensor for the ultrasensitive detection of CA242. Under the optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.005UmL -1 to 500UmL -1 with an ultralow detection limit of 0.067mUmL -1 . In comparison to previous works, the sensitivity of this method was 32.98μA (log 10 C CA242 ) -1 , which was a five-fold increase from the previous works. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Immunosensors using a quartz crystal microbalance

NASA Astrophysics Data System (ADS)

Kurosawa, Shigeru; Aizawa, Hidenobu; Tozuka, Mitsuhiro; Nakamura, Miki; Park, Jong-Won

2003-11-01

Better analytical technology has been demanded for accurate and rapid determination of trace amounts of chemical compounds, such as marker proteins for disease or endocrine disrupters like dioxin, which might be contained in blood, food and the environment. The study of immunosensors using a quartz crystal microbalance (QCM) has recently focused on conventional detection methods for the determination of chemical compounds together with the development of reagents and processes. This paper introduces the principle of the detection method of QCM immunosensors developed at AIST and its application to the detection of trace amounts of chemical compounds.

Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model.

PubMed

Zhong, Zhaoyang; Wu, Wei; Wang, Dong; Wang, Dan; Shan, Jinlu; Qing, Yi; Zhang, Zhimin

2010-06-15

A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA), as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays. The device consisted of a glassy carbon electrode coated with Prussian Blue (PB) on whose surface gold nanoparticles were electrochemically deposited to the further modified with the specific analyte-capturing molecule, anti-CEA antibodies. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN). The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s). The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. Importantly, the methodology provides a promising ultrasensitive assay strategy for clinical applications. Copyright 2010 Elsevier B.V. All rights reserved.

A piezoelectric immunosensor for the detection of alpha-fetoprotein using an interface of gold/hydroxyapatite hybrid nanomaterial.

PubMed

Ding, Yanjun; Liu, Jia; Wang, Hua; Shen, Guoli; Yu, Ruqin

2007-04-01

The ideal immobilization methods that are suitable for binding immuno-active materials with high efficiency onto the sensing surface are the key target to pursue in the current biosensor design. In this paper, a new hybrid material formed by assembling gold nanoparticles (GNP) onto nano-sized hydroxyapatite (HA) has been employed for the interface design of piezoelectric immunosensor, on which the antibodies were bound. The detection performances of the resulting immunosensor were investigated by use of the antibody-antigen model system of alpha-Fetoprotein (AFP), an important indicator in the diagnosis of clinical cancers. The hybrid material was characterized by the UV-vis spectroscopy, the SEM and TEM measurements. The frequency and electrochemical impedance responses characteristics for the processes of immobilization and immunoreaction of anchored anti-AFP antibodies were studied in detail. The immunoresponse of the proposed immunosensor was compared with those antibodies immobilized by using HA or GNP alone. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of AFP in the concentration range of 15.3-600.0ngml(-1). Such an interface design with the nano-sized hybrid materials should be tailored as a new alternative used for biosensor design.

A sandwich-type electrochemical immunosensor based on the biotin- streptavidin-biotin structure for detection of human immunoglobulin G

PubMed Central

Li, Yueyun; Zhang, Yihe; Jiang, Liping; Chu, Paul K.; Dong, Yunhui; Wei, Qin

2016-01-01

A sandwich-type immunosensor is designed and fabricated to detect the human immunoglobulin G (HIgG) using polyaniline and tin dioxide functionalized graphene (GS-SnO2-PAN) as the platform and biotin-functionalized amination magnetic nanoparticles composite (B-Fe3O4@APTES) as the label. GS-SnO2-PAN is used as the sensing agent to capture the primary anti-HIgG (Ab1) and SnO2 reduces the stack of GS. The B-Fe3O4@APTES with a large surface area and excellent biocompatibility captures second antibody (Ab2) efficiently based on the highly selective recognition of streptavidin to biotinylated antibody. The B-Fe3O4@APTES has better electro-catalytic activity in the reduction of hydrogen peroxide (H2O2) and the “biotin-streptavidin-biotin” (B-SA-B) strategy leads to signal amplification. Under optimal conditions, the immunosensor has a wide sensitivity range from 1 pg/L to 10 ng/L and low detection limit of 0.33 pg/L (S/N = 3) for HIgG. The immunosensor has high sensitivity, fast assay rate, as well as good reproducibility, specificity, and stability especially in the quantitative detection of biomolecules in serum samples. PMID:26948273

Label-free probing of genes by time-domain terahertz sensing.

PubMed

Haring Bolivar, P; Brucherseifer, M; Nagel, M; Kurz, H; Bosserhoff, A; Büttner, R

2002-11-07

A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a freespace analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

Using label-free screening technology to improve efficiency in drug discovery.

PubMed

Halai, Reena; Cooper, Matthew A

2012-02-01

Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

Ultrasensitive determination of human growth hormone (hGH) with a disposable electrochemical magneto-immunosensor.

PubMed

Serafín, V; Úbeda, N; Agüí, L; Yáñez-Sedeño, P; Pingarrón, J M

2012-05-01

In this paper, an electrochemical magneto-immunosensor for the detection of human growth hormone (hGH) is described for the first time. The immunosensor involves the use of tosyl-activated magnetic microparticles (TsMBs) to covalently immobilize a monoclonal mAbhHG antibody. A sandwich-type immunoassay with a secondary pAbhGH antibody and anti-IgG labelled with alkaline phosphatase (anti-IgG-AP) was employed. TsMBs–mAbhGH–hGH–pAbhGH–anti-IgG-AP conjugates were deposited onto the surface of a screen-printed gold electrode using a small neodymium magnet, and electrochemical detection was performed by square-wave voltammetry upon the addition of 4-aminophenyl phosphate as the AP substrate. All the variables involved in the preparation of immunoconjugates and in the immunoassay protocol were optimized. A calibration curve for hGH was constructed with a linear range between 0.01 and 100 ng/mL (r = 0.998) and a limit of detection of 0.005 ng/mL. This value is nearly three orders of magnitude lower than that obtained using surface plasmon resonance (Treviño et al., Talanta 78:1011-1016, 2009). Furthermore, good repeatability, with RSD = 3% (n = 10) at the 1-ng/mL hGH level, was obtained. Cross-reactivity studies with other hormones demonstrated good selectivity. The magneto-immunosensor was applied to the analysis of human serum spiked with hGH at the 4- and 0.1-ng/mL levels. Mean recoveries of 96 ± 6% and 99 ± 2%, respectively, were obtained.

Emerging applications of label-free optical biosensors

NASA Astrophysics Data System (ADS)

Zanchetta, Giuliano; Lanfranco, Roberta; Giavazzi, Fabio; Bellini, Tommaso; Buscaglia, Marco

2017-01-01

Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

DNAzyme-functionalized gold-palladium hybrid nanostructures for triple signal amplification of impedimetric immunosensor.

PubMed

Hou, Li; Gao, Zhuangqiang; Xu, Mingdi; Cao, Xia; Wu, Xiaoping; Chen, Guonan; Tang, Dianping

2014-04-15

A highly sensitive and selective impedimetric immunosensor with triple signal amplification was designed for ultrasensitive detection of prostate-specific antigen (PSA) by using anti-PSA antibody and DNAzyme-functionalized gold-palladium hybrid nanotags (Ab2-AuPd-DNA). The signal was amplified based on the Ab2-AuPd-DNA toward the catalytic precipitation of 4-choloro-1-naphthol (4-CN). DNAzyme (as a kind of peroxidase mimic) could catalyze the oxidation of 4-CN, whilst AuPd hybrid nanostructures could not only provide a large surface coverage for immobilization of biomolecules but also promote 4-CN oxidation to some extent. The produced insoluble benzo-4-chlorohexadienone via 4-CN was coated on the electrode surface, and hindered the electron transfer between the solution and the electrode, thereby increasing the Faradaic impedance of the base electrode. Three labeling strategies including Ab2-AuNP, Ab2-AuPd and Ab2-AuPd-DNA were investigated for determination of PSA, and improved analytical features were obtained with the Ab2-AuPd-DNA strategy. Under optimal conditions, the dynamic concentration range of the impedimetric immunosensor spanned from 1.0 pg mL(-1) to 50 ng mL(-1) PSA with a detection limit of 0.73 pg mL(-1). Intra- and inter-assay coefficients of variation were below 8.5% and 9.5%, respectively. Importantly, no significant differences at the 0.05 significance level were encountered in the analysis of 6 clinical serum specimens and 6 diluted standards between the impedimetric immunosensor and the commercialized electrochemiluminescent method for PSA detection. © 2013 Published by Elsevier B.V.

Ultrasensitive interdigitated capacitance immunosensor using gold nanoparticles.

PubMed

Alizadeh Zeinabad, Hojjat; Ghourchian, Hedayatollah; Falahati, Mojtaba; Fathipour, Morteza; Azizi, Marzieh; Boutorabi, Seyed Mehdi

2018-06-29

Immunosensors based on interdigitated electrodes (IDEs), have recently demonstrated significant improvements in the sensitivity of capacitance detection. Herein, a novel type of highly sensitive, compact and portable immunosensor based on a gold interdigital capacitor has been designed and developed for the rapid detection of hepatitis B surface antigen (HBsAg). To improve the efficiency of antibody immobilization and time-saving, a self-assembled monolayer (SAM) of 2-mercaptoethylamine film was coated on IDEs. Afterwards, carboxyl groups on primary antibodies were activated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and were immobilized on amino-terminated SAM for better control of the oriented immobilization of antibodies on gold IDEs. In addition, gold nanoparticles conjugated with a secondary antibody were used to enhance the sensitivity. Under optimal conditions, the immunosensor exhibited the sensitivity of 0.22 nF.pg ml -1 , the linear range from 5 pg ml -1 to 1 ng ml -1 and the detection limit of 1.34 pg ml -1 , at a signal-to-noise ratio of 3.

Ultrasensitive interdigitated capacitance immunosensor using gold nanoparticles

NASA Astrophysics Data System (ADS)

Alizadeh Zeinabad, Hojjat; Ghourchian, Hedayatollah; Falahati, Mojtaba; Fathipour, Morteza; Azizi, Marzieh; Boutorabi, Seyed Mehdi

2018-06-01

Immunosensors based on interdigitated electrodes (IDEs), have recently demonstrated significant improvements in the sensitivity of capacitance detection. Herein, a novel type of highly sensitive, compact and portable immunosensor based on a gold interdigital capacitor has been designed and developed for the rapid detection of hepatitis B surface antigen (HBsAg). To improve the efficiency of antibody immobilization and time-saving, a self-assembled monolayer (SAM) of 2-mercaptoethylamine film was coated on IDEs. Afterwards, carboxyl groups on primary antibodies were activated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and were immobilized on amino-terminated SAM for better control of the oriented immobilization of antibodies on gold IDEs. In addition, gold nanoparticles conjugated with a secondary antibody were used to enhance the sensitivity. Under optimal conditions, the immunosensor exhibited the sensitivity of 0.22 nF.pg ml–1, the linear range from 5 pg ml‑1 to 1 ng ml–1 and the detection limit of 1.34 pg ml‑1, at a signal-to-noise ratio of 3.

Application of Ionic Liquids in Amperometric Gas Sensors.

PubMed

Gębicki, Jacek; Kloskowski, Adam; Chrzanowski, Wojciech; Stepnowski, Piotr; Namiesnik, Jacek

2016-01-01

This article presents an analysis of available literature data on metrological parameters of the amperometric gas sensors containing ionic liquids as an electrolyte. Four mechanism types of signal generation in amperometric sensors with ionic liquid are described. Moreover, this article describes the influence of selected physico-chemical properties of the ionic liquids on the metrological parameters of these sensors. Some metrological parameters are also compared for amperometric sensors with GDE and SPE electrodes and with ionic liquids for selected analytes.

Ultrasensitive Nanoimmunosensor by coupling non-covalent functionalized graphene oxide platform and numerous ferritin labels on carbon nanotubes.

PubMed

Akter, Rashida; Jeong, Bongjin; Choi, Jong-Soon; Rahman, Md Aminur

2016-06-15

An ultrasensitive electrochemical nanostructured immunosensor for a breast cancer biomarker carbohydrate antigen 15-3 (CA 15-3) was fabricated using non-covalent functionalized graphene oxides (GO/Py-COOH) as sensor probe and multiwalled carbon nanotube (MWCNTs)-supported numerous ferritin as labels. The immunosensor was constructed by immobilizing a monoclonal anti-CA 15-3 antibody on the GO modified cysteamine (Cys) self-assembled monolayer (SAM) on an Au electrode (Au/Cys) through the amide bond formation between the carboxylic acid groups of GO/Py-COOH and amine groups of anti-CA 15-3. Secondary antibody conjugated MWCNT-supported ferritin labels (Ab2-MWCNT-Ferritin) were prepared through the amide bond formation between amine groups of Ab2 and ferritin and carboxylic acid groups of MWCNTs. The detection of CA 15-3 was based on the enhanced bioelectrocatalytic reduction of hydrogen peroxide mediated by hydroquinone (HQ) at the GO/Py-COOH-based sensor probe. The GO/Py-COOH-based sensor probe and Ab2-MWCNT-Ferritin labels were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscope (SEM), transmission electron microscope (TEM), and x-ray photoelectron spectroscopy (XPS) techniques. Using differential pulse voltammetry (DPV) technique, CA 15-3 can be selectively detected as low as 0.01 ± 0.07 U/mL in human serum samples. Additionally, the proposed CA 15-3 immunosensor showed excellent selectivity and better stability in human serum samples, which demonstrated that the proposed immunosensor has potentials in proteomic researches and diagnostics. Copyright © 2016 Elsevier B.V. All rights reserved.

Interdigitated Array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia coli O157:H7.

PubMed

Yang, Liju; Li, Yanbin; Erf, Gisela F

2004-02-15

A label-free electrochemical impedance immunosensor for rapid detection of Escherichia coli O157:H7 was developed by immobilizing anti-E. coli antibodies onto an indium-tin oxide interdigitated array (IDA) microelectrode. Based on the general electronic equivalent model of an electrochemical cell and the behavior of the IDA microelectrode, an equivalent circuit, consisting of an ohmic resistor of the electrolyte between two electrodes and a double layer capacitor, an electron-transfer resistor, and a Warburg impedance around each electrode, was introduced for interpretation of the impedance components of the IDA microelectrode system. The results showed that the immobilization of antibodies and the binding of E. coli cells to the IDA microelectrode surface increased the electron-transfer resistance, which was directly measured with electrochemical impedance spectroscopy in the presence of [Fe(CN)(6)](3-/4-) as a redox probe. The electron-transfer resistance was correlated with the concentration of E. coli cells in a range from 4.36 x 10(5) to 4.36 x 10(8) cfu/mL with the detection limit of 10(6) cfu/mL.

Enhanced peroxidase-like properties of Au@Pt DNs/NG/Cu2+ and application of sandwich-type electrochemical immunosensor for highly sensitive detection of CEA.

PubMed

Lv, Hui; Li, Yueyun; Zhang, Xiaobo; Gao, Zengqiang; Zhang, Chunyan; Zhang, Shuan; Dong, Yunhui

2018-07-30

Effective treatment of cancer depends upon the early detection of the tumor marker. Here, we report on the development of a new immunosensor for early detection of carcinoembryonic antigen (CEA). Cubic Au@Pt dendritic nanomaterials functionalized nitrogen-doped graphene loaded with copper ion (Au@Pt DNs/NG/Cu 2+ ) with enhanced peroxidase-like properties was synthesized as labels to effectively capture and immobilize secondary anti-CEA. The Au@Pt DNs with more active surface area could efficiently enhance electrocatalysis for reduction of hydrogen peroxide (H 2 O 2 ). Meanwhile, with good conductivity and large specific surface area, NG can immobilize a large amount of Au@Pt DNs. Furthermore, after adsorbed Cu 2+ can further promote the redox of H 2 O 2 and amplify the signal of the immunosensor. For the immobilization of primary antibodies, Au nanoparticles functionalized polydopamine (Au@PDA) were used as transducing materials to modify glassy carbon electrodes and enhance the electron transfer efficiently. Under optimal conditions, the immunosensor exhibited a satisfactory response to CEA with a limit detection of 0.167 pg/mL and linear detection range from 0.5 pg/mL to 50 ng/mL. Based on the high sensitivity and specificity of the immunosensor, we propose this multiple amplified biosensor for early detection of CEA. Copyright © 2018 Elsevier B.V. All rights reserved.

A high-sensitivity electrochemical immunosensor based on mobile crystalline material-41-polyvinyl alcohol nanocomposite and colloidal gold nanoparticles.

PubMed

Omidfar, Kobra; Zarei, Hajar; Gholizadeh, Fatemeh; Larijani, Bagher

2012-02-15

A novel competitive immunosensor was developed as a model system using anti-human serum albumin (HSA)-conjugated gold nanoparticles (AuNPs) as an electrochemical label and mobile crystalline material-41 (MCM-41)-polyvinyl alcohol (PVA) mesoporous nanocomposite as an immobilization platform. However, no attempt has yet been made to use the MCM-41 as the supporting electrolyte for the electrosynthesis of nonconducting polymer nanocomposite. This hybrid membrane was evaluated extensively by using field emission scanning electron microscopy (FESEM), cyclic voltammetry (CV), and differential pulse voltammetry (DPV) to determine its physicochemical and electrochemical properties in immunosensor application. FESEM revealed an appropriate and stable attachment between HSA and MCM-41 and also a dense layer deposition of MCM-41-HSA-PVA film onto the electrode surfaces. DPV was developed for quantitative determination of antigen in biological samples. A decrease in DPV responses was observed with increasing concentrations of HSA in standard and real samples. In optimal conditions, this immunosensor based on MCM-41-PVA nanocomposite film could detect HSA in a high linear range (0.5-200 μg ml⁻¹) with a low detection limit of 1 ng ml⁻¹. The proposed method showed acceptable reproducibility, stability, and reliability and could also be applied to detect the other antigens. Copyright © 2011 Elsevier Inc. All rights reserved.

A reusable piezoelectric immunosensor using antibody-adsorbed magnetic nanocomposite.

PubMed

Zhang, Yun; Wang, Hua; Yan, Bani; Zhang, Yuwei; Li, Jishan; Shen, Guoli; Yu, Ruqin

2008-03-20

This paper reports a simple, sensitive, and reusable piezoelectric immunosensor using magnetic hydroxyapatite (HAP)/gamma-Fe(2)O(3)/Au nanocomposite. Use of porous HAP nanocrystals embedded with gamma-Fe(2)O(3) and colloidal gold nanoparticles resulted in a multifunctional HAP/gamma-Fe(2)O(3)/Au nanocomposite. Under optimized conditions, the biocompatible nanocomposites were exploited for direct adsorption of large quantities of rabbit anti-human immunoglobulin G antibodies (anti-hIgG) with well-preserved immunoactivity. In a homogeneous bulk solution, the hIgG analytes were captured by the anti-hIgG-derivatized immunocomposites followed by magnetic separation/enrichment onto a bovine serum albumin (BSA)-sealed QCM probe before measuring. This QCM immunosensor can quantitatively determine concentrations of hIgG ranging from approximately 20 to 800 ng/ml, with a detection limit of approximately 15 ng/ml. Moreover, regeneration of the immunosensor can be simply realized by canceling the controllable magnetic field. With the possibility of performing the analysis automatically and considering its ease of use, high sensitivity, and good reusability, this magnetic separation-assisted QCM immunosensor may have great potential to be further tailored as a general and promising alternative for a broad range of practical applications.

A sensitive electrochemiluminescent biosensor based on AuNP-functionalized ITO for a label-free immunoassay of C-peptide.

PubMed

Liu, Xiang; Fang, Chen; Yan, Jilin; Li, Huiling; Tu, Yifeng

2018-05-23

The C-peptide is a co-product of pancreatic β-cells during insulin secretion; its content in body fluid is closely related to diabetes. This paper reports an immune-sensing strategy for a simple and effective assay of C-peptide based on label-free electrochemiluminescent (ECL) signaling, with high sensitivity and specificity. The basal electrode was constructed of an indium tin oxide (ITO) glass as a conductive substrate, which was decorated by Au nanoparticles (AuNPs) with hydrolysed (3-aminopropyl)trimethoxysilane as the linker. The characteristics of the fabricated electrode were investigated by electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. After immobilizing the C-peptide antibody, which takes great advantage of AuNPs' binding capacity, this immunosensor can quantify C-peptide using luminol as the ECL probe. By measuring ECL inhibition, calibration can be established to report the C-peptide concentration between 0.05 ng mL -1 and 100 ng mL -1 with a detection limit of 0.0142 ng mL -1 . As a proof of concept, the proposed strategy is a promising and versatile platform for the clinical diagnosis, classification, and research of diabetes. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

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.

Multianalyte detection using a capillary-based flow immunosensor.

PubMed

Narang, U; Gauger, P R; Kusterbeck, A W; Ligler, F S

1998-01-01

A highly sensitive, dual-analyte detection system using capillary-based immunosensors has been designed for explosive detection. This model system consists of two capillaries, one coated with antibodies specific for 2,4,6-trinitrotoluene (TNT) and the other specific for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) combined into a single device. The fused silica capillaries are prepared by coating anti-TNT and anti-RDX antibodies onto the silanized inner walls using a hetero-bifunctional crosslinker. After immobilization, the antibodies are saturated with a suitable fluorophorelabeled antigen. A "T" connector is used to continuously flow the buffer solution through the individual capillaries. To perform the assay, an aliquot of TNT or RDX or a mixture of the two analytes is injected into the continuous flow stream. In each capillary, the target analyte displaces the fluorophore-labeled antigen from the binding pocket of the antibody. The labeled antigen displaced from either capillary is detected downstream using two portable spectrofluorometers. The limits of detection for TNT and RDX in the multi-analyte formate are 44 fmol (100 microliters of 0.1 ng/ml TNT solution) and 224 fmol (100 microliters of 0.5 ng/ml RDX solution), respectively. The entire assay for both analytes can be performed in less than 3 min.

Deep Learning in Label-free Cell Classification

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

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

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

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

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

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.

Humidity-resistant ambient-temperature solid-electrolyte amperometric sensing apparatus

DOEpatents

Zaromb, S.

1994-06-21

Apparatus and methods for detecting selected chemical compounds in air or other gas streams at room or ambient temperature includes a liquid-free humidity-resistant amperometric sensor comprising a sensing electrode and a counter and reference electrode separated by a solid electrolyte. The sensing electrode preferably contains a noble metal, such as Pt black. The electrolyte is water-free, non-hygroscopic, and substantially water-insoluble, and has a room temperature ionic conductivity [>=]10[sup [minus]4] (ohm-cm)[sup [minus]1], and preferably [>=]0.01 (ohm-cm)[sup [minus]1]. The conductivity may be due predominantly to Ag[sup +] ions, as in Ag[sub 2]WO[sub 4], or to F[sup [minus

Redox and catalysis 'all-in-one' infinite coordination polymer for electrochemical immunosensor of tumor markers.

PubMed

Zhang, Bing; Liu, Bingqian; Chen, Guonan; Tang, Dianping

2015-02-15

Prostate-specific antigen (PSA), as a glycoprotein enzyme encoded in humans by the KLK3 gene, is one of the most important biomarkers for the diagnosis and prognosis of prostate cancer. Herein, a new electrochemical immunosensor for sensitive determination of PSA was designed by using redox and catalysis 'all-in-one' infinite coordination polymer (PtNP@ICP) as signal tag on the polyamidoamine dendrimers modified electrode interface. To construct such 'all-in-one' PtNP@ICP nanostructures, the coordination polymerization was fully carried between metal ions and polydentate bridging ligands, and the PtNP was encapsulated into the ICP in the process of polymerization. The prepared PtNP@ICP nanocatalyst was characterized by transmission electron microscope (TEM), energy dispersive X-ray spectrometry (EDX), ultraviolet and visible (UV-vis) spectrophotometry and Fourier transform infrared spectroscope (FTIR). And the synthesized PtNP@ICP was utilized as signal tag for the label of PSA. With a sandwich-type immunoassay format, the conjugated signal tag on the transducer increased with the increasing PSA concentration in the sample thus enhancing the signal of the electrochemical immunosensor due to the catalytic reduction toward H2O2 of the enveloped PtNP. Under optimal conditions, the current was proportional to the logarithm of PSA concentration ranging from 0.001 to 60 ng/mL. The detection limit (LOD) was 0.3 pg/mL at 3 sB. The immunosensor displayed an acceptable reproducibility, stability and selectivity. In addition, the methodology was evaluated with human serum specimens receiving good correlation with results from commercialized enzyme-linked immunosorbent assay (ELISA) method. Copyright © 2014 Elsevier B.V. All rights reserved.

Analytical Application of Flow Immunosensor in Detection of Thyroxine and Triiodothyronine in Serum.

PubMed

Wani, Tanveer A; Zargar, Seema; Majid, Salma; Darwish, Ibrahim A

2016-11-01

In this study, an immunosensor based on kinetic exclusion analysis (KinExA) was used for thyroxine (T4) and triiodothyronine (T3) estimation. A KinExA™ 3200 instrument was used for this analysis, which is an automated flow fluorimeter designed to separate free unbound antibody binding sites in reaction mixtures of antibody, antigen, and antibody-antigen complex. A T3-BSA- and T4-BSA-coated polymethyl methacrylate (PMMA) bead microcolumn is generated inside the flow cell of the instrument. A sample mixture containing T3 and T4 with their respective monoclonal antibodies and their complexes are drawn past the microbead column. The unbound T3 or T4 monoclonal antibody binding sites are captured by their respective T3 and T4 antigens coated on the PMMA beads as bovine serum albumin conjugates. Fluorescently labeled secondary antibodies bind to the T3 or T4 antigen-antibody complex to generate fluorescence intensity for analysis. The limit of detection for the T3 and T4 assays was found to be 0.06 and 1.9 ng mL -1 with acceptable precision values. The convenience of the automated KinExA format may be valuable in medical diagnostic laboratories.

Label-Free 3D Ag Nanoflower-Based Electrochemical Immunosensor for the Detection of Escherichia coli O157:H7 Pathogens

NASA Astrophysics Data System (ADS)

Huang, He; Liu, Minghuan; Wang, Xiangsheng; Zhang, Wenjie; Yang, Da-Peng; Cui, Lianhua; Wang, Xiansong

2016-11-01

It is highly desirable to develop a rapid and simple method to detect pathogens. Combining nanomaterials with electrochemical techniques is an efficient way for pathogen detection. Herein, a novel 3D Ag nanoflower was prepared via a biomineralization method by using bovine serum albumin (BSA) as a template. It was adopted as a sensing interface to construct an electrochemical bacteria immunosensor for the rapid detection of foodborne pathogens Escherichia coli ( E. coli) O157:H7. Bacterial antibody was immobilized onto the surface of Ag nanoflowers through covalent conjugation. Electrochemical impedance spectroscopy (EIS) was used to detect and validate the resistance changes, where [Fe(CN)6]3-/4- acted as the redox probe. A linear relation between R et and E. coli concentration was obtained in the E. coli concentration range of 3.0 × 102-3.0 × 108 cfu mL-1. The as-prepared biosensor gave rise to an obvious response to E. coli but had no distinct response to Cronobacter sakazakii, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus albus, Lactobacillus easei, and Shigella flexneri, revealing a high selectivity for the detection of the pathogens down to 100 cfu mL-1 in a short time. We believe that this BSA-conjugated 3D Ag nanoflowers could be used as a powerful interface material with good conductivity and biocompatibility for improving pathogen detection and treatment in the field of medicine, environment, and food safety.

Label-free resistive-pulse cytometry.

PubMed

Chapman, M R; Sohn, L L

2011-01-01

Numerous methods have recently been developed to characterize cells for size, shape, and specific cell-surface markers. Most of these methods rely upon exogenous labeling of the cells and are better suited for large cell populations (>10,000). Here, we review a label-free method of characterizing and screening cells based on the Coulter-counter technique of particle sizing: an individual cell transiting a microchannel (or "pore") causes a downward pulse in the measured DC current across that "pore". Pulse magnitude corresponds to the cell size, pulse width to the transit time needed for the cell to pass through the pore, and pulse shape to how the cell traverses across the pore (i.e., rolling or tumbling). When the pore is functionalized with an antibody that is specific to a surface-epitope of interest, label-free screening of a specific marker is possible, as transient binding between the two results in longer time duration than when the pore is unfunctionalized or functionalized with a nonspecific antibody. While this method cannot currently compete with traditional technology in terms of throughput, there are a number of applications for which this technology is better suited than current commercial cytometry systems. Applications include the rapid and nondestructive analysis of small cell populations (<100), which is not possible with current technology, and a platform for providing true point-of-care clinical diagnostics, due to the simplicity of the device, low manufacturing costs, and ease of use. Copyright © 2011 Elsevier Inc. All rights reserved.

Study and development of label-free optical biosensors for biomedical applications

NASA Astrophysics Data System (ADS)

Choi, Charles J.

For the majority of assays currently performed, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so that they may be readily visualized. The methods of using labels to track biomolecular binding events are very sensitive and effective, and are employed as standardized assay protocol across research labs worldwide. However, using labels induces experimental uncertainties due to the effect of the label on molecular conformation, active binding sites, or inability to find an appropriate label that functions equivalently for all molecules in an experiment. Therefore, the ability to perform highly sensitive biochemical detection without the use of fluorescent labels would further simplify assay protocols and would provide quantitative kinetic data, while removing experimental artifacts from fluorescent quenching, shelf-life, and background fluorescence phenomena. In view of the advantages mentioned above, the study and development of optical label-free sensor technologies have been undertaken here. In general, label-free photonic crystal (PC) biosensors and metal nanodome array surface-enhanced Raman scattering (SERS) substrates, both of which are fabricated by nanoreplica molding process, have been used as the method to attack the problem. Chapter 1 shows the work on PC label-free biosensor incorporated microfluidic network for bioassay performance enhancement and kinetic reaction rate constant determination. Chapter 2 describes the work on theoretical and experimental comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays. Chapter 3 shows the work on integration of PC biosensor with actuate-to-open valve microfluidic chip for pL-volume combinatorial mixing and screening application. In Chapter 4, the development and characterization of SERS nanodome array is shown. Lastly, Chapter 5 describes SERS nanodome sensor incorporated tubing for point-of-care monitoring of

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

PubMed Central

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

2014-01-01

Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP). PMID:25154394

Immunosensor development formatting for tungro disease detection using nano-gold antibody particles application

NASA Astrophysics Data System (ADS)

Uda, M. N. A.; Hasfalina, C. M.; Samsuzanaa, A. A.; Faridah, S.; Zamri, I.; Noraini, B. Siti; Sabrina, W. Nur; Hashim, U.; Gopinath, Subash C. B.

2017-03-01

The plant disease such as Rice tungro disease (RTD) becomes a major problem in rice production and also will effect in the economy loss in the country. Therefore, to tackle this problem at early stages, the immunosensor application is a most reliable sensor nowadays because of advantages towards detecting biological molecule. Thus, in order to deal with immunosensor development, it can be done by undergoing the formation of immunosensor format on screen-printed carbon electrode (SPCE). Results can be elaborated with the potential applications to detect the viruses.

Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy.

PubMed

You, Sixian; Tu, Haohua; Chaney, Eric J; Sun, Yi; Zhao, Youbo; Bower, Andrew J; Liu, Yuan-Zhi; Marjanovic, Marina; Sinha, Saurabh; Pu, Yang; Boppart, Stephen A

2018-05-29

Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.

Field-effect amperometric immuno-detection of protein biomarker.

PubMed

Wang, Jiapeng; Yau, Siu-Tung

2011-11-15

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work. Copyright © 2011 Elsevier B.V. All rights reserved.

Label-free photoacoustic nanoscopy

PubMed Central

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

2014-01-01

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

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

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

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

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

Developments in label-free microfluidic methods for single-cell analysis and sorting.

PubMed

Carey, Thomas R; Cotner, Kristen L; Li, Brian; Sohn, Lydia L

2018-04-24

Advancements in microfluidic technologies have led to the development of many new tools for both the characterization and sorting of single cells without the need for exogenous labels. Label-free microfluidics reduce the preparation time, reagents needed, and cost of conventional methods based on fluorescent or magnetic labels. Furthermore, these devices enable analysis of cell properties such as mechanical phenotype and dielectric parameters that cannot be characterized with traditional labels. Some of the most promising technologies for current and future development toward label-free, single-cell analysis and sorting include electronic sensors such as Coulter counters and electrical impedance cytometry; deformation analysis using optical traps and deformation cytometry; hydrodynamic sorting such as deterministic lateral displacement, inertial focusing, and microvortex trapping; and acoustic sorting using traveling or standing surface acoustic waves. These label-free microfluidic methods have been used to screen, sort, and analyze cells for a wide range of biomedical and clinical applications, including cell cycle monitoring, rapid complete blood counts, cancer diagnosis, metastatic progression monitoring, HIV and parasite detection, circulating tumor cell isolation, and point-of-care diagnostics. Because of the versatility of label-free methods for characterization and sorting, the low-cost nature of microfluidics, and the rapid prototyping capabilities of modern microfabrication, we expect this class of technology to continue to be an area of high research interest going forward. New developments in this field will contribute to the ongoing paradigm shift in cell analysis and sorting technologies toward label-free microfluidic devices, enabling new capabilities in biomedical research tools as well as clinical diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices. © 2018 Wiley Periodicals, Inc.

Nano and Microparticle-Enhanced Immunosensor Approaches for the Detection of Cancer Biomarker Proteins

NASA Astrophysics Data System (ADS)

Mani, Vigneshwaran

Accurate, sensitive, point-of-care multiplexed protein measurements are critical for early disease detection and monitoring, impacting biomarker and drug discovery, and personalized medicine. Significant application involves monitoring panels of proteins in the blood that are biomarkers for diagnosing cancer. However, measurements of biomarker panels in blood or other bodily fluids have been slow to integrate into current practice of cancer diagnostics partly due to the lack of technically simple, low-cost, sensitive, point-of-care multiplexed measurement devices, as well as the lack of rigorously validated protein panels. The present thesis in part addresses these limitations by the development of electrochemical and surface plasmon resonance (SPR) immunosensors utilizing 1mum superparamagnetic labels for accurate detection of prostate cancer biomarker proteins in patient serum samples. Electrochemical discrete immunosensors featuring nanostructured surface with densely packed 5 nm glutathione-coated gold nanoparticles coupled with multi-enzyme magnetic particle (MP) labels enabled measurement of prostate specific antigen (PSA) with a detection limit (DL) of 0.5 pg mL-1 in undiluted serum. Such low DLs are attributed to high surface area, conductivity of nanostructured surface, and multi-enzyme signal amplification. DLs are further improved by utilizing MP bioconjugated with more than 100,000 antibody labels to offline capture proteins from the serum sample matrix, minimizing nonspecific binding of interfering proteins on sensor surface before detection. This approach provided an unprecedented 10 fg DL mL-1 for PSA in undiluted serum using a flow SPR biosensor. Finally electrochemical microfluidic immunoarrays featuring nanostructured surface and offline protein capture by multi-label MPs enabled multiplexed detection of prostate cancer biomarkers PSA and interleukin-6 (IL-6). These approaches provided up to 1000-fold lower DLs compared to commercial bead based

Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

PubMed

Terada, Takaho; Yokoyama, Shigeyuki

2015-01-01

This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

Labeling of indocyanine green with carrier-free iodine-123

DOEpatents

Ansari, Azizullah N.; Lambrecht, Richard M.; Redvanly, Carol S.; Wolf, Alfred P.

1976-01-01

The method of labeling indocyanine green (ICG) with carrier-free iodine-123 comprising the steps of condensing xenon-123 on crystals of ICG followed by permitting decay of the .sup.123 Xe a sufficient length of time to produce .sup.123 I-electronically excited ions and atoms which subsequently label ICG.

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen.

PubMed

Guo, Jinjin; Han, Xiaowei; Wang, Junchun; Zhao, Junqing; Guo, Zilin; Zhang, Yuzhong

2015-12-15

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP-Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP-Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen-antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1-100 ng ml(-1), and the limit of detection was 30 pg ml(-1) (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Graphene oxide-labeled sandwich-type impedimetric immunoassay with sensitive enhancement based on enzymatic 4-chloro-1-naphthol oxidation.

PubMed

Hou, Li; Cui, Yuling; Xu, Mingdi; Gao, Zhuangqiang; Huang, Jianxin; Tang, Dianping

2013-09-15

A new sandwich-type impedimetric immunosensor based on functionalized graphene oxide nanosheets with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for the detection of carcinoembryonic antigen (CEA) by coupling with enzymatic biocatalytic precipitation of 4-chloro-1-naphthol (4-CN) on the captured antibody-modified glassy carbon electrode. Two molecular tags (with and without the graphene oxide nanosheets) were investigated for the detection of CEA and improved analytical features were acquired with the graphene-based labeling. With the labeling method, the performance and factors influencing the properties of the impedimetric immunosensors were also studied and evaluated. Under the optimal conditions, the dynamic concentration range of the impedimetric immunosensors spanned from 1.0pgmL(-1) to 80ngmL(-1) CEA with a detection limit (LOD) of 0.64pgmL(-1). Intra- and inter-assay coefficients of variation were less than 7.5% and 11%, respectively. Additionally, the methodology was evaluated for CEA analysis of 10 clinical serum samples and 5 diluted serum samples, receiving in a good accordance with the results obtained by the impedimetric immunoassay and the commercialized electrochemiluminescent method. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Label and label-free based surface-enhanced Raman scattering for pathogen bacteria detection: A review.

PubMed

Liu, Yu; Zhou, Haibo; Hu, Ziwei; Yu, Guangxia; Yang, Danting; Zhao, Jinshun

2017-08-15

Rapid, accurate detection of pathogen bacteria is a highly topical research area for the sake of food safety and public health. Surface-enhanced Raman scattering (SERS) is being considered as a powerful and attractive technique for pathogen bacteria detection, due to its sensitivity, high speed, comparatively low cost, multiplexing ability and portability. This contribution aims to give a comprehensive overview of SERS as a technique for rapid detection of pathogen bacteria based on label and label-free strategies. A brief tutorial on SERS is given first of all. Then we summarize the recent trends and developments of label and label-free based SERS applied to detection of pathogen bacteria, including the relatively complete interpretation of SERS spectra. In addition, multifunctional SERS platforms for pathogen bacteria in matrix are discussed as well. Furthermore, an outlook of the work done and a perspective on the future directions of SERS as a reliable tool for real-time pathogen bacteria detection are given. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free capacitive biosensor for sensitive detection of multiple biomarkers using gold interdigitated capacitor arrays.

PubMed

Qureshi, Anjum; Niazi, Javed H; Kallempudi, Saravan; Gurbuz, Yasar

2010-06-15

In this study, a highly sensitive and label-free multianalyte capacitive immunosensor was developed based on gold interdigitated electrodes (GID) capacitor arrays to detect a panel of disease biomarkers. C-reactive protein (CRP), TNFalpha, and IL6 have strong and consistent relationships between markers of inflammation and future cardiovascular risk (CVR) events. Early detection of a panel of biomarkers for a disease could enable accurate prediction of a disease risk. The detection of protein biomarkers was based on relative change in capacitive/dielectric properties. Two different lab-on-a-chip formats were employed for multiple biomarker detection on GID-capacitors. In format I, capacitor arrays were immobilized with pure forms of anti-CRP, -TNFalpha, and -IL6 antibodies in which each capacitor array contained a different immobilized antibody. Here, the CRP and IL6 were detected in the range 25 pg/ml to 25 ng/ml and 25 pg/ml to 1 ng/ml for TNFalpha in format I. Sensitive detection was achieved with chips co-immobilized (diluted) with equimolar mixtures of anti-CRP, -IL6, and -TNFalpha antibodies (format II) in which all capacitors in an array were identical and tested for biomarkers with sequential incubation. The resulting response to CRP, IL6, and TNFalpha in format II for all biomarkers was found to be within 25 pg/ml to 25 ng/ml range. The capacitive biosensor for panels of inflammation and CVR markers show significant clinical value and provide great potential for detection of biomarker panel in suspected subjects for early diagnosis. Copyright 2010 Elsevier B.V. All rights reserved.

Nanomaterial-Based Electrochemical Immunosensors for Clinically Significant Biomarkers

PubMed Central

Ronkainen, Niina J.; Okon, Stanley L.

2014-01-01

Nanotechnology has played a crucial role in the development of biosensors over the past decade. The development, testing, optimization, and validation of new biosensors has become a highly interdisciplinary effort involving experts in chemistry, biology, physics, engineering, and medicine. The sensitivity, the specificity and the reproducibility of biosensors have improved tremendously as a result of incorporating nanomaterials in their design. In general, nanomaterials-based electrochemical immunosensors amplify the sensitivity by facilitating greater loading of the larger sensing surface with biorecognition molecules as well as improving the electrochemical properties of the transducer. The most common types of nanomaterials and their properties will be described. In addition, the utilization of nanomaterials in immunosensors for biomarker detection will be discussed since these biosensors have enormous potential for a myriad of clinical uses. Electrochemical immunosensors provide a specific and simple analytical alternative as evidenced by their brief analysis times, inexpensive instrumentation, lower assay cost as well as good portability and amenability to miniaturization. The role nanomaterials play in biosensors, their ability to improve detection capabilities in low concentration analytes yielding clinically useful data and their impact on other biosensor performance properties will be discussed. Finally, the most common types of electroanalytical detection methods will be briefly touched upon. PMID:28788700

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.

AuNPs/CNOs/SWCNTs/chitosan-nanocomposite modified electrochemical sensor for the label-free detection of carcinoembryonic antigen.

PubMed

Rizwan, Mohammad; Elma, Syazwani; Lim, Syazana Abdullah; Ahmed, Minhaz Uddin

2018-06-01

In this work, a nanocomposite of gold nanoparticles (AuNPs), carbon nano-onions (CNOs), single-walled carbon nanotubes (SWCNTs) and chitosan (CS) (AuNPs/CNOs/SWCNTs/CS) was prepared for the development of highly sensitive electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA), clinical tumor marker. Firstly, layer-by-layer fabrication of the CEA-immunosensors was studied using cyclic voltammetry (CV) and square wave voltammetry (SWV). By combining the advantages of large surface area and electronic properties of AuNPs, CNOs, SWCNTs, and film forming properties of CS, AuNPs/CNOs/SWCNTs/CS-nanocomposite-modified glassy carbon electrode showed a 200% increase in effective surface area and electronic conductivity. The calibration plot gave a negative linear relationship between log[concentration] of CEA and electrical current with a correlation coefficient of 0.9875. The CEA-immunosensor demonstrated a wide linear detection range of 100 fg mL -1 to 400 ng mL -1 with a low detection limit of 100 fg mL -1 . In addition to high sensitivity, reproducibility and large stability, CEA-immunosensor provided an excellent selectivity and resistant-to-interference in the presence of other antigens in serum and hence a potential to be used with real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

PubMed

Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

2015-02-15

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Paper-based immunosensor with signal amplification by enzyme-labeled anti-p16INK4a multifunctionalized gold nanoparticles for cervical cancer screening.

PubMed

Yokchom, Ruchuon; Laiwejpithaya, Somsak; Maneeprakorn, Weerakanya; Tapaneeyakorn, Satita; Rabablert, Jundee; Dharakul, Tararaj

2018-04-01

The aim of this study was to develop a paper-based immunosensor for cervical cancer screening, with signal amplification by multifunctionalized gold nanoparticles (AuNPs). The AuNPs were functionalized with a highly specific antibody to the p16 INK4a cancer biomarker. The signal was amplified using a combination of the peroxidase activity of horseradish peroxidase (HRP) enzyme-antibody conjugate and the peroxidase-like activity of the AuNPs. The immune complex of p16 INK4a protein and multifunctionalized AuNPs was deposited on the nitrocellulose membrane, and a positive result was generated by catalytic oxidation of peroxidase enzyme substrate 3,3',5,5'-Tetramethylbenzidine (TMB). The entire reaction occurred on the membrane within 30 min. Evaluation in clinical samples revealed 85.2% accuracy with a kappa coefficient of 0.69. This proof of concept study demonstrates the successful development of a highly accurate, paper-based immunosensor that is easy to interpret using the naked eye and that is suitable for cervical cancer screening in low-resource settings. Copyright © 2018 Elsevier Inc. All rights reserved.

Functionalized vertically aligned ZnO nanorods for application in electrolyte-insulator-semiconductor based pH sensors and label-free immuno-sensors

NASA Astrophysics Data System (ADS)

Kumar, Narendra; Senapati, Sujata; Kumar, Satyendra; Kumar, Jitendra; Panda, Siddhartha

2016-04-01

Vertically aligned ZnO nanorods were grown on a SiO2/Si surface by optimization of the temperature and atmosphere for annealing of the seed. The seed layer annealed at 500 °C in vacuum provided well separated and uniform seeds which also provided the best condition to get densely packed, uniformly distributed, and vertically aligned nanorods. These nanorods grown on the substrates were used to fabricate electrolyte-insulator-semiconductor (EIS) devices for pH sensing. Etching of ZnO at acidic pH prevents the direct use of nanorods for pH sensing. Therefore, the nanorods functionalised with 3-aminopropyltriethoxysilane (APTES) were utilized for pH sensing and showed the pH sensitivity of 50.1 mV/pH. APTES is also known to be used as a linker to immobilize biomolecules (such as antibodies). The EIS device with APTES functionalized nanorods was used for the label free detection of prostate-specific antigen (PSA). Finally, voltage shifts of 23 mV and 35 mV were observed with PSA concentrations of 1 ng/ml and 100 ng/ml, respectively.

Detection of small molecules with a flow immunosensor

NASA Technical Reports Server (NTRS)

Kusterbeck, Anne W.; Ligler, Frances S.

1991-01-01

We describe the development of an easy-to-use sensor with widespread applications for detecting small molecules. The flow immunosensor can analyze discrete samples in under one minute or continuously monitor a flowing stream for the presence of specific analytes. This detection system is extremely specific, and achieves a level of sensitivity which meets or exceeds the detection limits reported for rival assays. Because the system is also compact, transportable, and automated, it has the potential to impact diverse areas. For example, the flow immunosensor has successfully detected drugs of abuse and explosives, and may well address many of the needs of the environmental community with respect to continuous monitoring for pollutants. Efforts are underway to engineer a portable device in the field.

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.

Amplified impedimetric immunosensor based on instant catalyst for sensitive determination of ochratoxin A.

PubMed

Tang, Juan; Huang, Yapei; Zhang, Cengceng; Liu, Huiqiong; Tang, Dianping

2016-12-15

A new impedimetric immunosensor for the fast determination of ochratoxin A (OTA) in food samples was developed based on the instant catalyst as enhancer. Initially, the signal tags were prepared via co-immobilization of anti-OTA antibody and amine-terminated dendrimer (PAMAM) on the graphene oxide nanosheets through the covalent interaction, which were utilized as a good platform for combining manganese ion (anti-OTA-GO-PAMAM-Mn(2+)). Upon target OTA introduction, a competitive-type immunoreaction was implemented between the analyte and the immobilized OTA-BSA on the electrode for the anti-OTA antibody on the graphene oxide nanosheets labels. After a competitive immunoassay format, the anti-OTA-GO-PAMAM-Mn(2+) were captured onto the electrode surface, which could induce the in situ formation of MnO2via classical redox reaction between Mn(2+) and KMnO4 on the immunesensing platform. Moreover, the generated MnO2 nanoparticles act as efficient catalyst could catalyze the 4-chloro-1-naphthol (4-CN) oxidation without H2O2 to generate an insoluble precipitation on the platform. Under the optimal conditions, the instant catalyst based impedimetric immunosensor displayed a wide dynamic working range between 0.1pgmL(-1) and 30ngmL(-1). The detection limit (LOD) of the assay was 0.055pgmL(-1). The developed method exhibited high selectivity and can be used for the determination of OTA in real red wine samples. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Nanoparticle-Based Electrochemical Immunosensor for the Detection of Phosphorylated Acetylcholinesterase: An Exposure Biomarker of Organophosphate Pesticides and Nerve AgentsOrganophosphate Pesticides and Nerve Agents

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

Liu, Guodong; Wang, Jun; Barry, Richard C.

A nanoparticle-based electrochemical immunosensor has been developed for the detection of phosphorylated acetylcholinesterase (AChE) adducts, which is a potential exposure biomarker for organophosphate pesticides (OP) and chemical warfare nerve agent exposures. Zirconia nanoparticles (ZrO2 NPs) were used as selective sorbents to capture the phosphorylated AChE adduct, and quantum dots (ZnS@CdS, QDs) were used as tags to label monoclonal anti-AChE antibody to track the immunorecognition events. The sandwich-like immunoreactions were performed among the ZrO2 NPs, which were pre-coated on a screen printed electrode (SPE) by electrodeposition, phosphorylated AChE and QD-anti-AChE. The captured QD tags were determined on the SPE by electrochemicalmore » stripping analysis of its metallic component (cadmium) after an acid-dissolution step. Paraoxon was used as a model OP insecticide to prepare the phosphorylated AChE adduct to demonstrate the proof of principle for this sensor technology. The paraoxon-AChE adduct was characterized by Fourier Transform Infrared Spectrum, and the binding affinity of anti-AChE to the paraoxon-AChE was validated with an enzyme-linked immunosorbent assay. The parameters (e.g., amount of ZrO2 NP, QD-anti-AChE concentration,) that govern the electrochemical response of immunosensors were optimized. The voltammetric response of the immunosensor is highly linear over the range of 10 pM to 4 nM paraoxon-AChE, and the limit of detection is estimated to be 8 pM. This new nanoparticle-based electrochemical immunosensor thus provides a sensitive and quantitative tool for biomonitoring exposure to OP pesticides and nerve agents.« less

Simulation Study of Nano Aqueous Flow Sensor Based on Amperometric Measurement

PubMed Central

Wu, Jian; Zhou, Qingli; Liu, Jun; Lou, Zhengguo

2006-01-01

In this paper, a novel nano aqueous flow sensor which consists of two closely spaced amperometric sensors is investigated by digital simulation. The simulation results indicate that the ratio of the responses of two closely spaced amperometric sensors is only related to flow rates in the channel, insensitive to the analyte concentration in the solution. By comparing the output of two amperometric sensors, the flow rate in the channel can be deduced. It is not necessary to determine the analyte concentration in advance. The simulation results show it is able to detect flow rate by in the range of several nano-liters per minute when the distance between the working electrodes of two amperometric sensors is 200 nm and the cross-section of the channel is 1 μm × 1 μm.

High-frequency phase shift measurement greatly enhances the sensitivity of QCM immunosensors.

PubMed

March, Carmen; García, José V; Sánchez, Ángel; Arnau, Antonio; Jiménez, Yolanda; García, Pablo; Manclús, Juan J; Montoya, Ángel

2015-03-15

In spite of being widely used for in liquid biosensing applications, sensitivity improvement of conventional (5-20MHz) quartz crystal microbalance (QCM) sensors remains an unsolved challenging task. With the help of a new electronic characterization approach based on phase change measurements at a constant fixed frequency, a highly sensitive and versatile high fundamental frequency (HFF) QCM immunosensor has successfully been developed and tested for its use in pesticide (carbaryl and thiabendazole) analysis. The analytical performance of several immunosensors was compared in competitive immunoassays taking carbaryl insecticide as the model analyte. The highest sensitivity was exhibited by the 100MHz HFF-QCM carbaryl immunosensor. When results were compared with those reported for 9MHz QCM, analytical parameters clearly showed an improvement of one order of magnitude for sensitivity (estimated as the I50 value) and two orders of magnitude for the limit of detection (LOD): 30μgl(-1) vs 0.66μgL(-1)I50 value and 11μgL(-1) vs 0.14μgL(-1) LOD, for 9 and 100MHz, respectively. For the fungicide thiabendazole, I50 value was roughly the same as that previously reported for SPR under the same biochemical conditions, whereas LOD improved by a factor of 2. The analytical performance achieved by high frequency QCM immunosensors surpassed those of conventional QCM and SPR, closely approaching the most sensitive ELISAs. The developed 100MHz QCM immunosensor strongly improves sensitivity in biosensing, and therefore can be considered as a very promising new analytical tool for in liquid applications where highly sensitive detection is required. Copyright © 2014 Elsevier B.V. All rights reserved.

Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c) Using Differential Pulse Voltammetry (DPV).

PubMed

Molazemhosseini, Alireza; Magagnin, Luca; Vena, Pasquale; Liu, Chung-Chiun

2016-07-01

A single-use disposable in vitro electrochemical immunosensor for the detection of HbA1c in undiluted human serum using differential pulse voltammetry (DPV) was developed. A three-electrode configuration electrochemical biosensor consisted of 10-nm-thin gold film working and counter electrodes and a thick-film printed Ag/AgCl reference electrode was fabricated on a polyethylene terephthalate (PET) substrate. Micro-fabrication techniques including sputtering vapor deposition and thick-film printing were used to fabricate the biosensor. This was a roll-to-roll cost-effective manufacturing process making the single-use disposable in vitro HbA1c biosensor a reality. Self-assembled monolayers of 3-Mercaptopropionic acid (MPA) were employed to covalently immobilize anti-HbA1c on the surface of gold electrodes. Electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) confirmed the excellent coverage of MPA-SAM and the upward orientation of carboxylic groups. The hindering effect of HbA1c on the ferricyanide/ferrocyanide electron transfer reaction was exploited as the HbA1c detection mechanism. The biosensor showed a linear range of 7.5-20 µg/mL of HbA1c in 0.1 M PBS. Using undiluted human serum as the test medium, the biosensor presented an excellent linear behavior (R² = 0.999) in the range of 0.1-0.25 mg/mL of HbA1c. The potential application of this biosensor for in vitro measurement of HbA1c for diabetic management was demonstrated.

Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c) Using Differential Pulse Voltammetry (DPV)

PubMed Central

Molazemhosseini, Alireza; Magagnin, Luca; Vena, Pasquale; Liu, Chung-Chiun

2016-01-01

A single-use disposable in vitro electrochemical immunosensor for the detection of HbA1c in undiluted human serum using differential pulse voltammetry (DPV) was developed. A three-electrode configuration electrochemical biosensor consisted of 10-nm-thin gold film working and counter electrodes and a thick-film printed Ag/AgCl reference electrode was fabricated on a polyethylene terephthalate (PET) substrate. Micro-fabrication techniques including sputtering vapor deposition and thick-film printing were used to fabricate the biosensor. This was a roll-to-roll cost-effective manufacturing process making the single-use disposable in vitro HbA1c biosensor a reality. Self-assembled monolayers of 3-Mercaptopropionic acid (MPA) were employed to covalently immobilize anti-HbA1c on the surface of gold electrodes. Electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) confirmed the excellent coverage of MPA-SAM and the upward orientation of carboxylic groups. The hindering effect of HbA1c on the ferricyanide/ferrocyanide electron transfer reaction was exploited as the HbA1c detection mechanism. The biosensor showed a linear range of 7.5–20 µg/mL of HbA1c in 0.1 M PBS. Using undiluted human serum as the test medium, the biosensor presented an excellent linear behavior (R2 = 0.999) in the range of 0.1–0.25 mg/mL of HbA1c. The potential application of this biosensor for in vitro measurement of HbA1c for diabetic management was demonstrated. PMID:27376299

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.

Chemiluminescent optical fiber immunosensor for detection of autoantibodies to ovarian and breast cancer-associated antigens.

PubMed

Salama, Orly; Herrmann, Sebastien; Tziknovsky, Alina; Piura, Benjamin; Meirovich, Michael; Trakht, Ilya; Reed, Brent; Lobel, Leslie I; Marks, Robert S

2007-02-15

We report herein the development of an optical fiber based chemiluminescent immunosensor for detection of the native autoimmune response to GIPC-1, a PDZ containing protein involved in regulation of G-protein signaling. The recombinant protein GIPC-1 was expressed in bacteria, purified, refolded and conjugated to the tip of an optical fiber. A human monoclonal 27.B1 IgM isolated from a breast cancer patient, which targets the GIPC-1 protein, was used for calibration of the immunosensor and was detected down to a concentration of 30 pg/ml. We determined that the fiber-optic immunosensor had a detection limit 50 times lower than chemiluminescent ELISA, and approximately 500 times lower than colorimetric ELISA. In addition, sera from 11 ovarian cancer patients, 22 breast cancer patients and asymptomatic controls were tested for the presence of IgM anti-GIPC-1 autoantibodies in their serum using the two methods. The immunosensor assay detected 54% and 77% GIPC-1 positive sera within ovarian and breast cancer patients, respectively, as compared to chemiluminescent ELISA, which only detected 18% and 27%, respectively. We envision that this immunosensor may serve as a diagnostic tool for screening women for ovarian and breast cancer at an early stage, thus increasing their chance of survival.

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

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

DOE PAGES

Lu, Fa-Ke; Basu, Srinjan; Igras, Vivien; ...

2015-08-31

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 onmore » 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. Moreover, 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. In conclusion, 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.« less

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

Label-free and live cell imaging by interferometric scattering microscopy.

PubMed

Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

2018-03-14

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

Highly sensitive immunosensing of prostate specific antigen using poly cysteine caped by graphene quantum dots and gold nanoparticle: A novel signal amplification strategy.

PubMed

Malekzad, Hediyeh; Hasanzadeh, Mohammad; Shadjou, Nasrin; Jouyban, Abolghasem

2017-12-01

A mediator-free electrochemical immunosensor for quantitation of prostate specific antigen (PSA) based on dual signal amplification strategy was fabricated. In this work, PSA-antibody (anti-PSA) was immobilized onto a green and biocompatible nanocomposite containing poly l-cysteine (P-Cys) as conductive matrix and graphene quantum dots (GQDs)/gold nanoparticles (GNPs) as dual signal amplification elements. Therefore, a novel multilayer film based on P-Cys, GQDs, and GNPs was exploited to develop a highly sensitive amperometric immunosensor for detection of PSA. Fully electrochemical methodology was used to prepare a new transducer on a gold surface which provided a high surface area to immobilize a high amount of the anti-PSA. Importantly, GNPs prepared by soft template synthesized method lead to compact morphology was achieved. The surface morphology of electrode surface was characterized by high-resolution field emission scanning electron microscope (FE-SEM) and energy dispersive spectroscopy (EDX). Chemical compositions of the gold nanoparticles were analysed by an EDX. The immunosensor was employed for the detection of PSA in physiological pH. Under optimized condition the calibration curve for PSA concentration was linear up to 2-9pgmL -1 with lower limit of quantification of 1.8pgmL -1 . Copyright © 2017 Elsevier B.V. All rights reserved.

Amperometric, Bipotentiometric, and Coulometric Titration.

ERIC Educational Resources Information Center

Stock, John T.

1984-01-01

Reviews literature on amperometric, bipotentiometric, and coulometric titration methods examining: apparatus and methodology; acid-base reactions; precipitation and complexing reactions (considering methods involving silver, mercury, EDTA or analogous reagents, and other organic compounds); and oxidation-reduction reactions (considering methods…

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.

OAM-labeled free-space optical flow routing.

PubMed

Gao, Shecheng; Lei, Ting; Li, Yangjin; Yuan, Yangsheng; Xie, Zhenwei; Li, Zhaohui; Yuan, Xiaocong

2016-09-19

Space-division multiplexing allows unprecedented scaling of bandwidth density for optical communication. Routing spatial channels among transmission ports is critical for future scalable optical network, however, there is still no characteristic parameter to label the overlapped optical carriers. Here we propose a free-space optical flow routing (OFR) scheme by using optical orbital angular moment (OAM) states to label optical flows and simultaneously steer each flow according to their OAM states. With an OAM multiplexer and a reconfigurable OAM demultiplexer, massive individual optical flows can be routed to the demanded optical ports. In the routing process, the OAM beams act as data carriers at the same time their topological charges act as each carrier's labels. Using this scheme, we experimentally demonstrate switching, multicasting and filtering network functions by simultaneously steer 10 input optical flows on demand to 10 output ports. The demonstration of data-carrying OFR with nonreturn-to-zero signals shows that this process enables synchronous processing of massive spatial channels and flexible optical network.

Label-free nano-biosensing on the road to tuberculosis detection.

PubMed

Golichenari, Behrouz; Velonia, Kelly; Nosrati, Rahim; Nezami, Alireza; Farokhi-Fard, Aref; Abnous, Khalil; Behravan, Javad; Tsatsakis, Aristidis M

2018-08-15

Tuberculosis, an ailment caused by the bacterium Mycobacterium tuberculosis (Mtb) complex, is one of the catastrophic transmittable diseases that affect human. Reports published by WHO indicate that in 2017 about 6.3 million people progressed to TB and 53 million TB patients died from 2000 to 2016. Therefore, early diagnosis of the disease is of great importance for global health care programs. Common diagnostics like the traditional PPD test and antibody-assisted assays suffer the lack of sensitivity, long processing time and cumbersome post-test proceedings. These shortcomings restrict their use and encourage innovations in TB diagnostics. In recent years, the biosensor concept opened up new horizons in sensitive and fast detection of the disease, reducing the interval time between sampling and diagnostic result. Among new diagnostics, label-free nano-biosensors are highly promising for sensitive and accessible detection of tuberculosis. Various specific label-free nano-biosensors have been recently reported detecting the whole cell of M. tuberculosis, mycobacterial proteins and IFN-γ as crucial markers in early diagnosis of TB. This article provides a focused overview on nanomaterial-based label-free biosensors for tuberculosis detection. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

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

PubMed

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

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.

A sensitive capacitive immunosensor for direct detection of human heart fatty acid-binding protein (h-FABP).

PubMed

Mihailescu, Carmen-Marinela; Stan, Dana; Iosub, Rodica; Moldovan, Carmen; Savin, Mihaela

2015-01-01

The fabrication of a capacitive interdigitated immunosensor (CID) based on a mixed self-assembled monolayer (mSAM) film for the direct detection of heart fatty-acid binding protein (h-FABP) without any labeling is described. The capacitance changes of mSAMs vs. homogenous ordered self-assembled monolayers (hSAMs) on gold work electrodes/covalently bonded antibodies/buffered medium are utilized for monitoring the specific antibody-antigen interaction. Capacitance measurements in the absence and presence of Faradaic currents were performed. The electrochemical properties of mixed monolayers were compared with those of a pure monolayer of 11-mercaptoundecanoic acid (MUA) self-assembled on gold surfaces. Taking into account the stability of the studied monolayers during the electrochemical experiments with the Faradaic process, the best SAM functionalization method was used for developing a sensitive capacitive immunosensor with a non-Faradaic process for direct immune detection of human h-FABP. Under the optimized conditions, the proposed mixed self-assembled monolayer (mSAM1) on gold electrode exhibited good insulating properties such as a capacitive behavior when detecting h-FABP from human serum in the range of 98 pg ml(-1)-100 ng ml(-1), with a detection limit of 0.836 ng ml(-1) comparative with a homogenous self-assembled monolayer (hSAM). Copyright © 2014 Elsevier B.V. All rights reserved.

A sensitive, colorimetric immunosensor based on Cu-MOFs and HRP for detection of dibutyl phthalate in environmental and food samples.

PubMed

Zhu, Nuanfei; Zou, Yanmin; Huang, Menglu; Dong, Shuaibing; Wu, Xiangyang; Liang, Guoxi; Han, Zhixiang; Zhang, Zhen

2018-08-15

A sensitive and artful colorimetric immunosensor based on horseradish peroxidase (HRP) was designed by labelling metal-organic frameworks (Cu-MOFs) on the second antibody (Cu-MOFs@Ab 2 ) as signal amplification for the detection of trace dibutyl phthalate (DBP). In this system, when Cu-MOFs@Ab 2 was captured by antigen- primary antibody (Ab 1 ) complex, tremendous Cu(II) will be released from Cu-MOFs in the presence of nitric acid (HNO 3 ), and Cu(II) will be further reduced to Cu(I) after the addition of sodium ascorbate (SA), consequently, inhibiting the HRP to catalyse the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (ox TMB). Under the optimized conditions, the limit of detection (LOD) was 1 μg L -1 , which was almost 60 times lower than that using a conventional ELISA with the same antibody. In addition, our method showed good accuracy and reproducibility (recoveries of 87.73-103.4%; CV values of 1.46-5.95%) through a spike-recovery analysis. The proposed immunosensor indicated great potential for trace DBP determination from environmental and food samples. Copyright © 2018. Published by Elsevier B.V.

Microchannel DNA Sequencing by End-Labelled Free Solution Electrophoresis

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

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.

Detection of staphylococcal enterotoxin A (SEA) at picogram level by a capacitive immunosensor.

PubMed

Jantra, Jongjit; Kanatharana, Proespichaya; Asawatreratanakul, Punnee; Wongkittisuksa, Booncharoen; Limsakul, Chusak; Thavarungkul, Panote

2011-01-01

This work presents the use of a flow injection capacitive immunosensor to detect staphylococcal enterotoxin A (SEA). The study was based on the direct detection of a capacitance change due to the binding between SEA and anti-SEA immobilized on a gold electrode. The optimal regeneration solution, flow rate, sample volume and buffer conditions were studied. Under the optimum conditions, this label-free biosensor provided linearity between 1 × 10(-12) g L(-1) and 1 × 10(-8) g L(-1) of SEA and the limit of detection was 1 × 10(-12) g L(-1) which was much lower than the infectious dose (0.5 × 10(-6) - 1 × 10(-6) g L(-1)). Using the regeneration solution of, 15.0 mM glycine-HCl pH 2.20, to break the binding between SEA and the immobilized anti-SEA enabled the electrode to be reused up to 39 times. This technique was applied to analyze SEA in liquid and solid food samples. Any matrix effect can be eliminated by simple dilution. SEA contamination was found in three samples, iced tea with milk (28 ± 1 ng L(-1)), orange juice (113 ± 6 ng L(-1)) and fried chicken (1.1 ± 0.2 ng g(-1)); however, the concentrations were much lower than the infectious dose. The proposed method would be useful for rapid screening of SEA in various matrices.

Novel photoelectrochemical immunosensor for disease-related protein assisted by hemin/G-quadruplex-based DNAzyme on gold nanoparticles to enhance cathodic photocurrent on p-CuBi2O4 semiconductor.

PubMed

Lv, Shuzhen; Zhang, Kangyao; Lin, Zhenzhen; Tang, Dianping

2017-10-15

A novel p-type semiconductor material (p-CuBi 2 O 4 ) is designed for the construction of split-type photoelectrochemical (PEC) immunosensor for alpha-fetoprotein (AFP) with the hemin assistant to enhance the cathodic photocurrent. Initially, the photocathode of PEC immunosensor is fabricated by p-CuBi 2 O 4 on a layer of gold nanoparticles (AuNPs, as a front contact of p-CuBi 2 O 4 ) to enhance the efficiency of charge separation. In the presence of target AFP, a sandwich-type immunoreaction was carried out in capture antibody-coated microplate by using detection antibody and hemin-based G-quadruplex (labeled on the AuNP) as the signal probe. Upon exonuclease I (Exo I) introduction, the enzyme digested the hemin/G-quadruplex-based DNAzyme to release the hemin[Fe(III)], which captured the generated electrons of p-CuBi 2 O 4 -based photocathode to enhance photocurrent via the reduction of hemin[Fe(III)] to hemin[Fe(II)] in PEC detection system. Under the optimal conditions, the split-type photocathodic immunosensor showed a wide linear dynamic range from 50pgmL -1 to 20ngmL -1 at a limit of detection (LOD) of 14.7pgmL -1 toward target AFP. Moreover, the PEC immunosensor also displayed high specificity and good reproducibility. Favorably, method accuracy was evaluated to analyze human serum specimens, and gave matched-well results in comparison with commercially available enzyme-linked immunosorbent assay (ELISA) method. Copyright © 2017 Elsevier B.V. All rights reserved.

DIRECT ELECTROCHEMICAL IMMUNOSENSOR FOR POLYCHLORINATED BIPHENYLS. (R825323)

EPA Science Inventory

A direct electrochemical immunosensor has been developed for the determination of polychlorinated biphenyls (PCBs) in water. The assay is based on the measurement of the current due to the specific binding between PCB and anti-PCB antibody-immobilized conducting polymer matrix. T...

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs.

PubMed

Vargas, Eva; Torrente-Rodríguez, Rebeca M; Ruiz-Valdepeñas Montiel, Víctor; Povedano, Eloy; Pedrero, María; Montoya, Juan J; Campuzano, Susana; Pingarrón, José M

2017-11-09

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA-RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H₂O₂/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at -0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA-RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNA t ) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.

Explosives detection in the marine environment using UUV-modified immunosensor

NASA Astrophysics Data System (ADS)

Charles, Paul T.; Adams, André A.; Deschamps, Jeffrey R.; Veitch, Scott P.; Hanson, Alfred; Kusterbeck, Anne W.

2011-05-01

Port and harbor security has rapidly become a point of interest and concern with the emergence of new improvised explosive devices (IEDs). The ability to provide physical surveillance and identification of IEDs and unexploded ordnances (UXO) at these entry points has led to an increased effort in the development of unmanned underwater vehicles (UUVs) equipped with sensing devices. Traditional sensors used to identify and locate potential threats are side scan sonar/acoustic methods and magnetometers. At the Naval Research Laboratory (NRL), we have developed an immunosensor capable of detecting trace levels of explosives that has been integrated into a REMUS payload for use in the marine environment. Laboratory tests using a modified PMMA microfluidic device with immobilized monoclonal antibodies specific for TNT and RDX have been conducted yielding detection levels in the low parts-per-billion (ppb) range. New designs and engineered improvements in microfluidic devices, fluorescence signal probes, and UUV internal fluidic and optical components have been investigated and integrated into the unmanned underwater prototype. Results from laboratory and recent field demonstrations using the prototype UUV immunosensor will be discussed. The immunosensor in combination with acoustic and other sensors could serve as a complementary characterization tool for the detection of IEDs, UXOs and other potential chemical or biological threats.

Humidity-resistant ambient-temperature solid-electrolyte amperometric sensing apparatus

DOEpatents

Zaromb, Solomon

1994-01-01

Apparatus and methods for detecting selected chemical compounds in air or other gas streams at room or ambient temperature includes a liquid-free humidity-resistant amperometric sensor comprising a sensing electrode and a counter and reference electrode separated by a solid electrolyte. The sensing electrode preferably contains a noble metal, such as Pt black. The electrolyte is water-free, non-hygroscopic, and substantially water-insoluble, and has a room temperature ionic conductivity .gtoreq.10.sup.-4 (ohm-cm).sup.-1, and preferably .gtoreq.0.01 (ohm-cm).sup.-1. The conductivity may be due predominantly to Ag+ ions, as in Ag.sub.2 WO.sub.4.4AgI, or to F- ions, as in Ce.sub.0.95 Ca.sub.0.05 F.sub.2.95. Electrical contacts serve to connect the electrodes to potentiostating and detecting circuitry which controls the potential of the sensing electrode relative to the reference electrode, detects the signal generated by the sensor, and indicates the detected signal.

A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection.

PubMed

Montoya, Angel; March, Carmen; Montagut, Yeison J; Moreno, Maria J; Manclus, Juan J; Arnau, Antonio; Jimenez, Yolanda; Jaramillo, Marisol; Marin, Paula A; Torres, Robinson A

2017-01-01

Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization. Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen. For the development of the HFF piezoelectric immunosensor, 100 MHz quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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.

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.

Label-Free Alignment of Nonmagnetic Particles in a Small Uniform Magnetic Field.

PubMed

Wang, Zhaomeng; Wang, Ying; Wu, Rui Ge; Wang, Z P; Ramanujan, R V

2018-01-01

Label-free manipulation of biological entities can minimize damage, increase viability and improve efficiency of subsequent analysis. Understanding the mechanism of interaction between magnetic and nonmagnetic particles in an inverse ferrofluid can provide a mechanism of label-free manipulation of such entities in a uniform magnetic field. The magnetic force, induced by relative magnetic susceptibility difference between nonmagnetic particles and surrounding magnetic particles as well as particle-particle interaction were studied. Label-free alignment of nonmagnetic particles can be achieved by higher magnetic field strength (Ba), smaller particle spacing (R), larger particle size (rp1), and higher relative magnetic permeability difference between particle and the surrounding fluid (Rμr). Rμr can be used to predict the direction of the magnetic force between both magnetic and nonmagnetic particles. A sandwich structure, containing alternate layers of magnetic and nonmagnetic particle chains, was studied. This work can be used for manipulation of nonmagnetic particles in lab-on-a-chip applications.

Detection of Explosives in a Dynamic Marine Environment Using a Moored TNT Immunosensor

PubMed Central

Charles, Paul T.; Adams, André A.; Deschamps, Jeffrey R.; Veitch, Scott; Hanson, Al; Kusterbeck, Anne W.

2014-01-01

A field demonstration and longevity assessment for long-term monitoring of the explosive 2,4,6-trinitrotoluene (TNT) in a marine environment using an anti-TNT microfluidic immunosensor is described. The TNT immunosensor is comprised of a microfluidic device with 39 parallel microchannels (2.5 cm × 250 μm × 500 μm, L × W × D) fabricated in poly(methylmethacrylate) (PMMA), then chemically functionalized with antibodies possessing a high affinity for TNT. Synthesized fluorescence reporter complexes used in a displacement-based assay format were used for TNT identification. For field deployment the TNT immunosensor was configured onto a submersible moored steel frame along with frame controller, pumps and TNT plume generator and deployed pier side for intermittent plume sampling of TNT (1h increments). Under varying current and tidal conditions trace levels of TNT in natural seawater were detected over an extended period (>18 h). Overnight operation and data recording was monitored via a web interface. PMID:24583970

Detection of explosives in a dynamic marine environment using a moored TNT immunosensor.

PubMed

Charles, Paul T; Adams, André A; Deschamps, Jeffrey R; Veitch, Scott; Hanson, Al; Kusterbeck, Anne W

2014-02-27

A field demonstration and longevity assessment for long-term monitoring of the explosive 2,4,6-trinitrotoluene (TNT) in a marine environment using an anti-TNT microfluidic immunosensor is described. The TNT immunosensor is comprised of a microfluidic device with 39 parallel microchannels (2.5 cm × 250 µm × 500 µm, L × W × D) fabricated in poly(methylmethacrylate) (PMMA), then chemically functionalized with antibodies possessing a high affinity for TNT. Synthesized fluorescence reporter complexes used in a displacement-based assay format were used for TNT identification. For field deployment the TNT immunosensor was configured onto a submersible moored steel frame along with frame controller, pumps and TNT plume generator and deployed pier side for intermittent plume sampling of TNT (1h increments). Under varying current and tidal conditions trace levels of TNT in natural seawater were detected over an extended period (>18 h). Overnight operation and data recording was monitored via a web interface.

A novel label-free cell-based assay technology using biolayer interferometry.

PubMed

Verzijl, D; Riedl, T; Parren, P W H I; Gerritsen, A F

2017-01-15

Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the β 2 -adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Fiber optic immunosensor for cross-linked fibrin concentration

NASA Astrophysics Data System (ADS)

Moskowitz, Samuel E.

2000-08-01

Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels <0.1 ng/ml. A capture monoclonal antibody to the antigen soluble cross-linked fibrin is employed. Immobilized at the tip of an optical fiber by avidin-biotin, the captured antigen is detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

ZnO thin film transistor immunosensor with high sensitivity and selectivity

NASA Astrophysics Data System (ADS)

Reyes, Pavel Ivanoff; Ku, Chieh-Jen; Duan, Ziqing; Lu, Yicheng; Solanki, Aniruddh; Lee, Ki-Bum

2011-04-01

A zinc oxide thin film transistor-based immunosensor (ZnO-bioTFT) is presented. The back-gate TFT has an on-off ratio of 108 and a threshold voltage of 4.25 V. The ZnO channel surface is biofunctionalized with primary monoclonal antibodies that selectively bind with epidermal growth factor receptor (EGFR). Detection of the antibody-antigen reaction is achieved through channel carrier modulation via pseudo double-gating field effect caused by the biochemical reaction. The sensitivity of 10 fM detection of pure EGFR proteins is achieved. The ZnO-bioTFT immunosensor also enables selectively detecting 10 fM of EGFR in a 5 mg/ml goat serum solution containing various other proteins.

Label-free SERS in biological and biomedical applications: Recent progress, current challenges and opportunities

NASA Astrophysics Data System (ADS)

Zheng, Xiao-Shan; Jahn, Izabella Jolan; Weber, Karina; Cialla-May, Dana; Popp, Jürgen

2018-05-01

To achieve an insightful look within biomolecular processes on the cellular level, the development of diseases as well as the reliable detection of metabolites and pathogens, a modern analytical tool is needed that is highly sensitive, molecular-specific and exhibits fast detection. Surface-enhanced Raman spectroscopy (SERS) is known to meet these requirements and, within this review article, the recent progress of label-free SERS in biological and biomedical applications is summarized and discussed. This includes the detection of biomolecules such as metabolites, nucleic acids and proteins. Further, the characterization and identification of microorganisms has been achieved by label-free SERS-based approaches. Eukaryotic cells can be characterized by SERS in order to gain information about the outer cell wall or to detect intracellular molecules and metabolites. The potential of SERS for medically relevant detection schemes is emphasized by the label-free detection of tissue, the investigation of body fluids as well as applications for therapeutic and illicit drug monitoring. The review article is concluded with an evaluation of the recent progress and current challenges in order to highlight the direction of label-free SERS in the future.

A label-free fluorescent aptamer sensor based on regulation of malachite green fluorescence

PubMed Central

Xu, Weichen; Lu, Yi

2009-01-01

We report a label-free fluorescent aptamer sensor for adenosine based on the regulation of malachite green (MG) fluorescence, with comparable sensitivity and selectivity to other labeled adenosine aptamer-based sensors. The sensor consists of free MG, an aptamer strand containing an adenosine aptamer next to an MG aptamer, and a bridging strand that partially hybridizes to the aptamer strand. Such a hybridization prevents MG from binding to MG aptamer, resulting in low fluorescence of MG in the absence of adenosine. Addition of adenosine causes the adenosine aptamer to bind adenosine, weakening the hybridization of the aptamer strand with the bridging strand, making it possible for MG to bind to the aptamer strand and exhibits high fluorescence intensity. Since this design is based purely on nucleic acid hybridization, it can be generally applied to other aptamers for the label-free detection of a broad range of analytes. PMID:20017558

Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers

PubMed Central

Riahi, Reza; Shaegh, Seyed Ali Mousavi; Ghaderi, Masoumeh; Zhang, Yu Shrike; Shin, Su Ryon; Aleman, Julio; Massa, Solange; Kim, Duckjin; Dokmeci, Mehmet Remzi; Khademhosseini, Ali

2016-01-01

There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips. PMID:27098564

Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers

NASA Astrophysics Data System (ADS)

Riahi, Reza; Shaegh, Seyed Ali Mousavi; Ghaderi, Masoumeh; Zhang, Yu Shrike; Shin, Su Ryon; Aleman, Julio; Massa, Solange; Kim, Duckjin; Dokmeci, Mehmet Remzi; Khademhosseini, Ali

2016-04-01

There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips.

A multi-center study benchmarks software tools for label-free proteome quantification

PubMed Central

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

2016-01-01

The 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, SWATH2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra), a method that uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test datasets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation windows 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 datasets 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. PMID:27701404

Electrochemical immunosensor based on poly (3,4-ethylenedioxythiophene) modified with gold nanoparticle to detect aflatoxin B1.

PubMed

Sharma, Arati; Kumar, A; Khan, Raju

2017-07-01

Gold nanoparticles (Au-NPs) functionalized Poly (3,4-ethylenedioxythiophene) (PEDOT) bioelectrodes were fabricated layer by layer deposition on ITO electrode for detection of aflatoxin B 1 . The monoclonal anti-aflatoxin antibodies (Anti-AFB 1 ) were immobilized over the surface of PEDOT/Au-NPs/ITO using EDC/NHS coupling. The surface morphology and characteristics of the modified electrodes were investigated by scanning electron microscope and contact angle measurements. The electrochemical analysis of the fabricated immunoelectrode and the immobilization of the antibodies have been evaluated and confirmed by performing Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Fourier Transform Infrared Spectroscopy (FTIR). Decrease in the value of electron transfer resistance (R et ) and increase in the peak current values after incorporation of Au-NPs signifies the enhanced properties of PEDOT embedded Au-NPs. The heterogeneous rate constant (k s ) and transfer coefficient (α) have been determined by using Laviron's method. The fabricated immunosensor exhibits high sensitive amperometric response of 3.72μAngmL -1 towards AFB 1 concentration in a linear range of 1-25ngmL -1 with detection limit (LOD) of 0.0045ngmL -1 and limit of quantification (LOQ) of 0.0156ngmL -1 . The fabricated immunoelectrode shows a reproducibility of 96.13% and 94.5% towards real maize sample spiked with AFB 1 of concentration 30ngmL -1 and 50ngmL -1 , respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

A dual marker label free electrochemical assay for Flavivirus dengue diagnosis.

PubMed

Santos, Adriano; Bueno, Paulo R; Davis, Jason J

2018-02-15

Dengue is a RNA viral illness of the genus Flavivirus which can cause, depending on the pervasiveness of the infection, hemorrhagic dengue fever or dengue shock syndrome. Herein we present an electrochemical label free approach enabling the rapid sensitive quantification of NS1 and IgG (supporting an ability to distinguish primary and secondary infections). Using a bifunctional SAM containing PEG moieties and a tethered redox thiol, both markers are detectable across clinically relevant levels by label free impedance derived redox capacitance. A subsequent frequency specific immittance function approach enables assaying (within seconds) with no impairment of analytical quality (linearity, sensitivity and variance). Copyright © 2017 Elsevier B.V. All rights reserved.

Specific labeling of zinc finger proteins using noncanonical amino acids and copper-free click chemistry.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A; Schroeder, Charles M

2012-09-19

Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays, and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry.

Comparing model-based and model-free analysis methods for QUASAR arterial spin labeling perfusion quantification.

PubMed

Chappell, Michael A; Woolrich, Mark W; Petersen, Esben T; Golay, Xavier; Payne, Stephen J

2013-05-01

Amongst the various implementations of arterial spin labeling MRI methods for quantifying cerebral perfusion, the QUASAR method is unique. By using a combination of labeling with and without flow suppression gradients, the QUASAR method offers the separation of macrovascular and tissue signals. This permits local arterial input functions to be defined and "model-free" analysis, using numerical deconvolution, to be used. However, it remains unclear whether arterial spin labeling data are best treated using model-free or model-based analysis. This work provides a critical comparison of these two approaches for QUASAR arterial spin labeling in the healthy brain. An existing two-component (arterial and tissue) model was extended to the mixed flow suppression scheme of QUASAR to provide an optimal model-based analysis. The model-based analysis was extended to incorporate dispersion of the labeled bolus, generally regarded as the major source of discrepancy between the two analysis approaches. Model-free and model-based analyses were compared for perfusion quantification including absolute measurements, uncertainty estimation, and spatial variation in cerebral blood flow estimates. Major sources of discrepancies between model-free and model-based analysis were attributed to the effects of dispersion and the degree to which the two methods can separate macrovascular and tissue signal. Copyright © 2012 Wiley Periodicals, Inc.

Mobile, Multi-modal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia

DTIC Science & Technology

2015-10-01

eyes and image choroidal vessels/capillaries using CARS intravital microscopy Subtask 3: Measure oxy-hemoglobin levels in PBI test and control eyes...AWARD NUMBER: W81XWH-14-1-0537 TITLE: Mobile, Multi-modal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia...4. TITLE AND SUBTITLE Mobile, Multimodal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia 5a. CONTRACT NUMBER W81XWH

Label-free, real-time interaction and adsorption analysis 1: surface plasmon resonance.

PubMed

Fee, Conan J

2013-01-01

A key requirement for the development of proteins for use in nanotechnology is an understanding of how individual proteins bind to other molecules as they assemble into larger structures. The introduction of labels to enable the detection of biomolecules brings the inherent risk that the labels themselves will influence the nature of biomolecular interactions. Thus, there is a need for label-free interaction and adsorption analysis. In this and the following chapter, two biosensor techniques are reviewed: surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM). Both allow real-time analysis of biomolecular interactions and both are label-free. The first of these, SPR, is an optical technique that is highly sensitive to the changes in refractive index that occur with protein (or other molecule) accumulation near an illuminated gold surface. Unlike QCM ( Chapter 18 ) SPR is not affected by the water that may be associated with the adsorbed layer nor by conformational changes in the adsorbed species. SPR thus provides unique information about the interaction of a protein with its binding partners.

Electrochemical immunosensor assay (EIA) for sensitive detection of E. coli O157:H7 with signal amplification on a SG-PEDOT-AuNPs electrode interface.

PubMed

Guo, Yuna; Wang, Yu; Liu, Su; Yu, Jinghua; Wang, Hongzhi; Cui, Min; Huang, Jiadong

2015-01-21

A novel electrochemical immunosensor assay (EIA) for highly sensitive and specific detection of Escherichia coli O157:H7 has been developed. This immunosensor is constructed by the assembly of capture antibody on SG-PEDOT-AuNPs composites modified glass carbon electrode. In the presence of target E. coli O157:H7, horseradish peroxidase (HRP)-labeled antibody is captured on the electrode surface to form a sandwich-type system via the specific identification. As a result, E. coli O157:H7 detection is realized by outputting a redox current from electro-reduction of hydrogen peroxide reaction catalyzed by HRP. In our assay, the combination of the unique properties of sulfonated graphene (SG) and gold nanoparticles (AuNPs) can not only accelerate electron transfer on electrode interface, but also provide an excellent scaffold for the conjugation of capture antibody that significantly improves the target capture efficiency and enhances the sensitivity of the biosensor. The results reveal the calibration plot obtained for E. coli O157:H7 is approximately linear from 7.8 × 10-7.8 × 10(6) colony-forming unit (cfu) mL(-1) with the limit of detection of 3.4 × 10 cfu mL(-1). In addition, the biosensor has been successfully applied to the quantitative assay of E. coli O157:H7 in synthetic samples (spring water and milk). Hence, the developed electrochemical-based immunosensor might provide a useful and practical tool for E. coli O157:H7 determination and related food safety analysis and clinical diagnosis.

Electrochemical immunosensor with nanocellulose-Au composite assisted multiple signal amplification for detection of avian leukosis virus subgroup J.

PubMed

Liu, Chao; Dong, Jing; Waterhouse, Geoffrey I N; Cheng, Ziqiang; Ai, Shiyun

2018-03-15

A sensitive sandwich-type electrochemical immunosensor was developed for the detection of avian leukosis virus subgroup J (ALV-J), which benefitted from multiple signal amplification involving graphene-perylene-3,4,9,10-tetracarboxylic acid nanocomposites (GR-PTCA), nanocellulose-Au NP composites (NC-Au) and the alkaline phosphatase (ALP) catalytic reaction. GR-PTCA nanocomposites on glassy carbon electrodes served as the immunosensor platform. Due to their excellent electrical conductivity and abundant polycarboxylic sites, the GR-PTCA nanocomposites allowed fast electron transfer and good immobilization of primary antibodies, thereby affording a strong immunosensor signal in the presence of ALV-J. The detected signal could be further amplified by the introduction of NC-Au composites as a carrier of secondary antibodies (Ab 2 ) and by harnessing the catalytic properties of Au and ALP. Under optimized testing conditions, the electrochemical immunosensor displayed excellent analytical performance for the detection of ALV-J, showing a linear current response from 10 2.08 to 10 4.0 TCID 50 /mL (TCID 50 : 50% tissue culture infective dose) with a low detection limit of 10 1.98 TCID 50 /mL (S/N = 3). In addition to high sensitivity, the immunosensor showed very good selectivity, reproducibility and operational stability, demonstrating potential application for the quantitative detection of ALV-J in clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

PSEA-Quant: a protein set enrichment analysis on label-free and label-based protein quantification data.

PubMed

Lavallée-Adam, Mathieu; Rauniyar, Navin; McClatchy, Daniel B; Yates, John R

2014-12-05

The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights.

PSEA-Quant: A Protein Set Enrichment Analysis on Label-Free and Label-Based Protein Quantification Data

PubMed Central

2015-01-01

The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights. PMID:25177766

Amperometric detector designs for capillary electrophoresis microchips.

PubMed

Castaño-Alvarez, Mario; Fernández-Abedul, M Teresa; Costa-García, Agustín

2006-03-24

Electrochemical (EC) detection is a sensitive and miniaturisable detection mode for capillary electrophoresis (CE) microchips. Detection cell design is very important in order to ensure electrical isolation from the high separation voltage. Amperometric detectors with different designs have been developed for coupling EC detection to CE-microchips. Different working electrode alignment: in-channel or end-channel has been tested in conjunction with several materials: gold, platinum or carbon. The end-channel detector was based on a platinum or gold wire manually aligned at the exit of the separation channel. Thick- (screen-printed carbon electrode) and thin-film (sputtered gold film) electrodes have also been employed with this configuration, but with a different design that allowed the rapid replacement of the electrode. The in-channel detector was based on a gold film within the separation channel. A gold-based dual electrode detector, which combined for the first time in- and end-channel detection, has been also tested. These amperometric detectors have been evaluated in combination to poly(methylmethacrylate) (PMMA) and Topas (thermoplastic olefin polymer of amorphous structure) CE-microchips. Topas is a new and promising cyclic olefin copolymer with high chemical resistance. Relevant parameters of the polymer microchip separation such as precision, efficiency or resolution and amperometric detection were studied with the different detector designs using p-aminophenol and L-ascorbic acid as model analytes in Tris-based buffer pH 9.0.

[Amperometric biosensor for ethanol analysis in wines and grape must during wine fermentation].

PubMed

Shkotova, L V; Slast'ia, E A; Zhyliakova, T A; Soldatkin, O P; Schuhmann, W; Dziadevych, S V

2005-01-01

The amperometric biosensor for ethanol determination based on alcohol oxidase immobilised by the method of electrochemical polymerization has been developed. The industrial screen-printed platinum electrodes were used as transducers for creation of amperometric alcohol biosensor. Optimal conditions for electrochemical deposition of an active membrane with alcohol oxidase has been determined. Biosensors are characterised by good reproducibility and operational stability with minimal detection limit of ethanol 8 x 10(-5) M. The good correlation of results for ethanol detection in wine and during wine fermentation by using the developed amperometric biosensor with the data obtained by the standard methods was shown (r = 0.995).

A Fumonisins Immunosensor Based on Polyanilino-Carbon Nanotubes Doped with Palladium Telluride Quantum Dots

PubMed Central

Masikini, Milua; Mailu, Stephen N.; Tsegaye, Abebaw; Njomo, Njagi; Molapo, Kerileng M.; Ikpo, Chinwe O.; Sunday, Christopher Edozie; Rassie, Candice; Wilson, Lindsay; Baker, Priscilla G. L.; Iwuoha, Emmanuel I.

2015-01-01

An impedimetric immunosensor for fumonisins was developed based on poly(2,5-dimethoxyaniline)-multi-wall carbon nanotubes doped with palladium telluride quantum dots onto a glassy carbon surface. The composite was assembled by a layer-by-layer method to form a multilayer film of quantum dots (QDs) and poly(2,5-dimethoxyaniline)-multi-wall carbon nanotubes (PDMA-MWCNT). Preparation of the electrochemical immunosensor for fumonisins involved drop-coating of fumonisins antibody onto the composite modified glassy carbon electrode. The electrochemical impedance spectroscopy response of the FB1 immunosensor (GCE/PT-PDMA-MWCNT/anti-Fms-BSA) gave a linear range of 7 to 49 ng L−1 and the corresponding sensitivity and detection limits were 0.0162 kΩ L ng−1 and 0.46 pg L−1, respectively, hence the limit of detection of the GCE/PT-PDMA-MWCNT immunosensor for fumonisins in corn certified material was calculated to be 0.014 and 0.011 ppm for FB1, and FB2 and FB3, respectively. These results are lower than those obtained by ELISA, a provisional maximum tolerable daily intake (PMTDI) for fumonisins (the sum of FB1, FB2, and FB3) established by the Joint FAO/WHO expert committee on food additives and contaminants of 2 μg kg−1 and the maximum level recommended by the U.S. Food and Drug Administration (FDA) for protection of human consumption (2–4 mg L−1). PMID:25558993

A fumonisins immunosensor based on polyanilino-carbon nanotubes doped with palladium telluride quantum dots.

PubMed

Masikini, Milua; Mailu, Stephen N; Tsegaye, Abebaw; Njomo, Njagi; Molapo, Kerileng M; Ikpo, Chinwe O; Sunday, Christopher Edozie; Rassie, Candice; Wilson, Lindsay; Baker, Priscilla G L; Iwuoha, Emmanuel I

2014-12-30

An impedimetric immunosensor for fumonisins was developed based on poly(2,5-dimethoxyaniline)-multi-wall carbon nanotubes doped with palladium telluride quantum dots onto a glassy carbon surface. The composite was assembled by a layer-by-layer method to form a multilayer film of quantum dots (QDs) and poly(2,5-dimethoxyaniline)-multi-wall carbon nanotubes (PDMA-MWCNT). Preparation of the electrochemical immunosensor for fumonisins involved drop-coating of fumonisins antibody onto the composite modified glassy carbon electrode. The electrochemical impedance spectroscopy response of the FB1 immunosensor (GCE/PT-PDMA-MWCNT/anti-Fms-BSA) gave a linear range of 7 to 49 ng L-1 and the corresponding sensitivity and detection limits were 0.0162 kΩ L ng-1 and 0.46 pg L-1, respectively, hence the limit of detection of the GCE/PT-PDMA-MWCNT immunosensor for fumonisins in corn certified material was calculated to be 0.014 and 0.011 ppm for FB1, and FB2 and FB3, respectively. These results are lower than those obtained by ELISA, a provisional maximum tolerable daily intake (PMTDI) for fumonisins (the sum of FB1, FB2, and FB3) established by the Joint FAO/WHO expert committee on food additives and contaminants of 2 μg kg-1 and the maximum level recommended by the U.S. Food and Drug Administration (FDA) for protection of human consumption (2-4 mg L-1).

Label-Free Bioanalyte Detection from Nanometer to Micrometer Dimensions-Molecular Imprinting and QCMs †.

PubMed

Mujahid, Adnan; Mustafa, Ghulam; Dickert, Franz L

2018-06-01

Modern diagnostic tools and immunoassay protocols urges direct analyte recognition based on its intrinsic behavior without using any labeling indicator. This not only improves the detection reliability, but also reduces sample preparation time and complexity involved during labeling step. Label-free biosensor devices are capable of monitoring analyte physiochemical properties such as binding sensitivity and selectivity, affinity constants and other dynamics of molecular recognition. The interface of a typical biosensor could range from natural antibodies to synthetic receptors for example molecular imprinted polymers (MIPs). The foremost advantages of using MIPs are their high binding selectivity comparable to natural antibodies, straightforward synthesis in short time, high thermal/chemical stability and compatibility with different transducers. Quartz crystal microbalance (QCM) resonators are leading acoustic devices that are extensively used for mass-sensitive measurements. Highlight features of QCM devices include low cost fabrication, room temperature operation, and most importantly ability to monitor extremely low mass shifts, thus potentially a universal transducer. The combination of MIPs with quartz QCM has turned out as a prominent sensing system for label-free recognition of diverse bioanalytes. In this article, we shall encompass the potential applications of MIP-QCM sensors exclusively label-free recognition of bacteria and virus species as representative micro and nanosized bioanalytes.

Skin protection by sunscreens is improved by explicit labeling and providing free sunscreen.

PubMed

Nicol, Isabelle; Gaudy, Caroline; Gouvernet, Joanny; Richard, Marie A; Grob, Jean J

2007-01-01

Whatever the improvement in the protection spectrum of sunscreens (SCs), actual skin protection mainly depends on the way they are used, especially on the quantity applied. This prospective randomized study assessed how much sun protection factor (SPF) labeling, which is hardly understandable by a layman, and high cost account for misuse of SCs. In three beach resorts in France, 364 individuals were blindly randomized during their holidays to three arms (1) free SCs intervention (FS) = four types of SCs with their usual SPF label (60B-A, 20B-A, 12B-A, 6B-3A) at free disposal; (2) same free SCs with an explicit labeling (FNL), including sunburn protection, likely protection against long-term effects of UV, and possibility to get a tan; and (3) no intervention (NI). As compared to FS, FNL increased the quantity of SCs applied, mainly in the minority of people who were not "tan-seekers", reduced sunburns particularly in sun-sensitive individuals (25.6 vs 58.3%, P=0.005), and induced a shift in the level of SCs chosen. Free delivery SCs were associated with a more systematic application of SCs in case of exposure, and a decreased sunburn occurrence, without increase of exposure. These results suggest that a labeling more explicit for the public would result in a better protection in SC users and that cost could be a limiting factor to use SC as often as necessary.

Label-enhanced surface plasmon resonance applied to label-free interaction analysis of small molecules and fragments.

PubMed

Eng, Lars; Nygren-Babol, Linnéa; Hanning, Anders

2016-10-01

Surface plasmon resonance (SPR) is a well-established method for studying interactions between small molecules and biomolecules. In particular, SPR is being increasingly applied within fragment-based drug discovery; however, within this application area, the limited sensitivity of SPR may constitute a problem. This problem can be circumvented by the use of label-enhanced SPR that shows a 100-fold higher sensitivity as compared with conventional SPR. Truly label-free interaction data for small molecules can be obtained by applying label-enhanced SPR in a surface competition assay format. The enhanced sensitivity is accompanied by an increased specificity and inertness toward disturbances (e.g., bulk refractive index disturbances). Label-enhanced SPR can be used for fragment screening in a competitive assay format; the competitive format has the added advantage of confirming the specificity of the molecular interaction. In addition, label-enhanced SPR extends the accessible kinetic regime of SPR to the analysis of very fast fragment binding kinetics. In this article, we demonstrate the working principles and benchmark the performance of label-enhanced SPR in a model system-the interaction between carbonic anhydrase II and a number of small-molecule sulfonamide-based inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

Ohio Appalachian residents' views on smoke-free laws and cigarette warning labels.

PubMed

Reiter, Paul L; Wewers, Mary E; Paskett, Electra D; Klein, Elizabeth G; Katz, Mira L

2012-01-01

Smoke-free laws and the addition of graphic warning labels to cigarette packages represent public health policies that can potentially reduce smoking and smoking-related disease. The attitudes and beliefs relating to these policies were examined among residents of Ohio Appalachia, a mostly rural region with high smoking prevalence among its residents. Focus groups were conducted with participants from Ohio Appalachia during the summer of 2007. Groups included healthcare providers (n=37), community leaders (n=31), parents (n=19), and young adult women aged 18-26 years (n=27). Most participants were female (94%), non-Hispanic White (94%), and married (65%). Participants believed that most non-smokers supported Ohio's enforced statewide comprehensive smoke-free law that began in 2007, while some smokers opposed the law due to a perceived infringement of their rights. They also reported that most residents and local businesses were abiding by and enforcing the law. Participants supported the addition of graphic warning labels to cigarette packages in the USA. They believed that such warning labels could help deter adolescents and adult non-smokers from smoking initiation, particularly if the negative aesthetic effects of smoking were emphasized. However, they felt the labels would be less effective among current smokers and older individuals living in their communities. Participants generally held positive views about both the smoke-free law and the addition of graphic warning labels to cigarette packages in the USA. These tobacco-related public health policies are promising strategies for potentially reducing smoking and its associated diseases among residents living in Appalachia. Additional research is needed to further examine support for these policies among more diverse Appalachian populations.

Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone.

PubMed

Lee, Kuang-Li; You, Meng-Lin; Tsai, Chia-Hsin; Lin, En-Hung; Hsieh, Shu-Yi; Ho, Ming-Hsun; Hsu, Ju-Chun; Wei, Pei-Kuen

2016-01-15

The widespread and intensive use of neonicotinoid insecticides induces negative cascading effects on ecosystems. It is desirable to develop a portable sensitive sensing platform for on-site screening of high-risk pesticides. We combined an indirect competitive immunoassay, highly sensitive surface plasmon resonance (SPR) biochip and a simple portable imaging setup for label-free detection of imidacloprid pesticides. The SPR biochip consists of several capped nanoslit arrays with different periods which form a spectral image on the chip. The qualitative and semiquantitative analyses of pesticides can be directly observed from the spot shift on the chip. The precise semiquantitative analyses can be further completed by using image processing in a smartphone. We demonstrate simultaneous detection of four different concentrations of imidacloprid pesticides. The visual detection limit is about 1ppb, which is well below the maximum residue concentration permitted by law (20ppb). Compared to the one-step strip assay, the proposed chip is capable of performing semiquantitative analyses and multiple detection. Compared to the enzyme-linked immunosorbent assay, our method is label-free and requires simple washing steps and short reaction time. In addition, the label-free chip has a comparable sensitivity but wider working range than those labeling techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-Free Raman Imaging to Monitor Breast Tumor Signatures

NASA Astrophysics Data System (ADS)

Ciubuc, John

Methods built on Raman spectroscopy have shown major potential in describing and discriminating between malignant and benign specimens. Accurate, real-time medical diagnosis benefits in substantial improvements through this vibrational optical method. Not only is acquisition of data possible in milliseconds and analysis in minutes, Raman allows concurrent detection and monitoring of all biological components. Besides validating a significant Raman signature distinction between non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, this study reveals a label-free method to assess overexpression of epidermal growth factor receptors (EGFR) in tumor cells. EGFR overexpression sires Raman features associated with phosphorylated threonine and serine, and modifications of DNA/RNA characteristics. Investigations by gel electrophoresis reveal EGF induction of phosphorylated Akt, agreeing with the Raman results. The analysis presented is a vital step toward Raman-based evaluation of EGF receptors in breast cancer cells. With the goal of clinically applying Raman-guided methods for diagnosis of breast tumors, the current results lay the basis for proving label-free optical alternatives in making prognosis of the disease.

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7.

PubMed

Su, Xiao-Li; Li, Yanbin

2004-01-15

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.

Enzyme-free ethanol sensor based on electrospun nickel nanoparticle-loaded carbon fiber paste electrode.

PubMed

Liu, Yang; Zhang, Lei; Guo, Qiaohui; Hou, Haoqing; You, Tianyan

2010-03-24

We have developed a novel nickel nanoparticle-loaded carbon fiber paste (NiCFP) electrode for enzyme-free determination of ethanol. An electrospinning technique was used to prepare the NiCF composite with large amounts of spherical nanoparticles firmly embedded in carbon fibers (CF). In application to electroanalysis of ethanol, the NiCFP electrode exhibited high amperometric response and good operational stability. The calibration curve was linear up to 87.5 mM with a detection limit of 0.25 mM, which is superior to that obtained with other transition metal based electrodes. For detection of ethanol present in liquor samples, the values obtained with the NiCFP electrode were in agreement with the ones declared on the label. The attractive analytical performance and simple preparation method make this novel material promising for the development of effective enzyme-free sensors. Copyright 2010 Elsevier B.V. All rights reserved.

21 CFR 101.91 - Gluten-free labeling of food.

Code of Federal Regulations, 2014 CFR

2014-04-01

... that has not been processed to remove gluten (e.g., wheat flour); or (3) An ingredient that is derived... is below 20 ppm gluten (i.e., below 20 mg gluten per kg of food). (b) Requirements. (1) A food that... Nutrient Content Claims nor Health Claims § 101.91 Gluten-free labeling of food. (a) Definitions. (1) The...

Patterns of free amino acids in German convenience food products: marked mismatch between label information and composition.

PubMed

Hermanussen, M; Gonder, U; Jakobs, C; Stegemann, D; Hoffmann, G

2010-01-01

Free amino acids affect food palatability. As information on amino acids in frequently purchased pre-packaged food is virtually absent, we analyzed free amino acid patterns of 17 frequently purchased ready-to-serve convenience food products, and compared them with the information obtained from the respective food labels. Quantitative amino acid analysis was performed using ion-exchange chromatography. gamma-Aminobutyric acid (GABA) concentrations were verified using a stable isotope dilution gas chromatography/mass spectrometry (GC-MS) method. The patterns of free amino acids were compared with information obtained from food labels. An obvious mismatch between free amino acid patterns and food label information was detected. Even on considering that tomatoes and cereal proteins are naturally rich in glutamate, the concentrations of free glutamate outranged the natural concentration of this amino acid in several products, and strongly suggested artificial enrichment. Free glutamate was found to be elevated even in dishes that explicitly state 'no glutamate added'. Arginine was markedly elevated in lentils. Free cysteine was generally low, possibly reflecting thermal destruction of this amino acid during food processing. The meat and brain-specific dipeptide carnosine (CARN) was present in most meat-containing products. Some products did not contain detectable amounts of CARN in spite of meat content being claimed on the food labels. We detected GABA at concentrations that contribute significantly to the taste sensation. This investigation highlights a marked mismatch between food label information and food composition.

Humidity-resistant ambient-temperature solid-electrolyte amperometric sensing apparatus and methods

DOEpatents

Zaromb, Solomon

2001-01-01

Apparatus and methods for detecting selected chemical compounds in air or other gas streams at room or ambient temperature includes a liquid-free humidity-resistant amperometric sensor comprising a sensing electrode and a counter and reference electrode separated by a solid electrolyte. The sensing electrode preferably contains a noble metal, such as Pt black. The electrolyte is water-free, non-hygroscopic, and substantially water-insoluble, and has a room temperature ionic conductivity .gtoreq.10.sup.-4 (ohm-cm).sup.-1, and preferably .gtoreq.0.01 (ohm-cm).sup.-1. The conductivity may be due predominantly to Ag+ ions, as in Ag.sub.2 WO.sub.4.4AgI, or to F- ions, as in Ce.sub.0.95 Ca.sub.0.05 F.sub.2.95. Electrical contacts serve to connect the electrodes to potentiostating and detecting circuitry which controls the potential of the sensing electrode relative to the reference electrode, detects the signal generated by the sensor, and indicates the detected signal.

Specific Labeling of Zinc Finger Proteins using Non-canonical Amino Acids and Copper-free Click Chemistry

PubMed Central

Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A.

2012-01-01

Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry. PMID:22871171

Polyamidoamine dendrimers-capped carbon dots/Au nanocrystal nanocomposites and its application for electrochemical immunosensor.

PubMed

Gao, Qi; Han, Jingman; Ma, Zhanfang

2013-11-15

In this work, polyamidoamine dendrimers capped-carbon dots (PAMAM-CDs) were fabricated by one-step microwave assisted pyrolysis of citric acid (CA) and PAMAM, where the formation of CDs and the surface passivation were accomplished simultaneously. The obtained graphitic PAMAM-CDs, with abundant amine groups, were employed as reducing and capping agents for the formation of PAMAM-CDs/Au nanocrystal nanocomposites. The resulting nanocomposites exhibited excellent conductivity, stability and biocompatibility on the surface of electrode and were designed as an immobilized matrix for sensitive immunosensing of alpha-fetoprotein (AFP). The proposed immunosensor showed a wide linear detection range from 100 fg mL(-1) to 100 ng mL(-1). The detection limit for AFP was 0.025 pg mL(-1). Importantly, the immunosensor was evaluated for the analysis of clinical serum samples, obtaining a good correlation with enzyme-linked immunosorbent assay (ELISA). The results indicated that the immunosensor provided a possible application for the detection of AFP in clinical diagnosis. Copyright © 2013 Elsevier B.V. All rights reserved.

Prussian blue-gold nanoparticles-ionic liquid functionalized reduced graphene oxide nanocomposite as label for ultrasensitive electrochemical immunoassay of alpha-fetoprotein.

PubMed

Gao, Qi; Liu, Na; Ma, Zhanfang

2014-06-04

In this work, poly(diallyldimethylammonium chloride) (PDDA) protected Prussian blue/gold nanoparticles/ionic liquid functionalized reduced graphene oxide (IL-rGO-Au-PDDA-PB) nanocomposite was fabricated. The resulting nanocomposite exhibited high biocompatibility, conductivity and catalytic activity. To assess the performance of the nanocomposite, a sensitive sandwich-type immunosensor was constructed for detecting alpha-fetoprotein (AFP). Greatly enhanced sensitivity for this immunosensor was based on triple signal amplification strategies. Firstly, IL-rGO modified electrode was used as biosensor platform to capture a large amount of antibody due to its increased surface area, thus amplifying the detection response. Secondly, a large number of Au-PDDA-PB was conjugated on the surface of IL-rGO, which meant the enrichment of the signal and the more immobilization of label antibody. Finally, the catalytic reaction between H2O2 and the IL-rGO-Au-PDDA-PB nanocomposite further enhanced the signal response. The signals increased linearly with AFP concentrations in the range of 0.01-100 ng mL(-1). The detection limit for AFP was 4.6 pg mL(-1). The immunosensor showed high sensitivity, excellent selectivity and good stability. Moreover, the immunosensor was applied to the analysis of AFP in serum sample with satisfactory result. Copyright © 2014 Elsevier B.V. All rights reserved.

Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset.

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; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne

2016-01-30

Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in

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.

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

Dynamic and label-free high-throughput detection of biomolecular interactions based on phase-shift interferometry

NASA Astrophysics Data System (ADS)

Li, Qiang; Huang, Guoliang; Gan, Wupeng; Chen, Shengyi

2009-08-01

Biomolecular interactions can be detected by many established technologies such as fluorescence imaging, surface plasmon resonance (SPR)[1-4], interferometry and radioactive labeling of the analyte. In this study, we have designed and constructed a label-free, real-time sensing platform and its operating imaging instrument that detects interactions using optical phase differences from the accumulation of biological material on solid substrates. This system allows us to monitor biomolecular interactions in real time and quantify concentration changes during micro-mixing processes by measuring the changes of the optical path length (OPD). This simple interferometric technology monitors the optical phase difference resulting from accumulated biomolecular mass. A label-free protein chip that forms a 4×4 probe array was designed and fabricated using a commercial microarray robot spotter on solid substrates. Two positive control probe lines of BSA (Bovine Serum Albumin) and two experimental human IgG and goat IgG was used. The binding of multiple protein targets was performed and continuously detected by using this label-free and real-time sensing platform.

Integrated amperometric affinity biosensors using Co2+-tetradentate nitrilotriacetic acid modified disposable carbon electrodes: application to the determination of β-lactam antibiotics.

PubMed

Conzuelo, Felipe; Gamella, María; Campuzano, Susana; Martínez-Ruiz, Paloma; Esteban-Torres, María; de las Rivas, Blanca; Reviejo, A Julio; Muñoz, Rosario; Pingarrón, José M

2013-03-19

A novel strategy for the construction of disposable amperometric affinity biosensors is described in this work. The approach uses a recombinant bacterial penicillin binding protein (PBP) tagged by an N-terminal hexahistidine tail which was immobilized onto Co(2+)-tetradentate nitrilotriacetic acid (NTA)-modified screen-printed carbon electrodes (SPCEs). The biosensor was employed for the specific detection and quantification of β-lactam antibiotics residues in milk, which was accomplished by means of a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling. The amperometric response measured at -0.20 V versus the Ag pseudoreference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as redox mediator was used as the transduction signal. The developed affinity sensor allowed limits of detection to be obtained in the low part-per-billion level for the antibiotics tested in untreated milk samples. Moreover, the biosensor exhibited a good selectivity against other antibiotics residues frequently detected in milk and dairy products. The analysis time was of approximately 30 min.

Copper oxide assisted cysteine hierarchical structures for immunosensor application

NASA Astrophysics Data System (ADS)

Pandey, Chandra Mouli; Sumana, Gajjala; Tiwari, Ida

2014-09-01

The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 μm have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38 × 10-4 cm s-1. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.

Highly stable porous silicon-carbon composites as label-free optical biosensors.

PubMed

Tsang, Chun Kwan; Kelly, Timothy L; Sailor, Michael J; Li, Yang Yang

2012-12-21

A stable, label-free optical biosensor based on a porous silicon-carbon (pSi-C) composite is demonstrated. The material is prepared by electrochemical anodization of crystalline Si in an HF-containing electrolyte to generate a porous Si template, followed by infiltration of poly(furfuryl) alcohol (PFA) and subsequent carbonization to generate the pSi-C composite as an optically smooth thin film. The pSi-C sensor is significantly more stable toward aqueous buffer solutions (pH 7.4 or 12) compared to thermally oxidized (in air, 800 °C), hydrosilylated (with undecylenic acid), or hydrocarbonized (with acetylene, 700 °C) porous Si samples prepared and tested under similar conditions. Aqueous stability of the pSi-C sensor is comparable to related optical biosensors based on porous TiO(2) or porous Al(2)O(3). Label-free optical interferometric biosensing with the pSi-C composite is demonstrated by detection of rabbit IgG on a protein-A-modified chip and confirmed with control experiments using chicken IgG (which shows no affinity for protein A). The pSi-C sensor binds significantly more of the protein A capture probe than porous TiO(2) or porous Al(2)O(3), and the sensitivity of the protein-A-modified pSi-C sensor to rabbit IgG is found to be ~2× greater than label-free optical biosensors constructed from these other two materials.

In Vivo Integrity and Biological Fate of Chelator-Free Zirconium-89-Labeled Mesoporous Silica Nanoparticles

PubMed Central

2015-01-01

Traditional chelator-based radio-labeled nanoparticles and positron emission tomography (PET) imaging are playing vital roles in the field of nano-oncology. However, their long-term in vivo integrity and potential mismatch of the biodistribution patterns between nanoparticles and radio-isotopes are two major concerns for this approach. Here, we present a chelator-free zirconium-89 (89Zr, t1/2 = 78.4 h) labeling of mesoporous silica nanoparticle (MSN) with significantly enhanced in vivo long-term (>20 days) stability. Successful radio-labeling and in vivo stability are demonstrated to be highly dependent on both the concentration and location of deprotonated silanol groups (−Si–O–) from two types of silica nanoparticles investigated. This work reports 89Zr-labeled MSN with a detailed labeling mechanism investigation and long-term stability study. With its attractive radio-stability and the simplicity of chelator-free radio-labeling, 89Zr-MSN offers a novel, simple, and accurate way for studying the in vivo long-term fate and PET image-guided drug delivery of MSN in the near future. PMID:26213260

In Vivo Integrity and Biological Fate of Chelator-Free Zirconium-89-Labeled Mesoporous Silica Nanoparticles.

PubMed

Chen, Feng; Goel, Shreya; Valdovinos, Hector F; Luo, Haiming; Hernandez, Reinier; Barnhart, Todd E; Cai, Weibo

2015-08-25

Traditional chelator-based radio-labeled nanoparticles and positron emission tomography (PET) imaging are playing vital roles in the field of nano-oncology. However, their long-term in vivo integrity and potential mismatch of the biodistribution patterns between nanoparticles and radio-isotopes are two major concerns for this approach. Here, we present a chelator-free zirconium-89 ((89)Zr, t1/2 = 78.4 h) labeling of mesoporous silica nanoparticle (MSN) with significantly enhanced in vivo long-term (>20 days) stability. Successful radio-labeling and in vivo stability are demonstrated to be highly dependent on both the concentration and location of deprotonated silanol groups (-Si-O(-)) from two types of silica nanoparticles investigated. This work reports (89)Zr-labeled MSN with a detailed labeling mechanism investigation and long-term stability study. With its attractive radio-stability and the simplicity of chelator-free radio-labeling, (89)Zr-MSN offers a novel, simple, and accurate way for studying the in vivo long-term fate and PET image-guided drug delivery of MSN in the near future.

Label-free and substrate-free potentiometric aptasensing using polycation-sensitive membrane electrodes.

PubMed

Ding, Jiawang; Chen, Yan; Wang, Xuewei; Qin, Wei

2012-02-21

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

A novel electrochemical immunosensor based on ITO modified by carboxyl-ended silane agent for ultrasensitive detection of MAGE-1 in human serum.

PubMed

Gündoğdu, Aslı; Aydın, Elif Burcu; Sezgintürk, Mustafa Kemal

2017-11-15

A new, low-cost electrochemical immunosensor was developed for rapid detection of Melanoma-associated antigen 1 (MAGE-1), a cancer biomarker. The fabrication procedure of immunosensor was based on the covalent immobilization of anti-MAGE-1, biorecognition molecule, on ITO electrode by carboxyethylsilanetriol (CTES) monolayer. The biosensing MAGE-1 antigen was monitored by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) technique. Apart from these techniques, single frequency impedance (SFI) was used for investigation of antibody-antigen interactions. Scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) were utilized for characterization of the proposed biosensor. To fabricate highly sensitive, good stability immunosensor, some parameters were optimized. Under optimal conditions, the developed electrochemical immunosensor for MAGE-1 exhibited a dynamic range of 4 fg/mL and 200 fg/mL with a low detection limit of 1.30 fg/mL. It had acceptable repeatability (5.05%, n = 20) and good storage stability (3.58% loss after 10 weeks). Moreover, this electrochemical immunosensor has been successfully applied to the determination of MAGE-1 in human serum samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Label-free optical biosensing with slot-waveguides.

PubMed

Barrios, Carlos A; Bañuls, María José; González-Pedro, Victoria; Gylfason, Kristinn B; Sánchez, Benito; Griol, Amadeu; Maquieira, A; Sohlström, H; Holgado, M; Casquel, R

2008-04-01

We demonstrate label-free molecule detection by using an integrated biosensor based on a Si(3)N(4)/SiO(2) slot-waveguide microring resonator. Bovine serum albumin (BSA) and anti-BSA molecular binding events on the sensor surface are monitored through the measurement of resonant wavelength shifts with varying biomolecule concentrations. The biosensor exhibited sensitivities of 1.8 and 3.2 nm/(ng/mm(2)) for the detection of anti-BSA and BSA, respectively. The estimated detection limits are 28 and 16 pg/mm(2) for anti-BSA and BSA, respectively, limited by wavelength resolution.

An electrochemical immunosensor for efficient detection of uropathogenic E. coli based on thionine dye immobilized chitosan/functionalized-MWCNT modified electrode.

PubMed

Gayathri, Chandran Hema; Mayuri, Pinapeddavari; Sankaran, Krishnan; Kumar, Annamalai Senthil

2016-08-15

Uropathogenic Escherichia coli (UPEC) is the major cause of 150 million Urinary Tract Infections (UTI) reported annually world-wide. High prevalence of multi-drug-resistance makes it dangerous and difficult to cure. Therefore simple, quick and early diagnostic tools are essential for effective treatment and control. We report an electrochemical immunosensor based on thionine dye (Th) immobilized on functionalized-multiwalled carbon nanotube+chitosan composite coated on glassy carbon electrode (GCE/f-MWCNT-Chit@Th) for quick and sensitive detection of UPEC in aqueous solution. This immunosensor was constructed by sequential immobilization of UPEC, bovine serum albumin, primary antibody and Horse Radish Peroxidase (HRP) tagged secondary antibody on the surface of GCE/f-MWCNT-Chit@Th. When analyzed using 2.5mM of hydrogen peroxide reduction reaction using cyclic voltammetry in phosphate buffer, pH 7.0, the immunosensor showed excellent linearity in a range of 10(2)-10(9)cfu of UPEC mL(-1) with a current sensitivity of 7.162μA {log(cfumL(-1))}(-1). The specificity of this immunosensor was tested using other UTI and non-UTI bacteria, Staphylococcus, Klebsiella, Proteus and Shigella. The clinical applicability of the immunosensor was also successfully tested directly in UPEC spiked urine samples (simulated sample). Copyright © 2016 Elsevier B.V. All rights reserved.

Ultrasensitive luminol electrochemiluminescence for protein detection based on in situ generated hydrogen peroxide as coreactant with glucose oxidase anchored AuNPs@MWCNTs labeling.

PubMed

Cao, Yaling; Yuan, Ruo; Chai, Yaqin; Mao, Li; Niu, Huan; Liu, Huijing; Zhuo, Ying

2012-01-15

In this study, an ultrasensitive luminol electrochemiluminescence (ECL) immunosensor was constructed using carboxyl group functionalized multi-walled carbon nanotubes (MWCNTs) as platform and glucose oxidase (GOD) supported on Au nanoparticles (AuNPs) decorated MWCNTs (AuNPs@MWCNTs-GOD) as labels. Firstly, using poly(ethylenimine) (PEI) as linkage reagents, AuNPs@MWCNTs were prepared and introduced for binding of the secondary antibody (Ab(2)) and glucose oxidase (GOD) with high loading amount and good biological activity due to the improved surface area of AuNPs@MWCNTs and excellent biocompatibility of AuNPs. Then the GOD and Ab(2) labeled AuNPs@MWCNTs were linked to the electrode surface via sandwich immunoreactions. These localized GOD and AuNPs amplified luminol ECL signals dramatically, which was achieved by efficient catalysis of the GOD and AuNPs towards the oxidation of glucose to in situ generate improved amount of hydrogen peroxide (H(2)O(2)) as coreactant and the enhancement of AuNPs to the ECL reaction of luminol-H(2)O(2). The experimental results demonstrated that the proposed immunosensor exhibited sensitive and stable response for the detection of α-1-fetoprotein (AFP), ranging from 0.0001 to 80 ng mL(-1) with a limit of detection down to 0.03 pg mL(-1) (S/N=3). With excellent stability, sensitivity, selectivity and simplicity, the proposed luminol ECL immunosensor showed great potential in clinical applications. Copyright © 2011 Elsevier B.V. All rights reserved.

Advantages and application of label-free detection assays in drug screening.

PubMed

Cunningham, Brian T; Laing, Lance G

2008-08-01

Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein-protein binding assays and protein-small molecule binding assays to be performed with high-throughput and high sensitivity.

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

PubMed Central

Schmidt, Carla; Grønborg, Mads; Deckert, Jochen; Bessonov, Sergey; Conrad, Thomas; Lührmann, Reinhard; Urlaub, Henning

2014-01-01

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique. PMID:24448447

Homemade Bienzymatic-Amperometric Biosensor for Beverages Analysis

ERIC Educational Resources Information Center

Blanco-Lopez, M. C.; Lobo-Castanon, M. J.; Miranda-Ordieres, A. J.

2007-01-01

The construction of an amperometric biosensor for glucose analysis is described demonstrating that the analysis is easy to perform and the biosensor gives good analytical performance. This experiment helped the students to acquire problem-solving and teamwork skills, allowing them to reach a high level of independent and critical thought.

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.

Label-free biosensing of Salmonella enterica serovars at single-cell level

USDA-ARS?s Scientific Manuscript database

Nanotechnology has greatly facilitated the development of label-free biosensors. The atomic force microscopy (AFM) has been used to study the molecular mechanism of the reactions for protein and aptamers. The surface plasmon resonance (SPR) have been used in fast detection of various pathogenic bact...

Luminescence-Functionalized Metal-Organic Frameworks Based on a Ruthenium(II) Complex: A Signal Amplification Strategy for Electrogenerated Chemiluminescence Immunosensors.

PubMed

Xiong, Cheng-Yi; Wang, Hai-Jun; Liang, Wen-Bin; Yuan, Ya-Li; Yuan, Ruo; Chai, Ya-Qin

2015-06-26

Novel luminescence-functionalized metal-organic frameworks (MOFs) with superior electrogenerated chemiluminescence (ECL) properties were synthesized based on zinc ions as the central ions and tris(4,4'-dicarboxylicacid-2,2'-bipyridyl)ruthenium(II) dichloride ([Ru(dcbpy)3](2+)) as the ligands. For potential applications, the synthesized MOFs were used to fabricate a "signal-on" ECL immunosensor for the detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP). As expected, enhanced ECL signals were obtained through a simple fabrication strategy because luminescence-functionalized MOFs not only effectively increased the loading of [Ru(dcbpy)3](2+), but also served as a loading platform in the ECL immunosensor. Furthermore, the proposed ECL immunosensor had a wide linear range from 5 pg mL(-1) to 25 ng mL(-1) and a relatively low detection limit of 1.67 pg mL(-1) (signal/noise=3). The results indicated that luminescence-functionalized MOFs provided a novel amplification strategy in the construction of ECL immunosensors and might have great prospects for application in bioanalysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A simple architecture with self-assembled monolayers to build immunosensors for detecting the pancreatic cancer biomarker CA19-9.

PubMed

Soares, Andrey Coatrini; Soares, Juliana Coatrini; Shimizu, Flavio Makoto; Rodrigues, Valquiria da Cruz; Awan, Iram Taj; Melendez, Matias Eliseo; Piazzetta, Maria Helena Oliveira; Gobbi, Angelo Luiz; Reis, Rui Manuel; Fregnani, José Humberto T G; Carvalho, André Lopes; Oliveira, Osvaldo N

2018-05-14

The challenge of the early diagnosis of pancreatic cancer in routine clinical practice requires low-cost means of detection, and this may be achieved with immunosensors based on electrical or electrochemical principles. In this paper, we report a potentially low-cost immunosensor built with interdigitated gold electrodes coated with a self-assembled monolayer and a layer of anti-CA19-9 antibodies, which is capable of detecting the pancreatic cancer biomarker CA19-9 using electrical impedance spectroscopy. Due to specific, irreversible adsorption of CA19-9 onto its corresponding antibody, according to data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), the immunosensor is highly sensitive and selective. It could detect CA19-9 in commercial samples with a limit of detection of 0.68 U mL-1, in addition to distinguishing between blood serum samples from patients with different concentrations of CA19-9. Furthermore, by treating the capacitance data with information visualization methods, we were able to verify the selectivity and robustness of the immunosensor with regard to false positives, as the samples containing higher CA19-9 concentrations, including those from tumor cells, could be distinguished from those with possible interferents.

A graphene-based electrochemical competitive immunosensor for the sensitive detection of okadaic acid in shellfish

NASA Astrophysics Data System (ADS)

Eissa, Shimaa; Zourob, Mohammed

2012-11-01

A novel graphene-based voltammetric immunosensor for sensitive detection of okadaic acid (OA) was developed. A simple and efficient electrografting method was utilized to functionalize graphene-modified screen-printed carbon electrodes (GSPE) by the electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt in acidic aqueous solution. Next, the okadaic acid antibody was covalently immobilized on the carboxyphenyl modified graphene electrodes via carbodiimide chemistry. Square wave voltammetry (SWV) was used to investigate the stepwise assembly of the immunosensor. A competitive assay between OA and a fixed concentration of okadaic acid-ovalbumin conjugate (OA-OVA) for the immobilized antibodies was employed for the detection of okadaic acid. The decrease of the [Fe(CN)6]3-/4- reduction peak current in the square wave voltammetry for various concentrations of okadaic acid was used for establishing the calibration curve. A linear relationship between the SWV peak current difference and OA concentration was obtained up to ~5000 ng L-1. The developed immunosensor allowed a detection limit of 19 ng L-1 of OA in PBS buffer. The matrix effect studied with spiked shellfish tissue extracts showed a good percentage of recovery and the method was also validated with certified reference mussel samples.A novel graphene-based voltammetric immunosensor for sensitive detection of okadaic acid (OA) was developed. A simple and efficient electrografting method was utilized to functionalize graphene-modified screen-printed carbon electrodes (GSPE) by the electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt in acidic aqueous solution. Next, the okadaic acid antibody was covalently immobilized on the carboxyphenyl modified graphene electrodes via carbodiimide chemistry. Square wave voltammetry (SWV) was used to investigate the stepwise assembly of the immunosensor. A competitive assay between OA and a fixed concentration of okadaic acid

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

PubMed Central

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

2015-01-01

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. PMID:26525841

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

PubMed

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

2015-11-03

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.

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.

Development of protein A functionalized microcantilever immunosensors for the analyses of small molecules at parts per trillion levels.

PubMed

Tan, Weiming; Huang, Yuan; Nan, Tiegui; Xue, Changguo; Li, Zhaohu; Zhang, Qingchuan; Wang, Baomin

2010-01-15

Development of microcantilever biosensors for small molecules was exemplified with the beta-adrenergic agonist clenbuterol and the antibiotic chloramphenicol. In this paper, antibody sulfhydrylation and protein A were used to modify the microcantilever Au surface, and the antibody activities on the microcantilever were evaluated with direct competitive enzyme-linked immunosorbent assay (dcELISA). The activity of the antibodies immobilized on the microcantilever via protein A was 1.7-fold of that via the sulfhydrylation reagent 2-iminothiolane hydrochloride. A microcantilever immunosensor method with protein A as the functionalization reagent was established to detect the residues of clenbuterol and chloramphenicol at limits of detection (LOD) of approximately 0.1 and 0.2 ng/mL, respectively. Such LODs were better than that of the corresponding dcELISAs. The concentration of clenbuterol in a fortified feed sample detected with the microcantilever immunosensor after thorough extraction and purification agreed well with that detected with the dcELISA. Protein A showed to be simple and reproducible for functionalization of the antibodies on the Au surface and, thus, has common application values in microcantilever immunosensor development. The results suggest that microcantilever immunosensors be suitable for detection of small molecules, and the assay sensitivity is mainly related to the quality and activities of the antibodies.

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.

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.

Current Technologies of Electrochemical Immunosensors: Perspective on Signal Amplification.

PubMed

Cho, Il-Hoon; Lee, Jongsung; Kim, Jiyeon; Kang, Min-Soo; Paik, Jean Kyung; Ku, Seockmo; Cho, Hyun-Mo; Irudayaraj, Joseph; Kim, Dong-Hyung

2018-01-12

An electrochemical immunosensor employs antibodies as capture and detection means to produce electrical charges for the quantitative analysis of target molecules. This sensor type can be utilized as a miniaturized device for the detection of point-of-care testing (POCT). Achieving high-performance analysis regarding sensitivity has been one of the key issues with developing this type of biosensor system. Many modern nanotechnology efforts allowed for the development of innovative electrochemical biosensors with high sensitivity by employing various nanomaterials that facilitate the electron transfer and carrying capacity of signal tracers in combination with surface modification and bioconjugation techniques. In this review, we introduce novel nanomaterials (e.g., carbon nanotube, graphene, indium tin oxide, nanowire and metallic nanoparticles) in order to construct a high-performance electrode. Also, we describe how to increase the number of signal tracers by employing nanomaterials as carriers and making the polymeric enzyme complex associated with redox cycling for signal amplification. The pros and cons of each method are considered throughout this review. We expect that these reviewed strategies for signal enhancement will be applied to the next versions of lateral-flow paper chromatography and microfluidic immunosensor, which are considered the most practical POCT biosensor platforms.

Copper oxide assisted cysteine hierarchical structures for immunosensor application

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

Pandey, Chandra Mouli; Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005; Sumana, Gajjala, E-mail: sumanagajjala@gmail.com

2014-09-08

The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 μm have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constantmore » of 3.38 × 10{sup −4 }cm s{sup −1}. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.« less

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.

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.

Label-free imaging and spectroscopy for early detection of cervical cancer.

PubMed

Jing, Yueyue; Wang, Yulan; Wang, Xinyi; Song, Chuan; Ma, Jiong; Xie, Yonghui; Fei, Yiyan; Zhang, Qinghua; Mi, Lan

2018-05-01

The label-free imaging and spectroscopy method was studied on cervical unstained tissue sections obtained from 36 patients. The native fluorescence spectra of tissues are analyzed by the optical redox ratio (ORR), which is defined as fluorescence intensity ratio between NADH and FAD, and indicates the metabolism change with the cancer development. The ORRs of normal tissues are consistently higher than those of precancer or cancerous tissues. A criterion line of ORR at 5.0 can be used to discriminate cervical precancer/cancer from normal tissues. The sensitivity and specificity of the native fluorescence spectroscopy method for cervical cancer diagnosis are determined as 100% and 91%. Moreover, the native fluorescence spectroscopy study is much more sensitive on the healthy region of cervical precancer/cancer patients compared with the traditional clinical staining method. The results suggest label-free imaging and spectroscopy is a fast, highly sensitive and specific method on the detection of cervical cancer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A self-amplified transistor immunosensor under dual gate operation: highly sensitive detection of hepatitis B surface antigen

NASA Astrophysics Data System (ADS)

Lee, I.-K.; Jeun, M.; Jang, H.-J.; Cho, W.-J.; Lee, K. H.

2015-10-01

Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL-1) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases.Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor

Ultrasensitive direct impedimetric immunosensor for detection of serum HER2.

PubMed

Sharma, Shikha; Zapatero-Rodríguez, Julia; Saxena, Rahul; O'Kennedy, Richard; Srivastava, Sudha

2018-05-30

Assesment of human epidermal growth factor receptor 2 status is a key factor prompting definitive treatment decisions that help in reducing mortality rates associated with breast cancer. In this article, highly sensitive and low-cost impedimetric immunosensor using single-chain fragment variable antibody fragments was developed for quantitative detection of human epidermal growth factor receptor 2 from serum employing gold nanoparticle-modified disposable screen-printed carbon electrodes. The gold nanoparticles facilitate fast electron transfer and offer a biocompatible surface for immobilization of small antibody fragments in an oriented manner, resulting in improved antigen binding efficiency. The single-chain fragment variable antibody fragment-modified screen printed immunosensor exhibits wide dynamic range of 0.01-100 ng mL -1 and detection limit of 0.01 ng mL -1 . The advantages offered by this platform in terms of high sensitivity, broad dynamic range and low-cost demonstrates great potential for improved monitoring of human epidermal growth factor receptor 2 levels for the management of breast and other cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

Christmas-tree Derived Amplification Immuno-strategy for Sensitive Visual Detection of Vibrio parahaemolyticus Based on Gold Label Silver Stain Technology.

PubMed

Song, Xinxin; Wu, Yanjie; Wu, Lin; Hu, Yufang; Li, Wenrou; Guo, Zhiyong; Su, Xiurong; Jiang, Xiaohua

2017-01-01

A developed Christmas-tree derived immunosensor based on a gold label silver stain (GLSS) technique was fabricated for a highly sensitive analysis of Vibrio parahaemolyticu (VP). In this strategy, captured VP antibody (cAb) was immobilized on a solid substrate; then, the VPs were sequentially tagged with a signal probe by incubating the assay with a detection VP antibody (dAb) conjugated gold nanoparticles (AuNPs)-labeled graphite-like carbon nitride (g-C 3 N 4 ). Finally, the attached signal probe could harvest a visible signal by the silver meal deposition, and then followed by homebrew Matlab 6.0 as a grey value acquisition. In addition, the overall design of the biosensor was established in abundant AuNPs and g-C 3 N 4 with a two-dimensional structure, affording a bulb-decorated Christmas-tree model. Moreover, with the optimized conditions, the detection limit of the as-proposed biosensor is as low as 10 2 CFU (Colony-Forming Units) mL -1 , exhibiting an increase of two orders of magnitude compared with the traditional immune-gold method. Additionally, the developed visible immunosensor was also successfully applied to the analysis of complicated samples.

Amperometric biosensor for Salmonella typhimurium detection in milk

USDA-ARS?s Scientific Manuscript database

This paper reports an amperometric biosensor for rapid and sensitive Salmonella Typhimurium detection in milk. The biosensor was assembled from the self-assembled monolayers technique on a gold surface. In this device, polyclonal antibodies were oriented by protein A. The biosensor structure was cha...

A free-air system for long-term stable carbon isotope labeling of adult forest trees

EPA Science Inventory

Stable carbon (C) isotopes, in particular employed in labeling experiments, are an ideal tool to broaden our understanding of C dynamics in trees and forest ecosystems. Here, we present a free-air exposure system, named isoFACE, designed for long-term stable C isotope labeling in...

Robust label-free biosensing using microdisk laser arrays with on-chip references.

PubMed

Wondimu, S F; Hippler, M; Hussal, C; Hofmann, A; Krämmer, S; Lahann, J; Kalt, H; Freude, W; Koos, C

2018-02-05

Whispering-gallery mode (WGM) microdisk lasers show great potential for highly sensitive label-free detection in large-scale sensor arrays. However, when used in practical applications under normal ambient conditions, these devices suffer from temperature fluctuations and photobleaching. Here we demonstrate that these challenges can be overcome by a novel referencing scheme that allows for simultaneous compensation of temperature drift and photobleaching. The technique relies on reference structures protected by locally dispensed passivation materials, and can be scaled to extended arrays of hundreds of devices. We prove the viability of the concept in a series of experiments, demonstrating robust and sensitive label-free detection over a wide range of constant or continuously varying temperatures. To the best of our knowledge, these measurements represent the first demonstration of biosensing in active WGM devices with simultaneous compensation of both photobleaching and temperature drift.

Side-polished fiber immunosensor based on surface plasmon resonance for detection of Legionella pneumophila

NASA Astrophysics Data System (ADS)

Tsao, Yu-Chia; Yang, Yi-Wen; Tsai, Woo-Hu; Yan, Tsong-Rong

2008-02-01

Side-polished fiber immunosensor based on surface plasmon resonance (SPR) onto self-assembled protein A layer was proposed for the detection of Legionella pneumophila. A self-assembled protein A layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and activated by N-Ethyl-N'-(3-dimethylaminopropyl) carbodiimide/ N-Hydroxysuccinimide (EDC/NHS). The formation of self-assembled protein A and gold layer on side-polished surface and the binding of antibody and antigen in series were confirmed by SPR response on spectrum. The binding protein A layer can improve the sensitivity, which indirectly supports the configurations that antibody layer is immobilized on the binding protein A layer with a well-ordered orientation. The surface morphology analyses of self-assembled protein A layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein A were demonstrated by SPR dip shifts on optical spectrum analyzer. The SPR fiber immunosensor for detection of L. pneumophila was developed and the detection limit was 10 CFU/ml with the SPR dip shift in wavelength from 1070 to 1105nm. The current fabrication technique of a SPR immunosensor using optical fiber for the detection of Legionella pneumophila could be applied to construct other biosensor.

An electrochemical immunosensor for sensitive detection of Escherichia coli O157:H7 by using chitosan, MWCNT, polypyrrole with gold nanoparticles hybrid sensing platform.

PubMed

Güner, Ahmet; Çevik, Emre; Şenel, Mehmet; Alpsoy, Lokman

2017-08-15

An electrochemical immunosensor for the common food pathogen Escherichia coli O157:H7 was developed. This novel immunosensor based on the PPy/AuNP/MWCNT/Chi hybrid bionanocomposite modified pencil graphite electrode (PGE). This hybrid bionanocomposite platform was modified with anti-E. coli O157:H7 monoclonal antibody. The prepared bionanocomposite platform and immunosensor was characterized by using cyclic voltammetry (CV). Under the optimum conditions, the results have shown the order of the preferential selectivity of the method is gram negative pathogenic species E. coli O157:H7. Concentrations of E. coli O157:H7 from 3×10 1 to 3×10 7 cfu/mL could be detected. The detection limit was ∼30cfu/mL in PBS buffer. Briefly, we developed a high sensitive electrochemical immunosensor for specific detection of E. coli O157:H7 contamination with the use of sandwich assay evaluated in this study offered a reliable means of quantification of the bacteria. For the applications in food quality and safety control, our immunosensor showed reproducibility and stability. Copyright © 2017. Published by Elsevier Ltd.

An ultra-sensitive impedimetric immunosensor for detection of the serum oncomarker CA-125 in ovarian cancer patients

NASA Astrophysics Data System (ADS)

Johari-Ahar, M.; Rashidi, M. R.; Barar, J.; Aghaie, M.; Mohammadnejad, D.; Ramazani, A.; Karami, P.; Coukos, G.; Omidi, Y.

2015-02-01

Effective treatment of ovarian cancer depends upon the early detection of the malignancy. Here, we report on the development of a new nanostructured immunosensor for early detection of cancer antigen 125 (CA-125). A gold electrode was modified with mercaptopropionic acid (MPA), and then consecutively conjugated with silica coated gold nanoparticles (AuNP@SiO2), CdSe quantum dots (QDs) and anti-CA-125 monoclonal antibody (mAb). The engineered MPA|AuNP@SiO2|QD|mAb immunosensor was characterised using transmission electron microscopy (TEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Successive conjugation of AuNP@SiO2, CdSe QD and anti-CA-125 mAb onto the gold electrode resulted in sensitive detection of CA-125 with a limit of detection (LOD) of 0.0016 U mL-1 and a linear detection range (LDR) of 0-0.1 U mL-1. Based on the high sensitivity and specificity of the immunosensor, we propose this highly stable and reproducible biosensor for the early detection of CA-125.Effective treatment of ovarian cancer depends upon the early detection of the malignancy. Here, we report on the development of a new nanostructured immunosensor for early detection of cancer antigen 125 (CA-125). A gold electrode was modified with mercaptopropionic acid (MPA), and then consecutively conjugated with silica coated gold nanoparticles (AuNP@SiO2), CdSe quantum dots (QDs) and anti-CA-125 monoclonal antibody (mAb). The engineered MPA|AuNP@SiO2|QD|mAb immunosensor was characterised using transmission electron microscopy (TEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Successive conjugation of AuNP@SiO2, CdSe QD and anti-CA-125 mAb onto the gold electrode resulted in sensitive detection of CA-125 with a limit of detection (LOD) of 0.0016 U mL-1 and a linear detection range (LDR) of 0-0.1 U mL-1. Based on the high sensitivity and specificity of the immunosensor, we propose

β-cyclodextrin-ferrocene host-guest complex multifunctional labeling triple amplification strategy for electrochemical immunoassay of subgroup J of avian leukosis viruses.

PubMed

Shang, Kun; Wang, Xindong; Sun, Bing; Cheng, Ziqiang; Ai, Shiyun

2013-07-15

A novel sandwich-type electrochemical immunosensor was fabricated for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) by employing β-cyclodextrin-ferrocene (CD-Fc) host-guest complex multifunctional Fe3O4 nanospheres as labels and β-cyclodextrin functional graphene sheets (CD-GS) nanocomposite as sensor platform. The sensitivity was greatly improved based on the triple amplification strategy. Firstly, the CD-GS improved the electron transfer rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab1). Secondly, the CD on the Fe3O4 surface with strong recognition capability could form stable CD-Fc host-guest inclusion complex and provided larger free room for the conjugation of secondary antibodies (Ab2) and glucose oxidase (GOD). Finally, the conjugated GOD exhibited extraordinary electrochemical biocatalysis towards the reduction reaction of Fc(+) by glucose. Under the optimized conditions, the electrochemical immunosensor exhibited a wide working range from 10(2.27)-10(3.50) TCID50/mL (TCID50: 50% tissue culture infective dose) with a low detection limit of 10(2.19) TCID50/mL (S/N=3). The selectivity, reproducibility, and stability are acceptable. The assay was evaluated for real avian serum sample, receiving satisfactory results. This new type of triple amplification strategy may provide potential applications for the clinic application. Copyright © 2013 Elsevier B.V. All rights reserved.

Novel amperometric glucose biosensor based on MXene nanocomposite.

PubMed

Rakhi, R B; Nayak, Pranati; Xia, Chuan; Alshareef, Husam N

2016-11-10

A biosensor platform based on Au/MXene nanocomposite for sensitive enzymatic glucose detection is reported. The biosensor leverages the unique electrocatalytic properties and synergistic effects between Au nanoparticles and MXene sheets. An amperometric glucose biosensor is fabricated by the immobilization of glucose oxidase (GOx) enzyme on Nafion solubilized Au/ MXene nanocomposite over glassy carbon electrode (GCE). The biomediated Au nanoparticles play a significant role in facilitating the electron exchange between the electroactive center of GOx and the electrode. The GOx/Au/MXene/Nafion/GCE biosensor electrode displayed a linear amperometric response in the glucose concentration range from 0.1 to 18 mM with a relatively high sensitivity of 4.2 μAmM -1 cm -2 and a detection limit of 5.9 μM (S/N = 3). Furthermore, the biosensor exhibited excellent stability, reproducibility and repeatability. Therefore, the Au/MXene nanocomposite reported in this work is a potential candidate as an electrochemical transducer in electrochemical biosensors.

Novel amperometric glucose biosensor based on MXene nanocomposite

PubMed Central

Rakhi, R. B.; Nayuk, Pranati; Xia, Chuan; Alshareef, Husam N.

2016-01-01

A biosensor platform based on Au/MXene nanocomposite for sensitive enzymatic glucose detection is reported. The biosensor leverages the unique electrocatalytic properties and synergistic effects between Au nanoparticles and MXene sheets. An amperometric glucose biosensor is fabricated by the immobilization of glucose oxidase (GOx) enzyme on Nafion solubilized Au/ MXene nanocomposite over glassy carbon electrode (GCE). The biomediated Au nanoparticles play a significant role in facilitating the electron exchange between the electroactive center of GOx and the electrode. The GOx/Au/MXene/Nafion/GCE biosensor electrode displayed a linear amperometric response in the glucose concentration range from 0.1 to 18 mM with a relatively high sensitivity of 4.2 μAmM−1 cm−2 and a detection limit of 5.9 μM (S/N = 3). Furthermore, the biosensor exhibited excellent stability, reproducibility and repeatability. Therefore, the Au/MXene nanocomposite reported in this work is a potential candidate as an electrochemical transducer in electrochemical biosensors. PMID:27830757

Label-free electrochemical genosensor based on mesoporous silica thin film.

PubMed

Saadaoui, Maroua; Fernández, Iñigo; Luna, Gema; Díez, Paula; Campuzano, Susana; Raouafi, Noureddine; Sánchez, Alfredo; Pingarrón, José M; Villalonga, Reynaldo

2016-10-01

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.

PubMed

Uckermann, Ortrud; Galli, Roberta; Leupold, Susann; Coras, Roland; Meinhardt, Matthias; Hallmeyer-Elgner, Susanne; Mayer, Thomas; Storch, Alexander; Schackert, Gabriele; Koch, Edmund; Blümcke, Ingmar; Steiner, Gerald; Kirsch, Matthias

2017-01-01

The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

An investigation of the well-water quality: immunosensor for pathogenic Pseudomonas aeruginosa detection based on antibody-modified poly(pyrrole-3 carboxylic acid) screen-printed carbon electrode.

PubMed

Bekir, Karima; Bousimma, Feriel; Barhoumi, Houcine; Fedhila, Kais; Maaref, Abderrazak; Bakhrouf, Amina; Ben Ouada, Hafedh; Namour, Philippe; Jaffrezic-Renault, Nicole; Ben Mansour, Hedi

2015-12-01

In this report, we describe a new immunosensor designed for the detection and the quantification of Pseudomonas aeruginosa bacteria in water. The developed biosensing system was based on the immobilization of purified polyclonal anti P. aeruginosa antibodies on electropolymerized poly(pyrrole-3-carboxylic acid)/glassy carbon electrode. The building of the immunosensor step by step was evaluated by electrochemical measurements such as cyclic voltammetry (CV) and impedance spectroscopy (EIS). The electrochemical signature of the immunosensor was established by the change of the charge transfer resistance when the bacteria suspended in solution became attached to the immobilized antibodies. As a result, stable and high sensitive impedimetric immunosensor was obtained with a sensitivity of 0.19 kΩ/decade defined in the linear range from 10(1) to 10(7) CFU/mL of cellular concentrations. A low detection limit was obtained for the P. aeruginosa bacteria and a high selectivity when other bacteria were occasioned as well as Escherichia coli. The developed immunosensor was applied in detecting pathogenic P. aeruginosa in well-water.

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.

Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au-Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels.

PubMed

Sharma, Mukesh Kumar; Narayanan, J; Pardasani, Deepak; Srivastava, Divesh N; Upadhyay, Sanjay; Goel, Ajay Kumar

2016-06-15

Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold-palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au-Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au-Pd NPs@BNNSs/Ab2. The resulting Au-Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5 pg/mL to 100 ng/mL with a minimum detection limit of 1 pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

Polymer dual ring resonators for label-free optical biosensing using microfluidics.

PubMed

Salleh, Muhammad H M; Glidle, Andrew; Sorel, Marc; Reboud, Julien; Cooper, Jonathan M

2013-04-18

We demonstrate a polymer resonator microfluidic biosensor that overcomes the complex manufacturing procedures required to fabricate traditional devices. In this new format, we show that a gapless light coupling photonic configuration, fabricated in SU8 polymer, can achieve high sensitivity, label-free chemical sensing in solution and high sensitivity biological sensing, at visible wavelengths.

Affinity Versus Label-Free Isolation of Circulating Tumor Cells: Who Wins?

PubMed

Murlidhar, Vasudha; Rivera-Báez, Lianette; Nagrath, Sunitha

2016-09-01

The study of circulating tumor cells (CTCs) has been made possible by many technological advances in their isolation. Their isolation has seen many fronts, but each technology brings forth a new set of challenges to overcome. Microfluidics has been a key player in the capture of CTCs and their downstream analysis, with the aim of shedding light into their clinical application in cancer and metastasis. Researchers have taken diverging paths to isolate such cells from blood, ranging from affinity-based isolation targeting surface antigens expressed on CTCs, to label-free isolation taking advantage of the size differences between CTCs and other blood cells. For both major groups, many microfluidic technologies have reported high sensitivity and specificity for capturing CTCs. However, the question remains as to the superiority among these two isolation techniques, specifically to identify different CTC populations. This review highlights the key aspects of affinity and label-free microfluidic CTC technologies, and discusses which of these two would be the highest benefactor for the study of CTCs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Current Technologies of Electrochemical Immunosensors: Perspective on Signal Amplification

PubMed Central

Cho, Il-Hoon; Kim, Jiyeon; Kang, Min-soo; Paik, Jean Kyung; Ku, Seockmo; Cho, Hyun-Mo; Irudayaraj, Joseph; Kim, Dong-Hyung

2018-01-01

An electrochemical immunosensor employs antibodies as capture and detection means to produce electrical charges for the quantitative analysis of target molecules. This sensor type can be utilized as a miniaturized device for the detection of point-of-care testing (POCT). Achieving high-performance analysis regarding sensitivity has been one of the key issues with developing this type of biosensor system. Many modern nanotechnology efforts allowed for the development of innovative electrochemical biosensors with high sensitivity by employing various nanomaterials that facilitate the electron transfer and carrying capacity of signal tracers in combination with surface modification and bioconjugation techniques. In this review, we introduce novel nanomaterials (e.g., carbon nanotube, graphene, indium tin oxide, nanowire and metallic nanoparticles) in order to construct a high-performance electrode. Also, we describe how to increase the number of signal tracers by employing nanomaterials as carriers and making the polymeric enzyme complex associated with redox cycling for signal amplification. The pros and cons of each method are considered throughout this review. We expect that these reviewed strategies for signal enhancement will be applied to the next versions of lateral-flow paper chromatography and microfluidic immunosensor, which are considered the most practical POCT biosensor platforms. PMID:29329274

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.

Sensitivity improvement of a sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen isoforms in human serum using electrochemical impedance spectroscopy and an ordered and hierarchically organized interfacial supramolecular architecture.

PubMed

Gutiérrez-Zúñiga, Gabriela Guadalupe; Hernández-López, José Luis

2016-01-01

A gold millielectrode (GME) functionalized with a mixed (16-MHA + EG3SH) self-assembled monolayer (SAM) was used to fabricate an indirect enzyme-linked immunosorbent assay (ELISA) immunosensor for the sensitive detection of prostate-specific antigen (PSA), a prostate cancer (PCa) biomarker, in human serum samples. To address and minimize the issue of non-specific protein adsorption, an organic matrix (amine-PEG3-biotin/avidin) was assembled on the previously functionalized electrode surface to build up an ordered and hierarchically organized interfacial supramolecular architecture: Au/16-MHA/EG3SH/amine-PEG3-biotin/avidin. The electrode was then exposed to serum samples at different concentrations of a sandwich-type immunocomplex molecule ((Btn)Ab-AgPSA-(HRP)Ab), and its interfacial properties were characterized using electrochemical impedance spectroscopy (EIS). Calibration curves for polarization resistance (RP) and capacitance (1/C) vs. total and free PSA concentrations were obtained and their analytical quality parameters were determined. This approach was compared with results obtained from a commercially available ELISA immunosensor. The results obtained in this work showed that the proposed immunosensor can be successfully applied to analyze serum samples of patients representative of the Mexican population. Copyright © 2015 Elsevier B.V. All rights reserved.

A 100K well screen for a muscarinic receptor using the Epic label-free system--a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors.

PubMed

Dodgson, K; Gedge, L; Murray, D C; Coldwell, M

2009-01-01

Seven-transmembrane receptors (7TMRs) are a family of proteins of great interest as therapeutic targets because of their abundance on the cell surface, diverse effects in modulating cell behavior and success as a key class of drugs. We have evaluated the Epic label-free system for the purpose of identifying antagonists of the muscarinic M3 receptor. We compared the data generated from the label-free technology with data for the same compounds in a calcium flux assay. We have shown that this technology can be used for high throughput screening (HTS) of 7TMRs and as an orthogonal approach to enable rapid evaluation of HTS outputs. A number of compounds have been identified which were not found in a functional HTS measuring the output from a single pathway, which may offer new approaches to inhibiting responses through this receptor.

Comparison between amperometric and true potentiometric end-point detection in the determination of water by the Karl Fischer method.

PubMed

Cedergren, A

1974-06-01

A rapid and sensitive method using true potentiometric end-point detection has been developed and compared with the conventional amperometric method for Karl Fischer determination of water. The effect of the sulphur dioxide concentration on the shape of the titration curve is shown. By using kinetic data it was possible to calculate the course of titrations and make comparisons with those found experimentally. The results prove that the main reaction is the slow step, both in the amperometric and the potentiometric method. Results obtained in the standardization of the Karl Fischer reagent showed that the potentiometric method, including titration to a preselected potential, gave a standard deviation of 0.001(1) mg of water per ml, the amperometric method using extrapolation 0.002(4) mg of water per ml and the amperometric titration to a pre-selected diffusion current 0.004(7) mg of water per ml. Theories and results dealing with dilution effects are presented. The time of analysis was 1-1.5 min for the potentiometric and 4-5 min for the amperometric method using extrapolation.

Nafion/lead nitroprusside nanoparticles modified carbon ceramic electrode as a novel amperometric sensor for L-cysteine.

PubMed

Razmi, H; Heidari, H

2009-05-01

This work describes the electrochemical and electrocatalytic properties of carbon ceramic electrode (CCE) modified with lead nitroprusside (PbNP) nanoparticles as a new electrocatalyst material. The structure of deposited film on the CCE was characterized by energy dispersive X-ray (EDX), Fourier transform infrared (FTIR), and scanning electron microscopy (SEM). The cyclic voltammogram (CV) of the PbNP modified CCE showed two well-defined redox couples due to [Fe(CN)5NO](3-)/[Fe(CN)5NO](2-) and Pb(IV)/Pb(II) redox reactions. The modified electrode showed electrocatalytic activity toward the oxidation of L-cysteine and was used as an amperometric sensor. Also, to reduce the fouling effect of L-cysteine and its oxidation products on the modified electrode, a thin film of Nafion was coated on the electrode surface. The sensor response was linearly changed with L-cysteine concentration in the range of 1 x 10(-6) to 6.72 x 10(-5)mol L(-1) with a detection limit (signal/noise ratio [S/N]=3) of 0.46 microM. The sensor sensitivity was 0.17 microA (microM)(-1), and some important advantages such as simple preparation, fast response, good stability, interference-free signals, antifouling properties, and reproducibility of the sensor for amperometric determination of L-cysteine were achieved.

Screen-printed immunosensor modified with carbon nanotubes in a continuous-flow system for the Botrytis cinerea determination in apple tissues.

PubMed

Fernández-Baldo, Martín A; Messina, Germán A; Sanz, Maria I; Raba, Julio

2009-08-15

Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. This paper describes the development of an immunosensor coupled to carbon-based screen-printed electrodes (SPCE) modified with multi-walled carbon nanotubes (CNTs), which show a rapid and sensitive determination of B. cinerea in apple tissues (Red-delicious) using a competitive immunoassay method. Both the infected plant tissue sample and the B. cinerea-specific monoclonal antibody are allowed to react immunologically with the B. cinerea purified antigens immobilized on a rotating disk. Then, the bound antibodies are quantified by a horseradish peroxidise (HRP) enzyme labeled second antibodies specific to mouse IgG, using 4-tertbutylcatechol (4-TBC) as enzymatic mediators. The HRP, in the presence of hydrogen peroxide, catalyses the oxidation of 4-TBC to 4-tertbutyl o-benzoquinone. The electrochemical reduction back to 4-TBC is detected on SPCE-CNT at -0.15 V. The response current is inversely proportional to the amount of the B. cinerea antigens present in the fruit sample. The time consumed per assay was 30 min and the calculated detection limits for electrochemical method and the ELISA procedure are 0.02 and 10 microg mL(-1), respectively. Moreover the intra- and inter-assay coefficients of variation were below 7%. This electrochemical immunosensor promises to be usefully suited to the detection and quantification of B. cinerea in apparently healthy plant prior to the development of the symptoms.

Construction of novel electrochemical immunosensor for detection of prostate specific antigen using ferrocene-PAMAM dendrimers.

PubMed

Çevik, Emre; Bahar, Özlem; Şenel, Mehmet; Abasıyanık, M Fatih

2016-12-15

In this study, an immunosensor was designed to utilize for the detection of prostate specific antigen (PSA) based on three different generations (G1, G2 and G3) of ferrocene (Fc) cored polyamidiamine dendrimers (Fc-PAMAM) gold (Au) electrode. The self-assembled monolayer principle (SAM) was used to fabricate the sensitive, selective and disposable immunosensor electrodes. In electrode fabrication cysteamine (Cys) was the first agent covalently linked on the Au electrode surface. Immobilized redox center (ferrocene) cored PAMAM dendrimers served as a layer for the further binding of biological components. The monoclonal antibody of PSA (anti-PSA) was covalently immobilized on dendrimers which were attached onto the modified Au surface (Au/Cys/Fc-PAMAMs/anti-PSA). PSA levels were quantitatively analyzed by using electrochemical differential pulse voltammetry (DPV) whose lowest detection limit was calculated as 0.001ngmL(-1). The Au/Cys/FcPAMAM/anti-PSA immunosensor showed excellent performance for PSA at the pulse amplitude; 50mV and the scan rate; 10mV/s in a wide linear concentration range of 0.01ng-100ngmL(-1). Analytical performance and specificity assays were carried out using human serum and different proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

An ultrasensitive luminol cathodic electrochemiluminescence immunosensor based on glucose oxidase and nanocomposites: graphene-carbon nanotubes and gold-platinum alloy.

PubMed

Jiang, Xinya; Chai, Yaqin; Yuan, Ruo; Cao, Yaling; Chen, Yingfeng; Wang, Haijun; Gan, Xianxue

2013-06-14

In the present study, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor based on luminol cathodic ECL was fabricated by using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene-carbon nanotubes nanocomposite as platform for the detection of carcinoembryonic antigen (CEA). For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi-GR-CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi-GR-CNTs modified electrode. Subsequently, glucose oxidase (GOD) was employed to block the non-specific sites of electrode surface. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate hydrogen peroxide (H2O2), which could subsequently promote the oxidation of luminol with an amplified cathodic ECL signal. The proposed immunosensor was performed at low potential (-0.1 to 0.4V) and low concentration of luminol. The CEA was determined in the range of 0.1 pg mL(-1) to 40 ng mL(-1) with a limit of detection down to 0.03 pg mL(-1) (SN(-1)=3). Moreover, with excellent sensitivity, selectivity, stability and simplicity, the as-proposed luminol-based ECL immunosensor provided great potential in clinical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Methylamine-Sensitive Amperometric Biosensor Based on (His)6-Tagged Hansenula polymorpha Methylamine Oxidase Immobilized on the Gold Nanoparticles

PubMed Central

Stasyuk, Nataliya Ye.; Smutok, Oleh V.; Zakalskiy, Andriy E.; Zakalska, Oksana M.; Gonchar, Mykhailo V.

2014-01-01

A novel methylamine-selective amperometric bienzyme biosensor based on recombinant primary amine oxidase isolated from the recombinant yeast strain Saccharomyces cerevisiae and commercial horseradish peroxidase is described. Two amine oxidase preparations were used: free enzyme (AMO) and covalently immobilized on the surface of gold nanoparticles (AMO-nAu). Some bioanalytical parameters (sensitivity, selectivity, and storage stability) of the developed biosensors were investigated. The sensitivity for both sensors is high: 1450 ± 113 and 700 ± 30 A−1 ·M−1 ·m−2 for AMO-nAu biosensor, respectively. The biosensors exhibit the linear range from 15 μM to 150 μM (AMO-nAu) and from 15 μM to 60 μM (AMO). The developed biosensor demonstrated a good selectivity toward methylamine (MA) (signal for dimethylamine and trimethylamine is less than 5% and for ethylamine 15% compared to MA output) and reveals a satisfactory storage stability. The constructed amperometric biosensor was used for MA assay in real samples of fish products in comparison with chemical method. The values obtained with both approaches different methods demonstrated a high correlation. PMID:25136590

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

PubMed

Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

2015-11-02

In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

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

PubMed

Actis, Paolo; Mak, Andy C; Pourmand, Nader

2010-08-01

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

Functionalized nanopipettes: toward label-free, single cell biosensors

PubMed Central

Actis, Paolo; Mak, Andy C.

2010-01-01

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

Label-free and non-contact optical biosensing of glucose with quantum dots.

PubMed

Khan, Saara A; Smith, Gennifer T; Seo, Felix; Ellerbee, Audrey K

2015-02-15

We present a label-free, optical sensor for biomedical applications based on changes in the visible photoluminescence (PL) of quantum dots in a thin polymer film. Using glucose as the target molecule, the screening of UV excitation due to pre-absorption by the product of an enzymatic assay leads to quenching of the PL of quantum dots (QDs) in a non-contact scheme. The irradiance changes in QD PL indicate quantitatively the level of glucose present. The non-contact nature of the assay prevents surface degradation of the QDs, which yields an efficient, waste-free, cost-effective, portable, and sustainable biosensor with attractive market features. The limit of detection of the demonstrated biosensor is ~3.5 µm, which is competitive with existing contact-based bioassays. In addition, the biosensor operates over the entire clinically relevant range of glucose concentrations of biological fluids including urine and whole blood. The comparable results achieved across a range of cost-affordable detectors, including a spectrophotometer, portable spectrometer, and iPhone camera, suggest that label-free and visible quantification of glucose with QD films can be applied to low-cost, point-of-care biomedical sensing as well as scientific applications in the laboratory for characterizing glucose or other analytes. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrochemical immunosensor with NiAl-layered double hydroxide/graphene nanocomposites and hollow gold nanospheres double-assisted signal amplification.

PubMed

Qiao, Lu; Guo, Yemin; Sun, Xia; Jiao, Yancui; Wang, Xiangyou

2015-08-01

A sensitive electrochemical immunosensor based on NiAl-layered double hydroxide/graphene nanocomposites (NiAl-LDH/G) and hollow gold nanospheres (HGNs) was proposed for chlorpyrifos detection. The NiAl-LDH/G was prepared using a conventional coprecipitation process and reduction of the supporting graphene oxide. Subsequently, the nanocomposites were dispersed with chitosan (CS). The NiAl-LDH/G possessed good electrochemical behavior and high binding affinity to the electrode. The high surface areas of HGNs and the vast aminos and hydroxyls of CS provided a platform for the covalently crosslinking of antibody. Under optimal conditions, the immunosensor exhibited a wide linear range from 5 to 150 μg/mL and from 150 to 2 μg/mL, with a detection limit of 0.052 ng/mL. The detection results showed good agreement with standard gas chromatography method. The constructed immunosensor exhibited good reproducibility, high specificity, acceptable stability and regeneration performance, which provided a new promising tool for chlorpyrifos detection in real samples.

Chelator-Free Labeling of Layered Double Hydroxide Nanoparticles for in Vivo PET Imaging

NASA Astrophysics Data System (ADS)

Shi, Sixiang; Fliss, Brianne C.; Gu, Zi; Zhu, Yian; Hong, Hao; Valdovinos, Hector F.; Hernandez, Reinier; Goel, Shreya; Luo, Haiming; Chen, Feng; Barnhart, Todd E.; Nickles, Robert J.; Xu, Zhi Ping; Cai, Weibo

2015-11-01

Layered double hydroxide (LDH) nanomaterial has emerged as a novel delivery agent for biomedical applications due to its unique structure and properties. However, in vivo positron emission tomography (PET) imaging with LDH nanoparticles has not been achieved. The aim of this study is to explore chelator-free labeling of LDH nanoparticles with radioisotopes for in vivo PET imaging. Bivalent cation 64Cu2+ and trivalent cation 44Sc3+ were found to readily label LDH nanoparticles with excellent labeling efficiency and stability, whereas tetravalent cation 89Zr4+ could not label LDH since it does not fit into the LDH crystal structure. PET imaging shows that prominent tumor uptake was achieved in 4T1 breast cancer with 64Cu-LDH-BSA via passive targeting alone (7.7 ± 0.1%ID/g at 16 h post-injection; n = 3). These results support that LDH is a versatile platform that can be labeled with various bivalent and trivalent radiometals without comprising the native properties, highly desirable for PET image-guided drug delivery.

Label-free optical biosensors based on aptamer-functionalized porous silicon scaffolds.

PubMed

Urmann, Katharina; Walter, Johanna-Gabriela; Scheper, Thomas; Segal, Ester

2015-02-03

A proof-of-concept for a label-free and reagentless optical biosensing platform based on nanostructured porous silicon (PSi) and aptamers is presented in this work. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensor design. Here we describe the fabrication and characterization of aptamer-conjugated PSi biosensors, where a previously characterized his-tag binding aptamer (6H7) is used as model system. Exposure of the aptamer-functionalized PSi to the target proteins as well as to complex fluids (i.e., bacteria lysates containing target proteins) results in robust and well-defined changes in the PSi optical interference spectrum, ascribed to specific aptamer-protein binding events occurring within the nanoscale pores, monitored in real time. The biosensors show exceptional stability and can be easily regenerated by a short rinsing step for multiple biosensing analyses. This proof-of-concept study demonstrates the possibility of designing highly stable and specific label-free optical PSi biosensors, employing aptamers as capture probes, holding immense potential for application in detection of a broad range of targets, in a simple yet reliable manner.

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

Multiplex and label-free screening of foodborne pathogens using surface plasmon resonance imaging

USDA-ARS?s Scientific Manuscript database

In order to protect outbreaks caused by foodborne pathogens, more rapid and efficient methods are needed for pathogen screening from food samples. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for label-free screening of multiple targets simultaneously with ...

Pt NPs and DNAzyme functionalized polymer nanospheres as triple signal amplification strategy for highly sensitive electrochemical immunosensor of tumour marker.

PubMed

Chang, Honghong; Zhang, Haochun; Lv, Jia; Zhang, Bing; Wei, Wenlong; Guo, Jingang

2016-12-15

Highly sensitive determination of tumour markers is the key for early diagnosis of cancer. Herein, triple signal amplification strategy resulting from polymer nanospheres, Pt NPs, and DNAzyme was proposed in the developed electrochemical immunosensor. First, electroactive polymer nanospheres were synthesized by infinite coordination polymerization of ferrocenedicarboxylic acid, which could generate strong electrochemical signals due to plentiful ferrocene molecules. Further, the polymer nanospheres were functionalized by Pt NPs and DNAzyme (hemin/G-quadruplex) with the ability of catalyzing H2O2, which contributes to enhance the electrochemical signals. The prepared conjugations were characterized by transmission electron microscope (TEM) and energy dispersive X-ray spectroscopy (EDX). And the process of preparation was monitored by zeta potential. Based on the sandwich-type immunoassay, the electrochemical immunosensor was constructed employing the conjugations as signal tags. Under optimal conditions, the DPV peak increased with the increasing of alpha fetal protein (AFP) concentration, and the linear range was from 0.1pgmL(-1) to 100ngmL(-1) with low detection limit of 0.086pgmL(-1). Meanwhile, the designed immunosensor exhibited excellent selectivity and anti-interference property, good reproducibility and stability. More importantly, there were no significant differences in analyzing real clinical samples between designed immunosensor and commercial ELISA. Copyright © 2016 Elsevier B.V. All rights reserved.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

PubMed

Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

2017-09-01

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Label-free and amplified quantitation of proteins in complex mixtures using diffractive optics technology.

PubMed

Cleverley, Steve; Chen, Irene; Houle, Jean-François

2010-01-15

Immunoaffinity approaches remain invaluable tools for characterization and quantitation of biopolymers. Their application in separation science is often limited due to the challenges of immunoassay development. Typical end-point immunoassays require time consuming and labor-intensive approaches for optimization. Real-time label-free analysis using diffractive optics technology (dot) helps guide a very effective iterative process for rapid immunoassay development. Both label-free and amplified approaches can be used throughout feasibility testing and ultimately in the final assay, providing a robust platform for biopolymer analysis over a very broad dynamic range. We demonstrate the use of dot in rapidly developing assays for quantitating (1) human IgG in complex media, (2) a fusion protein in production media and (3) protein A contamination in purified immunoglobulin preparations. 2009 Elsevier B.V. All rights reserved.

Controlled viable release of selectively captured label-free cells in microchannels.

PubMed

Gurkan, Umut Atakan; Anand, Tarini; Tas, Huseyin; Elkan, David; Akay, Altug; Keles, Hasan Onur; Demirci, Utkan

2011-12-07

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological

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

USDA-ARS?s Scientific Manuscript database

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...

Surface plasmon resonance imaging for label-free detection of foodborne pathogens and toxins

USDA-ARS?s Scientific Manuscript database

More rapid and efficient detection methods for foodborne pathogenic bacteria and toxins are needed to address the long assay time and limitations in multiplex capacity. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multi...

Ratiometric ultrasensitive electrochemical immunosensor based on redox substrate and immunoprobe

NASA Astrophysics Data System (ADS)

Tang, Zhongxue; Ma, Zhanfang

2016-10-01

In this work, we presented a ratiometric electrochemical immunosensor based on redox substrate and immunoprobe. Carboxymethyl cellulose-Au-Pb2+ (CMC-Au-Pb2+) and carbon-Au-Cu2+ (C-Au-Cu2+) nanocomposites were firstly synthesized and implemented as redox substrate and immunoprobe with strong current signals at -0.45 V and 0.15 V, respectively. Human immunoglobulin G (IgG) was used as a model analyte to examine the analytical performance of the proposed method. The current signals of CMC-Au-Pb2+ (Isubstrate) and C-Au-Cu2+ (Iprobe) were monitored. The effect of redox substrate and immunoprobe behaved as a better linear relationship between Iprobe/Isubstrate and Lg CIgG (ng mL-1). By measuring the signal ratio Iprobe/Isubstrate, the sandwich immunosensor for IgG exhibited a wide linear range from 1 fg mL-1 to 100 ng mL-1, which was two orders of magnitude higher than other previous works. The limit of detection reached 0.26 fg mL-1. Furthermore, for human serum samples, the results from this method were consistent with those of the enzyme linked immunosorbent assay (ELISA), demonstrating that the proposed immunoassay was of great potential in clinical diagnosis.

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.

A label-free impedimetric DNA sensing chip integrated with AC electroosmotic stirring.

PubMed

Wu, Ching-Chou; Yang, Dong-Jie

2013-05-15

AC electroosmosis (ACEO) flow and label-free electrochemical impedance spectroscopy are employed to increase the hybridization rate and specifically detect target DNA (tDNA) concentrations. A low-ionic-strength solution, 6.1μS/cm 1mM Tris (pH 9.3), was used to produce ACEO and proved the feasibility of hybridization. Adequate voltage parameters for the simultaneous ACEO driving and DNA hybridization in the 1mM Tris solution were 1.5 Vpp and 200Hz. Moreover, an electrode set with a 1:4 ring width-to-disk diameter ratio exhibited a larger ACEO velocity above the disk electrode surface to improve collecting efficiency. The ACEO-integrated DNA sensing chips could reach 90% saturation hybridization within 117s. The linear range and detection limit of the sensors was 10aM-10pM and 10aM, respectively. The label-free impedimetric DNA sensing chips with integrated ACEO stirring can perform rapid hybridization and highly-sensitive detections to specifically measure tDNA concentrations. Copyright © 2013 Elsevier B.V. All rights reserved.

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

A Quartz Crystal Microbalance Immunosensor for Stem Cell Selection and Extraction

PubMed Central

Costanzo, Salvatore; Zambrano, Gerardo; Mauro, Marco; Battaglia, Raffaele; Ferrini, Gianluca; Nastri, Flavia; Pavone, Vincenzo

2017-01-01

A cost-effective immunosensor for the detection and isolation of dental pulp stem cells (DPSCs) based on a quartz crystal microbalance (QCM) has been developed. The recognition mechanism relies on anti-CD34 antibodies, DPSC-specific monoclonal antibodies that are anchored on the surface of the quartz crystals. Due to its high specificity, real time detection, and low cost, the proposed technology has a promising potential in the field of cell biology, for the simultaneous detection and sorting of stem cells from heterogeneous cell samples. The QCM surface was properly tailored through a biotinylated self-assembled monolayer (SAM). The biotin–avidin interaction was used to immobilize the biotinylated anti-CD34 antibody on the gold-coated quartz crystal. After antibody immobilization, a cellular pellet, with a mixed cell population, was analyzed; the results indicated that the developed QCM immunosensor is highly specific, being able to detect and sort only CD34+ cells. Our study suggests that the proposed technology can detect and efficiently sort any kind of cell from samples with high complexity, being simple, selective, and providing for more convenient and time-saving operations. PMID:29182568

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

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Amperometric Carbon Fiber Nitrite Microsensor for In Situ Biofilm Monitoring

EPA Science Inventory

A highly selective needle type solid state amperometric nitrite microsensor based on direct nitrite oxidation on carbon fiber was developed using a simplified fabrication method. The microsensor’s tip diameter was approximately 7 µm, providing a high spatial resolution of at lea...

Passivated aluminum nanohole arrays for label-free biosensing applications.

PubMed

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

2014-01-22

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

On the Determination of Uncertainty and Limit of Detection in Label-Free Biosensors.

PubMed

Lavín, Álvaro; Vicente, Jesús de; Holgado, Miguel; Laguna, María F; Casquel, Rafael; Santamaría, Beatriz; Maigler, María Victoria; Hernández, Ana L; Ramírez, Yolanda

2018-06-26

A significant amount of noteworthy articles reviewing different label-free biosensors are being published in the last years. Most of the times, the comparison among the different biosensors is limited by the procedure used of calculating the limit of detection and the measurement uncertainty. This article clarifies and establishes a simple procedure to determine the calibration function and the uncertainty of the concentration measured at any point of the measuring interval of a generic label-free biosensor. The value of the limit of detection arises naturally from this model as the limit at which uncertainty tends when the concentration tends to zero. The need to provide additional information, such as the measurement interval and its linearity, among others, on the analytical systems and biosensor in addition to the detection limit is pointed out. Finally, the model is applied to curves that are typically obtained in immunoassays and a discussion is made on the application validity of the model and its limitations.

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.

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.

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.

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.

Electrochemical immunosensor for simultaneous detection of multiplex cancer biomarkers based on graphene nanocomposites.

PubMed

Chen, Xia; Jia, Xinle; Han, Jingman; Ma, Jie; Ma, Zhanfang

2013-12-15

In this work, a sandwich-format electrochemical immunosensor for simultaneous determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was fabricated using biofunctional carboxyl graphene nanosheets (CGS) as immunosensing probes, which were fabricated by means of immobilization of toluidine blue (TB) and labeled anti-CEA (Ab2,1), Prussian blue (PB) and anti-AFP (Ab2,2) successively on CGS. The capture anti-CEA (Ab1,1) and anti-AFP (Ab1,2) were immobilized onto the chitosan-Au nanoparticles (CHIT-AuNPs) modified electrode through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxy succinimide (EDC/NHS). Experimental results revealed that this sandwich-type immunoassay enabled simultaneous detection of CEA and AFP with linear range of 0.5-60 ng mL(-1) for both analytes. The detection limit was 0.1 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N=3). The assay results of serum samples with the proposed method were in a good agreement with the reference values from the standard ELISA method. And the negligible cross-reactivity between the two analytes allows it to possess potential promise in clinical diagnosis. Copyright © 2013 Elsevier B.V. All rights reserved.

Chemotaxis of cancer cells in three-dimensional environment monitored label-free by quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Schnekenburger, Jürgen; Ketelhut, Steffi

2017-02-01

We investigated the capabilities of digital holographic microscopy (DHM) for label-free quantification of the response of living single cells to chemical stimuli in 3D assays. Fibro sarcoma cells were observed in a collagen matrix inside 3D chemotaxis chambers with a Mach-Zehnder interferometer-based DHM setup. From the obtained series of quantitative phase images, the migration trajectories of single cells were retrieved by automated cell tracking and subsequently analyzed for maximum migration distance and motility. Our results demonstrate DHM as a highly reliable and efficient tool for label-free quantification of chemotaxis in 2D and 3D environments.

Whole-organ atlas imaged by label-free high-resolution photoacoustic microscopy assisted by a microtome

NASA Astrophysics Data System (ADS)

Wong, Terence T. W.; Zhang, Ruiying; Hsu, Hsun-Chia; Maslov, Konstantin I.; Shi, Junhui; Chen, Ruimin; Shung, K. Kirk; Zhou, Qifa; Wang, Lihong V.

2018-02-01

In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

Label-free SERS detection of Salmonella Typhimurium on DNA aptamer modified AgNR substrates

USDA-ARS?s Scientific Manuscript database

A straightforward label-free method based on aptamer binding and surface enhanced Raman specstroscopy (SERS) has been developed for the detection of Salmonella Typhimurium, an important foodborne pathogen that causes gastroenteritis in both humans and animals. Surface of the SERS-active silver nanor...

Recent Advances in Electrochemical Immunosensors

PubMed Central

Piro, Benoît; Reisberg, Steeve

2017-01-01

Immunosensors have experienced a very significant growth in recent years, driven by the need for fast, sensitive, portable and easy-to-use devices to detect biomarkers for clinical diagnosis or to monitor organic pollutants in natural or industrial environments. Advances in the field of signal amplification using enzymatic reactions, nanomaterials such as carbon nanotubes, graphene and graphene derivatives, metallic nanoparticles (gold, silver, various oxides or metal complexes), or magnetic beads show how it is possible to improve collection, binding or transduction performances and reach the requirements for realistic clinical diagnostic or environmental control. This review presents these most recent advances; it focuses first on classical electrode substrates, then moves to carbon-based nanostructured ones including carbon nanotubes, graphene and other carbon materials, metal or metal-oxide nanoparticles, magnetic nanoparticles, dendrimers and, to finish, explore the use of ionic liquids. Analytical performances are systematically covered and compared, depending on the detection principle, but also from a chronological perspective, from 2012 to 2016 and early 2017. PMID:28387718

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

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.

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…

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.

PubMed

Pirhonen, Juho; Arola, Johanna; Sädevirta, Sanja; Luukkonen, Panu; Karppinen, Sanna-Maria; Pihlajaniemi, Taina; Isomäki, Antti; Hukkanen, Mika; Yki-Järvinen, Hannele; Ikonen, Elina

2016-01-01

Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Quantitative label-free multimodality nonlinear optical imaging for in situ differentiation of cancerous lesions

NASA Astrophysics Data System (ADS)

Xu, Xiaoyun; Li, Xiaoyan; Cheng, Jie; Liu, Zhengfan; Thrall, Michael J.; Wang, Xi; Wang, Zhiyong; Wong, Stephen T. C.

2013-03-01

The development of real-time, label-free imaging techniques has recently attracted research interest for in situ differentiation of cancerous lesions from normal tissues. Molecule-specific intrinsic contrast can arise from label-free imaging techniques such as Coherent Anti-Stokes Raman Scattering (CARS), Two-Photon Excited AutoFluorescence (TPEAF), and Second Harmonic Generation (SHG), which, in combination, would hold the promise of a powerful label-free tool for cancer diagnosis. Among cancer-related deaths, lung carcinoma is the leading cause for both sexes. Although early treatment can increase the survival rate dramatically, lesion detection and precise diagnosis at an early stage is unusual due to its asymptomatic nature and limitations of current diagnostic techniques that make screening difficult. We investigated the potential of using multimodality nonlinear optical microscopy that incorporates CARS, TPEAF, and SHG techniques for differentiation of lung cancer from normal tissue. Cancerous and non-cancerous lung tissue samples from patients were imaged using CARS, TPEAF, and SHG techniques for comparison. These images showed good pathology correlation with hematoxylin and eosin (H and E) stained sections from the same tissue samples. Ongoing work includes imaging at various penetration depths to show three-dimensional morphologies of tumor cell nuclei using CARS, elastin using TPEAF, and collagen using SHG and developing classification algorithms for quantitative feature extraction to enable lung cancer diagnosis. Our results indicate that via real-time morphology analyses, a multimodality nonlinear optical imaging platform potentially offers a powerful minimally-invasive way to differentiate cancer lesions from surrounding non-tumor tissues in vivo for clinical applications.

Gluten Contamination in Naturally or Labeled Gluten-Free Products Marketed in Italy.

PubMed

Verma, Anil K; Gatti, Simona; Galeazzi, Tiziana; Monachesi, Chiara; Padella, Lucia; Baldo, Giada Del; Annibali, Roberta; Lionetti, Elena; Catassi, Carlo

2017-02-07

A strict and lifelong gluten-free diet is the only treatment of celiac disease. Gluten contamination has been frequently reported in nominally gluten-free products. The aim of this study was to test the level of gluten contamination in gluten-free products currently available in the Italian market. A total of 200 commercially available gluten-free products (including both naturally and certified gluten-free products) were randomly collected from different Italian supermarkets. The gluten content was determined by the R5 ELISA Kit approved by EU regulations. Gluten level was lower than 10 part per million (ppm) in 173 products (86.5%), between 10 and 20 ppm in 9 (4.5%), and higher than 20 ppm in 18 (9%), respectively. In contaminated foodstuff (gluten > 20 ppm) the amount of gluten was almost exclusively in the range of a very low gluten content. Contaminated products most commonly belonged to oats-, buckwheat-, and lentils-based items. Certified and higher cost gluten-free products were less commonly contaminated by gluten. Gluten contamination in either naturally or labeled gluten-free products marketed in Italy is nowadays uncommon and usually mild on a quantitative basis. A program of systematic sampling of gluten-free food is needed to promptly disclose at-risk products.

Gluten Contamination in Naturally or Labeled Gluten-Free Products Marketed in Italy

PubMed Central

Verma, Anil K.; Gatti, Simona; Galeazzi, Tiziana; Monachesi, Chiara; Padella, Lucia; Baldo, Giada Del; Annibali, Roberta; Lionetti, Elena; Catassi, Carlo

2017-01-01

Background: A strict and lifelong gluten-free diet is the only treatment of celiac disease. Gluten contamination has been frequently reported in nominally gluten-free products. The aim of this study was to test the level of gluten contamination in gluten-free products currently available in the Italian market. Method: A total of 200 commercially available gluten-free products (including both naturally and certified gluten-free products) were randomly collected from different Italian supermarkets. The gluten content was determined by the R5 ELISA Kit approved by EU regulations. Results: Gluten level was lower than 10 part per million (ppm) in 173 products (86.5%), between 10 and 20 ppm in 9 (4.5%), and higher than 20 ppm in 18 (9%), respectively. In contaminated foodstuff (gluten > 20 ppm) the amount of gluten was almost exclusively in the range of a very low gluten content. Contaminated products most commonly belonged to oats-, buckwheat-, and lentils-based items. Certified and higher cost gluten-free products were less commonly contaminated by gluten. Conclusion: Gluten contamination in either naturally or labeled gluten-free products marketed in Italy is nowadays uncommon and usually mild on a quantitative basis. A program of systematic sampling of gluten-free food is needed to promptly disclose at-risk products. PMID:28178205

Thin-film amperometric multibiosensor for simultaneous determination of lactate and glucose in wine.

PubMed

Shkotova, Lyudmyla V; Piechniakova, Nataliia Y; Kukla, Oleksandr L; Dzyadevych, Sergei V

2016-04-15

An amperometric multi-biosensor based on lactate and glucose oxidases has been developed for determination of lactate and glucose in wine. Gold thin-film amperometric electrodes were used as multi-transducers. Analytical characteristics of the multi-biosensor developed were studied. The minimum detectable concentration was 5×10(-6) mol/l for both glucose and lactate. High reproducibility and storage stability of the multi-biosensor are demonstrated in this paper. Lactate and glucose were determined in wine, and a good correlation was obtained with concentrations determined using high-performance liquid chromatography (correlation coefficient for glucose R(2)=0.998, for lactate R(2)=0.718). Copyright © 2015 Elsevier Ltd. All rights reserved.

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments.

PubMed

Povedano, Eloy; Vargas, Eva; Montiel, Víctor Ruiz-Valdepeñas; Torrente-Rodríguez, Rebeca M; Pedrero, María; Barderas, Rodrigo; Segundo-Acosta, Pablo San; Peláez-García, Alberto; Mendiola, Marta; Hardisson, David; Campuzano, Susana; Pingarrón, José M

2018-04-23

This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells.

An Ultrasensitive Electrochemical Immunosensor for HIV p24 Based on Fe₃O₄@SiO₂ Nanomagnetic Probes and Nanogold Colloid-Labeled Enzyme-Antibody Copolymer as Signal Tag.

PubMed

Gan, Ning; Du, Xiaowen; Cao, Yuting; Hu, Futao; Li, Tianhua; Jiang, Qianli

2013-03-25

An ultrasensitive portable electrochemical immunosensor for human immunodeficiency virus p24 (HIV p24) antigen detection has been developed, whereby the detection sensitivity was 1000 times higher than that of the ELISA method. Firstly, a novel HRP enzyme-antibody copolymer (EV-p24 Ab2) was synthesized through an EnVision regent (EV, a dextrin amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of anti IgG), then incubated in the secondary antibody of p24. Secondly, the copolymer was immobilized on the gold nanocolloids (AuNPs) to fabricate a novel signal tag (AuNPs/EV-p24 Ab2). Subsequently, a sandwich-type immunoreaction would take place between the capture probe (silicon dioxide-coated magnetic Fe₃O₄ nanoparticles (MNPs) labeled with the primary p24 antibody (MNPs-p24 Ab1)), p24 (different concentrations) and the signal tag [AuNPs/EV-p24 Ab2)] to form the immunocomplex. Finally, the immunocomplex was absorbed on the surface of screen printed carbon electrode (SPCE) by a magnet and immersed in the o-hydroxyl phenol (HQ) and H₂O₂. The large amounts of HRP on the signal tag can catalyze the oxidation of HQ by H₂O₂, which can induce an amplified reductive current. Moreover, the capture probe could improve the accumulation ability of p24 and facilitate its separation from the substrate through the magnet. Under optimal conditions, the proposed immunoassay exhibited good sensitivity to p24 within a certain concentration range from 0.001 to 10.00 ng/mL, with a detection limit of 0.5 pg/mL (S/N = 3). The proposed method can be used for real-time and early detection of HIV-infected people.

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.

Whispering Gallery Mode Resonators for Rapid Label-Free Biosensing in Small Volume Droplets

PubMed Central

Wildgen, Sarah M.; Dunn, Robert C.

2015-01-01

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. PMID:25806835

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.

Highly sensitive electrochemiluminescent immunosensor based on gold nanoparticles-functionalized zinc oxide nanorod and poly(amidoamine)-graphene for detecting brombuterol.

PubMed

Zhu, Qing; Cai, Fudong; Zhang, Jing; Zhao, Kang; Deng, Anping; Li, Jianguo

2016-12-15

β-adrenergic agonists (β-agonists) recognized as a growth promoter will reflect the health of human. Sensitive detection of β-agonists in foodstuff is valuable for the health of animals and human. A novel ultrasensitive competition-type electrochemiluminescent (ECL) immunosensor was developed for detecting brombuterol (Brom) based on CdTe Quantum dot (QDs) and polyamidoamine dendrimer (PAMAM, G2) modified graphene oxide (GO) (CdTe QDs-PAMAM-GO composite) as bioprobe for the first time. The surface of glassy carbon electrode (GCE) was coated with AuNPs-ZnO NRs composite film as the platform, which facilitated the electronic transmission rate to enhance the ECL intensity and provide enough active sites for capturing antibody. The resulting ECL immunosensor enabled the real samples detection of Brom with a lower detection limit of 0.3pgmL(-1) (S/N=3) and a wider linear range from 0.001 to 500ngmL(-1). The proposed immunosensor coupled with the excellent advantages of CdTe QDs-PAMAM-GO and AuNPs-ZnO NRs composite displayed high sensitivity and long-term stability, and provided an approach for determining other important biomarkers. Copyright © 2016 Elsevier B.V. All rights reserved.

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

PubMed

Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

2015-01-01

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements

PubMed Central

Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E.

2015-01-01

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles’ arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

Hairpin assembly circuit-based fluorescence cooperative amplification strategy for enzyme-free and label-free detection of small molecule.

PubMed

Feng, Chunjing; Zhu, Jing; Sun, Jiewei; Jiang, Wei; Wang, Lei

2015-10-01

Here, we developed an enzyme-free, label-free, and sensitive fluorescence cooperative amplification strategy based on a hairpin assembly circuit which coupled catalytic hairpin assembly (CHA) with hybridization chain reaction (HCR) for small molecule adenosine. A double-strand DNA probe with aptamer-catalysis strand (Apt-C) and inhibit strand (Inh) was designed for adenosine recognition and signal transduction which was named as Apt-C/Inh. Hairpins H1 and H2 were employed for constructing the CHA, and hairpins H3 and H4 for the HCR. Through the binding of adenosine and the Apt-C, the Inh was released from the Apt-C/Inh. Then the free Apt-C initiated the CHA through successively opening H1 and H2, generating H1/H2 complex and recyclable Apt-C. Next, the released Apt-C entered another CHA cycle, and the H1/H2 complex further initiated the HCR of H3 and H4 which induced the formation of the concatemers of H3/H4 complex. Such a process brought the two ends of hairpins H3 into close proximity, yielding numerous integrated G-quadruplexes which were initially sequestered in the stem and two terminals of H3. Finally, N-methyl mesoporphyrin IX (NMM) was added to generate an enhanced fluorescence signal. In the proposed strategy, driven only by the energy from hybridization, one target could trigger multiple HCR events via CHA-based target-cycle, leading to a remarkable enzyme-free amplification for adenosine. The detection limit could achieve as low as 9.7 × 10(-7) mol L(-1). Furthermore, G-quadruplexes were applied to construct label-free hairpin assembly circuit, which made it more simple and cost-effective. The satisfactory recoveries were obtained when detecting adenosine in spiked human serum and urine samples, demonstrating the feasibility of this detection strategy in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Ceria Doped Zinc Oxide Nanoflowers Enhanced Luminol-Based Electrochemiluminescence Immunosensor for Amyloid-β Detection.

PubMed

Wang, Jing-Xi; Zhuo, Ying; Zhou, Ying; Wang, Hai-Jun; Yuan, Ruo; Chai, Ya-Qin

2016-05-25

In this work, ceria doped ZnO nanomaterials with flower-structure (Ce:ZONFs) were prepared to construct a luminol-based electrochemiluminescence (ECL) immunosensor for amyloid-β protein (Aβ) detection. Herein, carboxyl groups (-COOH) covered Ce:ZONFs were synthesized by a green method with lysine as reductant. After that, Ce:ZONFs-based ECL nanocomposite was prepared by combining the luminophore of luminol and Ce:ZONFs via amidation and physical absorption. Luminol modified on Ce:ZONFs surface could generate a strong ECL signal under the assistance of reactive oxygen species (ROSs) (such as OH(•) and O2(•-)), which were produced by a catalytic reaction between Ce:ZONFs and H2O2. It was worth noticing that a quick Ce(4+) ↔ Ce(3+) reaction in this doped material could increase the rate of electron transfer to realize the signal amplification. Subsequently, the luminol functionalized Ce:ZONFs (Ce:ZONFs-Lum) were covered by secondary antibody (Ab2) and glucose oxidase (GOD), respectively, to construct a novel Ab2 bioconjugate (Ab2-GOD@Ce:ZONFs-Lum). The wire-structured silver-cysteine complex (AgCys NWs) with a large number of -COOH, which was synthesized by AgNO3 and l-cysteine, was used as substrate of the immunosensor to capture the primary antibody (Ab1). Under the optimal conditions, this proposed ECL immunosensor had exhibited high sensitivity for Aβ detection with a wide linear range from 80 fg/mL to 100 ng/mL and an ultralow detection limit of 52 fg/mL. Meanwhile, this biosensor had good specificity for Aβ, indicating that the provided strategy had a promising potential in the detection of Aβ.

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.

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.

Nanophotonic label-free biosensors for environmental monitoring.

PubMed

Chocarro-Ruiz, Blanca; Fernández-Gavela, Adrián; Herranz, Sonia; Lechuga, Laura M

2017-06-01

The field of environmental monitoring has experienced a substantial progress in the last years but still the on-site control of contaminants is an elusive problem. In addition, the growing number of pollutant sources is accompanied by an increasing need of having efficient early warning systems. Several years ago biosensor devices emerged as promising environmental monitoring tools, but their level of miniaturization and their fully operation outside the laboratory prevented their use on-site. In the last period, nanophotonic biosensors based on evanescent sensing have emerged as an outstanding choice for portable point-of-care diagnosis thanks to their capability, among others, of miniaturization, multiplexing, label-free detection and integration in lab-on-chip platforms. This review covers the most relevant nanophotonic biosensors which have been proposed (including interferometric waveguides, grating-couplers, microcavity resonators, photonic crystals and localized surface plasmon resonance sensors) and their recent application for environmental surveillance. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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

2017-08-01

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

A novel signal-on photoelectrochemical immunosensor for detection of alpha-fetoprotein by in situ releasing electron donor.

PubMed

Chen, Jiexia; Zhao, Guang-Chao

2017-12-15

A signal-on photoelectrochemical (PEC) immunosensor was constructed for detecting tumor marker in this work. α-fetoprotein (AFP) was chosen as a model analyte to investigate the prepared procedure and the analytical performance of the exploited sensor. In order to construct the sensor, CdSe QDs were used as photoactive material, biotin conjugated AFP antibody (Bio-anti-AFP) as detecting probe, streptavidin (SA) as signal capturing unit, biotin functionalized apoferritin encapsulated ascorbic acid (Bio-APOAA) as amplification unit, which were assembled onto the electrodes. The sensing strategy was based on in situ enzymatic hydrolysis of Bio-APOAA to release ascorbic acid (AA) as sacrificial electron donor to produce photocurrent. The photocurrent from the immunosensor was monitored as a result of AFP concentrations. The constructed sensing platform displayed high selectivity and good sensitivity for detecting AFP. Under optimal conditions, a wide linear range from 0.001 to 1000ng/mL and a low detection limit of 0.31pg/mL were obtained. The developed immunosensor is expected to be used to determine AFP and other tumor markers in human plasma in clinical laboratories either for pre-cancer screening or cancer monitoring. Moreover, this sensing platform further has the potential to use for the detection of trypsin activity and the corresponding inhibitor-screening. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

A SPR-based immunosensor for the detection of isoproturon.

PubMed

Gouzy, Marie-Françoise; Kess, Melanie; Krämer, Petra M

2009-02-15

The proof of principle of a reusable surface plasmon resonance (SPR)-based immunosensor for the monitoring of isoproturon (IPU), a selective and systemic herbicide, is presented. The detecting rat monoclonal anti-isoproturon antibody (mAb IOC 7E1) was reversibly immobilized through the use of a capture mouse anti-rat (kappa-chain) monoclonal antibody (mAb TIB 172), which was covalently immobilized on the sensor chip surface. Such strategy features a controlled binding of the captured detecting antibody as well as facilitates the surface regeneration. The capture of the anti-IPU mAb by the antibody (TIB 172) coated sensor surface could be carried out up to 120 times (immobilization/regeneration cycles) without any evidence of activity loss. With a high test midpoint and a low associated SPR signal, the direct detection format was shown to be unsuitable for the routine analysis of isoproturon. However, the limit of detection (LOD) could be easily enhanced by using a strategy based on a surface competition assay, which improved all immunosensor parameters. Moreover, the sensitivity and working range of the indirect format were found to be dependent on the surface density of the anti-IPU mAb IOC 7E1. As expected for competitive formats, the lowest surface coverage (0.5 ng/mm(2)) allowed a lower detection of the herbicide isoproturon with a calculated LOD of 0.1 microg/l, an IC(50) (50% inhibition) of 5.3+/-0.6 microg/l, and a working range (20-80% inhibition) of 1.3-16.3 microg/l.

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

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

Kausaite, A.; van Dijk, M.; Castrop, J.

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 antibodymore » 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.« less

Large area, label-free imaging of extracellular matrix using telecentricity

NASA Astrophysics Data System (ADS)

Visbal Onufrak, Michelle A.; Konger, Raymond L.; Kim, Young L.

2017-02-01

Subtle alterations in stromal tissue structures and organizations within the extracellular matrix (ECM) have been observed in several types of tissue abnormalities, including early skin cancer and wounds. Current microscopic imaging methods often lack the ability to accurately determine the extent of malignancy over a large area, due to their limited field of view. In this research we focus on the development of simple mesoscopic (i.e. between microscopic and macroscopic) biomedical imaging device for non-invasive assessment of ECM alterations over a large, heterogeneous area. In our technology development, a telecentric lens, commonly used in machine vision systems but rarely used in biomedical imaging, serves as a key platform to visualize alterations in tissue microenvironments in a label-free manner over a clinically relevant area. In general, telecentric imaging represents a simple, alternative method for reducing unwanted scattering or diffuse light caused by the highly anisotropic scattering properties of biological tissue. In particular, under telecentric imaging the light intensity backscattered from biological tissue is mainly sensitive to the scattering anisotropy factor, possibly associated with the ECM. We demonstrate the inherent advantages of combining telecentric lens systems with hyperspectral imaging for providing optical information of tissue scattering in biological tissue of murine models, as well as light absorption of hemoglobin in blood vessel tissue phantoms. Thus, we envision that telecentric imaging could potentially serve for simple site-specific, tissue-based assessment of stromal alterations over a clinically relevant field of view in a label-free manner, for studying diseases associated with disruption of homeostasis in ECM.

Determination of sucralose in Splenda and a sugar-free beverage using high-performance anion-exchange chromatography with pulsed amperometric detection.

PubMed

Hanko, Valoran P; Rohrer, Jeffrey S

2004-07-14

Sucralose is a chlorinated carbohydrate nonnutritive sweetener of food and beverage products. The determination of sucralose in food and beverages is important to ensure consistency in product quality. Sucralose was determined in two commercial products without sample preparation using high-performance anion-exchange (HPAE) chromatography coupled with pulsed amperometric detection (PAD). Sucralose was determined with a 10 min isocratic separation. To determine sucralose and other carbohydrates (e.g., dextrose) simultaneously, a gradient separation was developed. The linear range of electrochemical response extended over 3 orders of magnitude, from 0.01 (LOD) to 40 microM (16 microg/mL; 25 microL injection). High precision, high spike recovery, and method ruggedness were observed for both samples.

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study

PubMed Central

Pirhonen, Juho; Arola, Johanna; Sädevirta, Sanja; Luukkonen, Panu; Karppinen, Sanna-Maria; Pihlajaniemi, Taina; Isomäki, Antti; Hukkanen, Mika

2016-01-01

Background and Aims Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. Methods We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. Results We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. Conclusions This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading. PMID:26808140

LFQuant: a label-free fast quantitative analysis tool for high-resolution LC-MS/MS proteomics data.

PubMed

Zhang, Wei; Zhang, Jiyang; Xu, Changming; Li, Ning; Liu, Hui; Ma, Jie; Zhu, Yunping; Xie, Hongwei

2012-12-01

Database searching based methods for label-free quantification aim to reconstruct the peptide extracted ion chromatogram based on the identification information, which can limit the search space and thus make the data processing much faster. The random effect of the MS/MS sampling can be remedied by cross-assignment among different runs. Here, we present a new label-free fast quantitative analysis tool, LFQuant, for high-resolution LC-MS/MS proteomics data based on database searching. It is designed to accept raw data in two common formats (mzXML and Thermo RAW), and database search results from mainstream tools (MASCOT, SEQUEST, and X!Tandem), as input data. LFQuant can handle large-scale label-free data with fractionation such as SDS-PAGE and 2D LC. It is easy to use and provides handy user interfaces for data loading, parameter setting, quantitative analysis, and quantitative data visualization. LFQuant was compared with two common quantification software packages, MaxQuant and IDEAL-Q, on the replication data set and the UPS1 standard data set. The results show that LFQuant performs better than them in terms of both precision and accuracy, and consumes significantly less processing time. LFQuant is freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/lfquant/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free optical imaging of nonfluorescent molecules by stimulated radiation.

PubMed

Min, Wei

2011-12-01

Imaging contrasts other than fluorescence are highly desirable for label-free detection and interrogation of nonfluorescent molecular species inside live cells, tissues, and organisms. The recently developed stimulated Raman scattering (SRS) and stimulated emission microscopy techniques provide sensitive and specific contrast mechanisms for nonfluorescent species, by employing the light amplification aspect of stimulated radiation. Compared to their spontaneous counterparts, stimulated radiation can enhance the imaging performance significantly, making the previously 'dark' molecules observable. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques. Copyright © 2011 Elsevier Ltd. All rights reserved.

Label-free super-resolution with coherent nonlinear structured-illumination microscopy

NASA Astrophysics Data System (ADS)

Huttunen, Mikko J.; Abbas, Aazad; Upham, Jeremy; Boyd, Robert W.

2017-08-01

Structured-illumination microscopy enables up to a two-fold lateral resolution improvement by spatially modulating the intensity profile of the illumination beam. We propose a novel way to generalize the concept of structured illumination to nonlinear widefield modalities by spatially modulating, instead of field intensities, the phase of the incident field while interferometrically measuring the complex-valued scattered field. We numerically demonstrate that for second-order and third-order processes an almost four- and six-fold increase in lateral resolution is achievable, respectively. This procedure overcomes the conventional Abbe diffraction limit and provides new possibilities for label-free super-resolution microscopy.

Single-Step Incubation Determination of miRNAs in Cancer Cells Using an Amperometric Biosensor Based on Competitive Hybridization onto Magnetic Beads.

PubMed

Vargas, Eva; Povedano, Eloy; Montiel, Víctor Ruiz-Valdepeñas; Torrente-Rodríguez, Rebeca M; Zouari, Mohamed; Montoya, Juan José; Raouafi, Noureddine; Campuzano, Susana; Pingarrón, José M

2018-03-15

This work reports an amperometric biosensor for the determination of miRNA-21, a relevant oncogene. The methodology involves a competitive DNA-target miRNA hybridization assay performed on the surface of magnetic microbeads (MBs) and amperometric transduction at screen-printed carbon electrodes (SPCEs). The target miRNA competes with a synthetic fluorescein isothiocyanate (FITC)-modified miRNA with an identical sequence for hybridization with a biotinylated and complementary DNA probe (b-Cp) immobilized on the surface of streptavidin-modified MBs (b-Cp-MBs). Upon labeling, the FITC-modified miRNA attached to the MBs with horseradish peroxidase (HRP)-conjugated anti-FITC Fab fragments and magnetic capturing of the MBs onto the working electrode surface of SPCEs. The cathodic current measured at -0.20 V (versus the Ag pseudo-reference electrode) was demonstrated to be inversely proportional to the concentration of the target miRNA. This convenient biosensing method provided a linear range between 0.7 and 10.0 nM and a limit of detection (LOD) of 0.2 nM (5 fmol in 25 μL of sample) for the synthetic target miRNA without any amplification step. An acceptable selectivity towards single-base mismatched oligonucleotides, a high storage stability of the b-Cp-MBs, and usefulness for the accurate determination of miRNA-21 in raw total RNA (RNA t ) extracted from breast cancer cells (MCF-7) were demonstrated.

Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode.

PubMed

Gholivand, Mohammad Bagher; Khodadadian, Mehdi

2014-03-15

Cholesterol oxidase (ChOx) and catalase (CAT) were co-immobilized on a graphene/ionic liquid-modified glassy carbon electrode (GR-IL/GCE) to develop a highly sensitive amperometric cholesterol biosensor. The H2O2 generated during the enzymatic reaction of ChOx with cholesterol could be reduced electrocatalytically by immobilized CAT to obtain a sensitive amperometric response to cholesterol. The direct electron transfer between enzymes and electrode surface was investigated by cyclic voltammetry. Both enzymes showed well-defined redox peaks with quasi-reversible behaviors. An excellent sensitivity of 4.163 mA mM(-1)cm(-2), a response time less than 6s, and a linear range of 0.25-215 μM (R(2)>0.99) have been observed for cholesterol determination using the proposed biosensor. The apparent Michaelis-Menten constant (KM(app)) was calculated to be 2.32 mM. The bienzymatic cholesterol biosensor showed good reproducibility (RSDs<5%) with minimal interference from the coexisting electroactive compounds such as ascorbic acid and uric acid. The CAT/ChOx/GR-IL/GCE showed excellent analytical performance for the determination of free cholesterol in human serum samples. © 2013 Elsevier B.V. All rights reserved.

Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging*

PubMed Central

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers. PMID:24217021

Label-free quantitative protein profiling of vastus lateralis muscle during human aging.

PubMed

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.

Impedimetric immunosensor doped with reduced graphene sheets fabricated by controllable electrodeposition for the non-labelled detection of bacteria.

PubMed

Wan, Yi; Lin, Zhifeng; Zhang, Dun; Wang, Yi; Hou, Baorong

2011-01-15

A facile, sensitive and reliable impedimetric immunosensor doped with reduced graphene sheets (RGSs) and combined with a controllable electrodeposition technique was developed for the selective detection of marine pathogenic sulphate-reducing bacteria (SRB). The morphology of RGSs and the electrochemical properties of RGSs-doped chitosan (CS) nanocomposite film were investigated by atomic force microscopy, Fourier transform infrared spectroscopy, and cyclic voltammetry (CV). Electrochemical impedance spectroscopy and CV were used to verify the stepwise assembly of the sensor system. Faradic impedance spectroscopy for charge transfer for the redox probe Fe(CN)(6)(3-/4-) was done to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (R(ct)) increased with increasing SRB concentration. A linear relationship between R(ct) and SRB concentration was obtained in the SRB concentration range of 1.8×10(1) to 1.8×10(7) cfu/ml. The impedimetric biosensor gave a distinct response to SRB, but had no obvious response to Vibrio angillarum. It showed a high selectivity for the detection of the pathogen. Based on a combination of the biocompatibility of CS and good electrical conductivity of RGSs, a nanocomposite film with novel architecture was used to immobilize biological and chemical targets and to develop a new type of biosensor. Copyright © 2010 Elsevier B.V. All rights reserved.

Microcapillary-Based Flow-Through Immunosensor and Displacement Immunoassay Using the Same.

DTIC Science & Technology

1997-04-28

an antibody. If desired, an electroosmotic 24 pump may be used to flow fluid through the microcapillary or 25 microcapillaries in the chip...8 for field use. 9 Fig. 1C shows a flow immunosensor chip 100. Buffer flow 10 through microcapillary passage 102 by virtue of an electroosmotic ...Power for an 23 electroosmotic pump or other fluid pump, as well as any other on- 24 chip components, may be provided by a battery incorporated into

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry

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

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reactionmore » monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.« less

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry

DOE PAGES

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

2017-02-16

Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reactionmore » monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.« less

Assembling Amperometric Biosensors for Clinical Diagnostics

PubMed Central

Belluzo, María Soledad; Ribone, María Élida; Lagier, Claudia Marina

2008-01-01

Clinical diagnosis and disease prevention routinely require the assessment of species determined by chemical analysis. Biosensor technology offers several benefits over conventional diagnostic analysis. They include simplicity of use, specificity for the target analyte, speed to arise to a result, capability for continuous monitoring and multiplexing, together with the potentiality of coupling to low-cost, portable instrumentation. This work focuses on the basic lines of decisions when designing electron-transfer-based biosensors for clinical analysis, with emphasis on the strategies currently used to improve the device performance, the present status of amperometric electrodes for biomedicine, and the trends and challenges envisaged for the near future. PMID:27879771

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.

Three-Dimensional Hierarchical Plasmonic Nano-Architecture Enhanced Surface-Enhanced Raman Scattering Immuno-Sensor for Cancer Biomarker Detection in Blood Plasma

PubMed Central

Li, Ming; Cushing, Scott K.; Zhang, Jianming; Suri, Savan; Evans, Rebecca; Petros, William P.; Gibson, Laura F.; Ma, Dongling; Liu, Yuxin; Wu, Nianqiang

2013-01-01

A three-dimensional (3D) hierarchical plasmonic nano-architecture has been designed for a sensitive surface-enhanced Raman scattering (SERS) immuno-sensor for protein biomarker detection. The capture antibody molecules are immobilized on a plasmonic gold triangle nano-array pattern. On the other hand, the detection antibody molecules are linked to the gold nano-star@Raman-reporter@silica sandwich nanoparticles. When protein biomarkers are present, the sandwich nanoparticles are captured over the gold triangle nano-array, forming a confined 3D plasmonic field, leading to the enhanced electromagnetic field in intensity and in 3D space. As a result, the Raman reporter molecules are exposed to a high density of “hot spots”, which amplifies the Raman signal remarkably, improving the sensitivity of the SERS immuno-sensor. This SERS immuno-sensor exhibits a wide linear range (0.1 pg/mL to 10 ng/mL), and a low limit of detection (7 fg/mL) toward human immunoglobulin G (IgG) protein in the buffer solution. This biosensor has been successfully used for detection of the vascular endothelial growth factor (VEGF) in the human blood plasma from clinical breast cancer patient samples. PMID:23659430

Intra-operative label-free multimodal multiphoton imaging of breast cancer margins and microenvironment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sun, Yi; You, Sixian; Tu, Haohua; Spillman, Darold R.; Marjanovic, Marina; Chaney, Eric J.; Liu, George Z.; Ray, Partha S.; Higham, Anna; Boppart, Stephen A.

2017-02-01

Label-free multi-photon imaging has been a powerful tool for studying tissue microstructures and biochemical distributions, particularly for investigating tumors and their microenvironments. However, it remains challenging for traditional bench-top multi-photon microscope systems to conduct ex vivo tumor tissue imaging in the operating room due to their bulky setups and laser sources. In this study, we designed, built, and clinically demonstrated a portable multi-modal nonlinear label-free microscope system that combined four modalities, including two- and three- photon fluorescence for studying the distributions of FAD and NADH, and second and third harmonic generation, respectively, for collagen fiber structures and the distribution of micro-vesicles found in tumors and the microenvironment. Optical realignments and switching between modalities were motorized for more rapid and efficient imaging and for a light-tight enclosure, reducing ambient light noise to only 5% within the brightly lit operating room. Using up to 20 mW of laser power after a 20x objective, this system can acquire multi-modal sets of images over 600 μm × 600 μm at an acquisition rate of 60 seconds using galvo-mirror scanning. This portable microscope system was demonstrated in the operating room for imaging fresh, resected, unstained breast tissue specimens, and for assessing tumor margins and the tumor microenvironment. This real-time label-free nonlinear imaging system has the potential to uniquely characterize breast cancer margins and the microenvironment of tumors to intraoperatively identify structural, functional, and molecular changes that could indicate the aggressiveness of the tumor.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Label-Free Detection of Insulin and Glucagon within Human Islets of Langerhans Using Raman Spectroscopy

PubMed Central

Hilderink, Janneke; Otto, Cees; Slump, Cees; Lenferink, Aufried; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

2013-01-01

Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm-1 band assigned to disulfide bridges in insulin, and the 1552 cm-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans. PMID:24167603

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

A compact and portable optofluidic device for detection of liquid properties and label-free sensing

NASA Astrophysics Data System (ADS)

Lahoz, F.; Martín, I. R.; Walo, D.; Gil-Rostra, J.; Yubero, F.; Gonzalez-Elipe, A. R.

2017-06-01

Optofluidic lasers have been widely investigated over the last few years mainly because they can be easily integrated in sensor devices. However, high power pulse lasers are required as excitation sources, which, in practice, limit the portability of the system. Trying to overcome some of these limitations, in this paper we propose the combined use of a small CW laser with a Fabry-Perot optofluidic planar microcavity showing high sensitivity and versatility for detection of liquid properties and label-free sensing. Firstly, a fluorescein solution in ethanol is used to demonstrate the high performances of the FP microcavity as a temperature sensor both in the laser (high pump power above laser threshold) and in the fluorescence (low pump power) regimes. A shift in the wavelength of the resonant cavity modes is used to detect changes in the temperature and our results show that high sensitivities could be already obtained using cheap and portable CW diode lasers. In the second part of the paper, the demonstration of this portable device for label-free sensing is illustrated under low CW pumping. The wavelength positions of the optofluidic resonant modes are used to detect glucose concentrations in water solutions using a protein labelled with a fluorescent dye as the active medium.

Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.

PubMed

Acero Sánchez, Josep Ll; Fragoso, Alex; Joda, Hamdi; Suárez, Guillaume; McNeil, Calum J; O'Sullivan, Ciara K

2016-07-01

The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot.

Amperometric Solid Electrolyte Oxygen Microsensors with Easy Batch Fabrication

NASA Technical Reports Server (NTRS)

Hunter, Gary W.; Xu, Jennifer C.; Liu, ChungChiun

2011-01-01

An amperometric solid electrolyte oxygen (O2) microsensor using a novel and robust structure has been developed with a detection range of 0.025 to 21 percent of O2 concentration. The microsensor has a simple structure with a sensing area of 1.10 0.99 mm(exp 2), and is operated by applying voltage across the electrodes and measuring the resulting current flow at a temperature of 600 C.

Asymmetric split-ring resonator-based biosensor for detection of label-free stress biomarkers

NASA Astrophysics Data System (ADS)

Lee, Hee-Jo; Lee, Jung-Hyun; Choi, Suji; Jang, Ik-Soon; Choi, Jong-Soon; Jung, Hyo-Il

2013-07-01

In this paper, an asymmetric split-ring resonator, metamaterial element, is presented as a biosensing transducer for detection of highly sensitive and label-free stress biomarkers. In particular, the two biomarkers, cortisol and α-amylase, are used for evaluating the sensitivity of the proposed biosensor. In case of cortisol detection, the competitive reaction between cortisol-bovine serum albumin and free cortisol is employed, while alpha-amylase is directly detected by its antigen-antibody reaction. From the experimental results, we find that the limit of detection and sensitivity of the proposed sensing device are about 1 ng/ml and 1.155 MHz/ng ml-1, respectively.

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

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.

An electrochemical immunosensor for ochratoxin A determination in wines based on a monoclonal antibody and paramagnetic microbeads.

PubMed

Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Duato, Patricia; Castillo, Juan R

2012-06-01

We report a direct competitive immunosensor for the rapid determination of ochratoxin A (OTA) in wine samples. Magnetic beads (1 ± 0.5 μm diameter) covered with streptavidin were functionalized with a monoclonal antibody against OTA, and then left to incubate in a solution of tracer (ochratoxin conjugated to the enzyme peroxidase) and a range of OTA concentrations (10(-4) to 1,000 ng mL(-1)). After washing and separation steps helped with a magnetic field, a volume of the dispersion was put on screen-printed electrodes under a magnet, and after adding the substrate the p-benzoquinone generated enzymatically was detected by differential-pulse voltammetry. Wine samples (2 mL) were easily prepared simply by adjusting to pH = 7.5 with diluted NaOH and by adding polyvinylpyrrolidone for complexing polyphenols, without any other clean-up or preconcentration steps. The limit of detection for detecting OTA in wines was of 0.11 ± 0.01 ng L(-1), well below the permitted content of the mycotoxin by the European Union (<2 ng mL(-1)). Spiked wines were subjected to immunosensor calibrations to study the matrix effects. OTA concentrations measured with the immunosensor were compared with those obtained by high-performance liquid chromatography coupled to fluorescence detection (AOAC official method 2001.01). The OTA levels from two red wines of "Campo de Borja", Spain, ranged from about 0.027 to 0.033 ng mL(-1) of OTA.

Chemiluminescent optical fiber immunosensor for the detection of anti-West Nile virus IgG.

PubMed

Herrmann, Sebastien; Leshem, Boaz; Landes, Shimi; Rager-Zisman, Bracha; Marks, Robert S

2005-03-31

An ELISA-based optical fiber methodology developed for the detection of anti-West Nile virus IgG antibodies in serum was compared to standard colorimetric and chemiluminescent ELISA based on microtiter plates. Colorimetric ELISA was the least sensitive, especially at high titer dilutions. The fiber-optic immunosensor based on the same ELISA immunological rationale was the most sensitive technique.

Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma.

PubMed

Brito, Mayara F; Auler, Patrícia A; Tavares, Guilherme C; Rezende, Cristiana P; Almeida, Gabriel M F; Pereira, Felipe L; Leal, Carlos A G; Moura, Arlindo de Alencar; Figueiredo, Henrique C P; Henry, Marc

2018-06-11

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMS E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728. © 2018 Blackwell Verlag GmbH.

Label-free Raman observation of cytochrome c dynamics during apoptosis

PubMed Central

Okada, Masaya; Smith, Nicholas Isaac; Palonpon, Almar Flotildes; Endo, Hiromi; Kawata, Satoshi; Sodeoka, Mikiko; Fujita, Katsumasa

2012-01-01

We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750 cm-1 were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c distribution can be distinguished as release of cytochrome c from mitochondria. Our observation also revealed that the redox state of cytochrome c was maintained during the release from the mitochondria. Monitoring mitochondrial membrane potential with JC-1 dye confirmed that the observed cytochrome c release was associated with apoptosis. PMID:22184220

The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels

NASA Astrophysics Data System (ADS)

Malý, J.; Lampová, H.; Semerádtová, A.; Štofik, M.; Kováčik, L.

2009-09-01

This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH4 silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

Label-Free Biological and Chemical Sensing Using Whispering Gallery Mode Optical Resonators: Past, Present, and Future

PubMed Central

Su, Judith

2017-01-01

Sensitive and rapid label-free biological and chemical sensors are needed for a wide variety of applications including early disease diagnosis and prognosis, the monitoring of food and water quality, as well as the detection of bacteria and viruses for public health concerns and chemical threat sensing. Whispering gallery mode optical resonator based sensing is a rapidly developing field due to the high sensitivity and speed of these devices as well as their label-free nature. Here, we describe the history of whispering gallery mode optical resonator sensors, the principles behind detection, the latest developments in the fields of biological and chemical sensing, current challenges toward widespread adoption of these devices, and an outlook for the future. In addition, we evaluate the performance capabilities of these sensors across three key parameters: sensitivity, selectivity, and speed. PMID:28282881

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. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins ▿

PubMed Central

Carney, Paul J.; Lipatov, Aleksandr S.; Monto, Arnold S.; Donis, Ruben O.; Stevens, James

2010-01-01

During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization provided important information on the relative protection afforded by the population's cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability, and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. f-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities. PMID:20660137

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.

Solid surface fluorescence immunosensor for ultrasensitive detection of hepatitis B virus surface antigen using PAMAM/CdTe@CdS QDs nanoclusters.

PubMed

Babamiri, Bahareh; Hallaj, Rahman; Salimi, Abdollah

2018-06-20

In the present study, we constructed an ultrasensitive solid surface fluorescence-immunosensor based on highly luminescent CdTe@CdS-PAMAM structures as nanoprobe for determination of HBsAg by monitoring fluorescence intensity. This strategy was achieved by using PAMAM as a signal amplifier; the PAMAM dendrimer with the many functional amine groups can amplify the fluorescence signal of QDs by covalent attachment of CdTe@CdS on PAMAM and hence, improve the sensitivity of the proposed method significantly. A sandwich type immunosensor was formed after the addition of HBsAg and the PAMAM-QD-Ab 2 , respectively. Under optimal conditions, the designed immunosensor demonstrates a good analytical performance for the HBsAg detection in an excellent linear range from 5 fg ml -1 to 0.15 ng ml -1 with the detection limit (LOD) of 0.6 fg ml -1 at a S/N ratio of 3. In addition, the analysis of human serum samples shows that the fluorescent immunoassay has the great potential for early diagnosis of hepatitis B and can be used for the detection of other tumor markers in clinical applications.

Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

NASA Astrophysics Data System (ADS)

Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

2016-03-01

Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

Development of screen-printed tryptophan-kynurenine immunosensor for in vitro assay of kynurenine-mediated immunosuppression effect of cancer cells on activated T-cells.

PubMed

Karami, Pari; Majidi, Mir Reza; Johari-Ahar, Mohammad; Barar, Jaleh; Omidi, Yadollah

2017-06-15

The development of analytical methods that respond to the emerging need to perform rapid 'in situ' analyses of human metabolic pathways (HMPs) demonstrates disposable screen-printed electrodes (SPEs) as an alternative to the traditional tools. In the kynurenine pathway, one of the important HMPs, increased production of kynurenine (Kyn) as a main catabolite of tryptophan (Trp) degradation is involved in the immuno-editing process supporting cancer cells in escaping from the human immune system. In the current study, we demonstrate the development of a screen-printed potentiometric immunosensor for in vitro evaluation of Trp consumption and Kyn production controlled by cancer cells in response to the activated T-lymphocytes. To engineer this immunosensor, uniform layer of carboxylated multiwall carbon nanotubes (MWCNT) was deposited on gold screen-printed electrode (AuSPE), and afterwards monoclonal antibody (mAb) specific to l-kynurenine was covalently conjugated with the MWCNT modified AuSPE. The engineered immunosensor was examined in monitoring Trp consumption and Kyn production in metastatic (Calu-6, NCI-H1299, and HT29) and nonmetastatic (HepG2 and 1321NI) cancer cell lines. Without applying preparation and separation steps, this Trp-Kyn immunosensor offers an improved limit of detection (0.5nM and 120nM for Kyn and Trp detection, respectively) and a broad linear range of detection (LRD: 0.001-1µM and 1-100µM for Kyn, and 0.1-300µM for Trp detection). However, this immunosensor was successfully used for in situ analysis of Kyn that are produced during immuno-editing process in cell culture media, and could reveal that Trp consumption and Kyn production by highly metastatic cancer cells (HT29) were significantly higher than nonmetastatic HepG2 cancer cells. Owing to the screen-printed nature, such kind of biosensors have capability of being integrated into lab-on-a-chip (LOC), microfluidics, and micro total analysis systems. Copyright © 2016 Elsevier B

Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis.

PubMed

Dai, Peng; Wang, Qin; Wang, Weihua; Jing, Ruirui; Wang, Wei; Wang, Fengqin; Azadzoi, Kazem M; Yang, Jing-Hua; Yan, Zhen

2016-01-21

Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients' tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis.

Combining Raman spectroscopy and digital holographic microscopy for label-free classification of human immune cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

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

2017-02-01

Moving towards label-free techniques for cell identification is essential for many clinical and research applications. Raman spectroscopy and digital holographic microscopy (DHM) are both label-free, non-destructive optical techniques capable of providing complimentary information. We demonstrate a multi-modal system which may simultaneously take Raman spectra and DHM images to provide both a molecular and a morphological description of our sample. In this study we use Raman spectroscopy and DHM to discriminate between three immune cell populations CD4+ T cells, B cells, and monocytes, which together comprise key functional immune cell subsets in immune responses to invading pathogens. Various parameters that may be used to describe the phase images are also examined such as pixel value histograms or texture analysis. Using our system it is possible to consider each technique individually or in combination. Principal component analysis is used on the data set to discriminate between cell types and leave-one-out cross-validation is used to estimate the efficiency of our method. Raman spectroscopy provides specific chemical information but requires relatively long acquisition times, combining this with a faster modality such as DHM could help achieve faster throughput rates. The combination of these two complimentary optical techniques provides a wealth of information for cell characterisation which is a step towards achieving label free technology for the identification of human immune cells.

Comparison of Electrochemical Immunosensors and Aptasensors for Detection of Small Organic Molecules in Environment, Food Safety, Clinical and Public Security

PubMed Central

Piro, Benoit; Shi, Shihui; Reisberg, Steeve; Noël, Vincent; Anquetin, Guillaume

2016-01-01

We review here the most frequently reported targets among the electrochemical immunosensors and aptasensors: antibiotics, bisphenol A, cocaine, ochratoxin A and estradiol. In each case, the immobilization procedures are described as well as the transduction schemes and the limits of detection. It is shown that limits of detections are generally two to three orders of magnitude lower for immunosensors than for aptasensors, due to the highest affinities of antibodies. No significant progresses have been made to improve these affinities, but transduction schemes were improved instead, which lead to a regular improvement of the limit of detections corresponding to ca. five orders of magnitude over these last 10 years. These progresses depend on the target, however. PMID:26938570

Comparison of Electrochemical Immunosensors and Aptasensors for Detection of Small Organic Molecules in Environment, Food Safety, Clinical and Public Security.

PubMed

Piro, Benoit; Shi, Shihui; Reisberg, Steeve; Noël, Vincent; Anquetin, Guillaume

2016-02-29

We review here the most frequently reported targets among the electrochemical immunosensors and aptasensors: antibiotics, bisphenol A, cocaine, ochratoxin A and estradiol. In each case, the immobilization procedures are described as well as the transduction schemes and the limits of detection. It is shown that limits of detections are generally two to three orders of magnitude lower for immunosensors than for aptasensors, due to the highest affinities of antibodies. No significant progresses have been made to improve these affinities, but transduction schemes were improved instead, which lead to a regular improvement of the limit of detections corresponding to ca. five orders of magnitude over these last 10 years. These progresses depend on the target, however.

Label-free image-based detection of drug resistance with optofluidic time-stretch microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kobayashi, Hirofumi; Lei, Cheng; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

2017-02-01

Acquired drug resistance is a fundamental predicament in cancer therapy. Early detection of drug-resistant cancer cells during or after treatment is expected to benefit patients from unnecessary drug administration and thus play a significant role in the development of a therapeutic strategy. However, the development of an effective method of detecting drug-resistant cancer cells is still in its infancy due to their complex mechanism in drug resistance. To address this problem, we propose and experimentally demonstrate label-free image-based drug resistance detection with optofluidic time-stretch microscopy using leukemia cells (K562 and K562/ADM). By adding adriamycin (ADM) to both K562 and K562/ADM (ADM-resistant K562 cells) cells, both types of cells express unique morphological changes, which are subsequently captured by an optofluidic time-stretch microscope. These unique morphological changes are extracted as image features and are subjected to supervised machine learning for cell classification. We hereby have successfully differentiated K562 and K562/ADM solely with label-free images, which suggests that our technique is capable of detecting drug-resistant cancer cells. Our optofluidic time-stretch microscope consists of a time-stretch microscope with a high spatial resolution of 780 nm at a 1D frame rate of 75 MHz and a microfluidic device that focuses and orders cells. We compare various machine learning algorithms as well as various concentrations of ADM for cell classification. Owing to its unprecedented versatility of using label-free image and its independency from specific molecules, our technique holds great promise for detecting drug resistance of cancer cells for which its underlying mechanism is still unknown or chemical probes are still unavailable.

Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

PubMed

Zeng, Yan; Xu, Jin; Li, Dong; Li, Li; Wen, Zilong; Qu, Jianan Y

2012-07-01

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation.

PubMed

Azimzadeh, Omid; Scherthan, Harry; Yentrapalli, Ramesh; Barjaktarovic, Zarko; Ueffing, Marius; Conrad, Marcus; Neff, Frauke; Calzada-Wack, Julia; Aubele, Michaela; Buske, Christian; Atkinson, Michael J; Hauck, Stefanie M; Tapio, Soile

2012-04-18

Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives. Copyright © 2012 Elsevier B.V. All rights reserved.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Label-free SnO2 nanowire FET biosensor for protein detection

NASA Astrophysics Data System (ADS)

Jakob, Markus H.; Dong, Bo; Gutsch, Sebastian; Chatelle, Claire; Krishnaraja, Abinaya; Weber, Wilfried; Zacharias, Margit

2017-06-01

Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 μl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.

Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.

PubMed

de Juan-Franco, Elena; Rodríguez-Frade, J M; Mellado, M; Lechuga, Laura M

2013-09-30

We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL(-1) and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Fully integrated graphene electronic biosensor for label-free detection of lead (II) ion based on G-quadruplex structure-switching.

PubMed

Li, Yijun; Wang, Cheng; Zhu, Yibo; Zhou, Xiaohong; Xiang, Yu; He, Miao; Zeng, Siyu

2017-03-15

This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb 2+ ) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb 2+ ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb 2+ with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb 2+ monitoring is enabled with promising application potential. Copyright © 2016 Elsevier B.V. All rights reserved.

A Reagentless Amperometric Formaldehyde-Selective Chemosensor Based on Platinized Gold Electrodes

PubMed Central

Demkiv, Olha; Smutok, Oleh; Gonchar, Mykhailo; Nisnevitch, Marina

2017-01-01

Fabrication and characterization of a new amperometric chemosensor for accurate formaldehyde analysis based on platinized gold electrodes is described. The platinization process was performed electrochemically on the surface of 4 mm gold planar electrodes by both electrolysis and cyclic voltamperometry. The produced electrodes were characterized using scanning electron microscopy and X-ray spectral analysis. Using a low working potential (0.0 V vs. Ag/AgCl) enabled an essential increase in the chemosensor’s selectivity for the target analyte. The sensitivity of the best chemosensor prototype to formaldehyde is uniquely high (28180 A·M−1·m−2) with a detection limit of 0.05 mM. The chemosensor remained stable over a one-year storage period. The formaldehye-selective chemosensor was tested on samples of commercial preparations. A high correlation was demonstrated between the results obtained by the proposed chemosensor, chemical and enzymatic methods (R = 0.998). The developed formaldehyde-selective amperometric chemosensor is very promising for use in industry and research, as well as for environmental control. PMID:28772868

A Reagentless Amperometric Formaldehyde-Selective Chemosensor Based on Platinized Gold Electrodes.

PubMed

Demkiv, Olha; Smutok, Oleh; Gonchar, Mykhailo; Nisnevitch, Marina

2017-05-06

Fabrication and characterization of a new amperometric chemosensor for accurate formaldehyde analysis based on platinized gold electrodes is described. The platinization process was performed electrochemically on the surface of 4 mm gold planar electrodes by both electrolysis and cyclic voltamperometry. The produced electrodes were characterized using scanning electron microscopy and X-ray spectral analysis. Using a low working potential (0.0 V vs. Ag/AgCl) enabled an essential increase in the chemosensor's selectivity for the target analyte. The sensitivity of the best chemosensor prototype to formaldehyde is uniquely high (28180 A·M -1 ·m -2 ) with a detection limit of 0.05 mM. The chemosensor remained stable over a one-year storage period. The formaldehye-selective chemosensor was tested on samples of commercial preparations. A high correlation was demonstrated between the results obtained by the proposed chemosensor, chemical and enzymatic methods ( R = 0.998). The developed formaldehyde-selective amperometric chemosensor is very promising for use in industry and research, as well as for environmental control.

[Amperometric biosensor for lactate analysis in wines and grape must during fermentation].

PubMed

Shkotova, L V; Horiushkina, T B; Slast'ia, E A; Soldatkin, O P; Tranh-Minh, S; Chovelon, J M; Dziadevych, S V

2005-01-01

The amperometric biosensor based on lactate oxidase for determination of lactate has been developed, and two methods of immobilization of lactate oxidase on the surface of industrial screen-printed platinum electrodes SensLab were compared. A sensor with immobilized in the Resydrol polymer lactate oxidase by the method of physical adsorption is characterized of narrow dynamic range and greater response value in comparison with a biosensor based on immobilised in poly(3,4-ethylenedioxythiophene) lactate oxidase by the method of electrochemical polymerization. Operational stability of the biosensor developed was studied and it was shown, that the immobilization method does not influence their stability. The analysis of the lactate in wine and during wine fermentation has been conducted. High correlation of the data obtained by means of amperometric lactate biosensor and a standard method of an ionic chromatography has been shown. The developed biosensor could be applied in the food industry for the control and optimization of the wine fermentation process, and quality control of wine.

An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

PubMed

Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin

2014-08-15

Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. Copyright © 2014 Elsevier B.V. All rights reserved.

Organic electrochemical transistor based immunosensor for prostate specific antigen (PSA) detection using gold nanoparticles for signal amplification.

PubMed

Kim, Duck-Jin; Lee, Nae-Eung; Park, Joon-Shik; Park, In-Jun; Kim, Jung-Gu; Cho, Hyoung J

2010-07-15

We demonstrated a highly sensitive organic electrochemical transistor (OECT) based immunosensor with a low detection limit for prostate specific antigen/alpha1-antichymotrypsin (PSA-ACT) complex. The poly(styrenesulfonate) doped poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) based OECT with secondary antibody conjugated gold nanoparticles (AuNPs) provided a detection limit of the PSA-ACT complex as low as 1pg/ml, as well as improved sensitivity and a dynamic range, due to the role of AuNPs in the signal amplification. The sensor performances were particularly improved in the lower concentration range where the detection is clinically important for the preoperative diagnosis and screening of prostate cancer. This result shows that the OECT-based immunosensor can be used as a transducer platform acceptable to the point-of-care (POC) diagnostic systems and demonstrates adaptability of organic electronics to clinical applications. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Label-free Raman spectroscopy for accessing intracellular anticancer drug release on gold nanoparticles.

PubMed

Ock, Kwang-Su; Ganbold, Erdene Ochir; Park, Jin; Cho, Keunchang; Joo, Sang-Woo; Lee, So Yeong

2012-06-21

We investigated glutathione (GSH)-induced purine or pyrimidine anticancer drug release on gold nanoparticle (AuNP) surfaces by means of label-free Raman spectroscopy. GSH-triggered releases of 6-thioguanine (6TG), gemcitabine (GEM), acycloguanosine (ACY), and fadrozole (FAD) were examined in a comparative way by means of surface-enhanced Raman scattering (SERS). The GSH-induced dissociation constant of GEM (or ACY/FAD) from AuNPs was estimated to be larger by more than 38 times than that of 6TG from the kinetic relationship. Tripeptide control experiments were presented to check the turn-off Raman signalling mechanism. Dark-field microscopy (DFM) and transmission electron microscopy (TEM) indicated the intracellular AuNP loads. After their cellular uptake, GEM, ACY, and FAD would not show SERS intensities as strong as 6TG. This may be due to easier release of GEM, ACY, and FAD than 6TG by intracellular reducing species including GSH. We observed fairly strong SERS signals of GEM and 6TG in cell culture media solution. Our CCK-8 cytotoxicity assay data support that 6TG-AuNPs did not exhibit a substantial decrease in cell viability presumably due to strong binding. Label-free confocal Raman spectroscopy can be utilized as an effective tool to access intracellular anticancer drug release.

Amperometric Determination of Glucose at Parts per Million Levels with Immobilized Glucose Oxidase.

ERIC Educational Resources Information Center

Sittampalam, G.; Wilson, G. S.

1982-01-01

An experiment on the operation and utility of an amperometric immobilized enzyme electrode (or probe) is described, including advantages of the experiment, equipment, reagents, preparation of phosphate buffer, enzyme immobilization techniques, laboratory procedures, precautions, and discussion of experimental results. (SK)

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

To label or not to label: applications of quantitative proteomics in neuroscience research.

PubMed

Filiou, Michaela D; Martins-de-Souza, Daniel; Guest, Paul C; Bahn, Sabine; Turck, Christoph W

2012-02-01

Proteomics has provided researchers with a sophisticated toolbox of labeling-based and label-free quantitative methods. These are now being applied in neuroscience research where they have already contributed to the elucidation of fundamental mechanisms and the discovery of candidate biomarkers. In this review, we evaluate and compare labeling-based and label-free quantitative proteomic techniques for applications in neuroscience research. We discuss the considerations required for the analysis of brain and central nervous system specimens, the experimental design of quantitative proteomic workflows as well as the feasibility, advantages, and disadvantages of the available techniques for neuroscience-oriented questions. Furthermore, we assess the use of labeled standards as internal controls for comparative studies in humans and review applications of labeling-based and label-free mass spectrometry approaches in relevant model organisms and human subjects. Providing a comprehensive guide of feasible and meaningful quantitative proteomic methodologies for neuroscience research is crucial not only for overcoming current limitations but also for gaining useful insights into brain function and translating proteomics from bench to bedside. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel self-enhanced electrochemiluminescence immunosensor based on hollow Ru-SiO2@PEI nanoparticles for NSE analysis

NASA Astrophysics Data System (ADS)

Zhou, Limin; Huang, Jianshe; Yu, Bin; You, Tianyan

2016-02-01

Poly(ethylenimine) (PEI) and Ru(bpy)32+-doped silica (Ru-SiO2) nanoparticles were simply mixed together to prepare a novel self-enhanced electrochemiluminescence (ECL) composite of Ru-SiO2@PEI. The hollow Ru-SiO2@PEI nanoparticles were used to build an ECL immunosensor for the analysis of neuron specific enolase (NSE). PEI not only assembled on the surface of Ru-SiO2 nanoparticles through the electrostatic interaction to act as co-reactant for Ru(bpy)32+ ECL, but also provided alkaline condition to etch the Ru-SiO2 nanoparticles to form the hollow Ru-SiO2@PEI nanoparticles with porous shell. The unique structure of the Ru-SiO2@PEI nanoparticles loaded both a large amount of Ru(bpy)32+ and its co-reactant PEI at the same time, which shortened the electron-transfer distance, thereby greatly enhanced the luminous efficiency and amplified the ECL signal. The developed immunosensor showed a wide linear range from 1.0 × 10-11 to 1.0 × 10-5 mg mL-1 with a low detection limit of 1.0 × 10-11 mg mL-1 for NSE. When the immunosensor was used for the determination of NSE in clinical human serum, the results were comparable with those obtained by using enzyme-linked immunosorbent assay (ELISA) method. The proposed method provides a promising alternative for NSE analysis in clinical samples.

Separable Bilayer Microfiltration Device for Label-Free Enrichment of Viable Circulating Tumor Cells.

PubMed

Hao, Sijie; Nisic, Merisa; He, Hongzhang; Tai, Yu-Chong; Zheng, Si-Yang

2017-01-01

Analysis of rare circulating tumor cells enriched from metastatic cancer patients yields critical information on disease progression, therapy response, and the mechanism of cancer metastasis. Here we describe in detail a label-free enrichment process of circulating tumor cells based on its unique physical properties (size and deformability). Viable circulating tumor cells can be successfully enriched and analyzed, or easily released for further characterization due to the novel separable two-layer design.

Self-reference and random sampling approach for label-free identification of DNA composition using plasmonic nanomaterials.

PubMed

Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

2018-05-09

The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD 10.04 billion per year by 2020. However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor. Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy (SERS) in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations. We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition. The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error.

Real-Time Amperometric Recording of Extracellular H₂O₂ in the Brain of Immunocompromised Mice: An In Vitro, Ex Vivo and In Vivo Characterisation Study.

PubMed

Reid, Caroline H; Finnerty, Niall J

2017-07-08

We detail an extensive characterisation study on a previously described dual amperometric H₂O₂ biosensor consisting of H₂O₂ detection (blank) and degradation (catalase) electrodes. In vitro investigations demonstrated excellent H₂O₂ sensitivity and selectivity against the interferent, ascorbic acid. Ex vivo studies were performed to mimic physiological conditions prior to in vivo deployment. Exposure to brain tissue homogenate identified reliable sensitivity and selectivity recordings up to seven days for both blank and catalase electrodes. Furthermore, there was no compromise in pre- and post-implanted catalase electrode sensitivity in ex vivo mouse brain. In vivo investigations performed in anaesthetised mice confirmed the ability of the H₂O₂ biosensor to detect increases in amperometric current following locally perfused/infused H₂O₂ and antioxidant inhibitors mercaptosuccinic acid and sodium azide. Subsequent recordings in freely moving mice identified negligible effects of control saline and sodium ascorbate interference injections on amperometric H₂O₂ current. Furthermore, the stability of the amperometric current was confirmed over a five-day period and analysis of 24-h signal recordings identified the absence of diurnal variations in amperometric current. Collectively, these findings confirm the biosensor current responds in vivo to increasing exogenous and endogenous H₂O₂ and tentatively supports measurement of H₂O₂ dynamics in freely moving NOD SCID mice.

Analogous modified DNA probe and immune competition method-based electrochemical biosensor for RNA modification.

PubMed

Dai, Tao; Pu, Qinli; Guo, Yongcan; Zuo, Chen; Bai, Shulian; Yang, Yujun; Yin, Dan; Li, Yi; Sheng, Shangchun; Tao, Yiyi; Fang, Jie; Yu, Wen; Xie, Guoming

2018-08-30

N6-methyladenosine (m6A), one of the most abundant RNA methylation which is ubiquitous in eukaryotic RNA, plays vital roles in many biological progresses. Therefore, the rapid and accurate quantitative detection of m6A is particularly important for its functional research. Herein, a label-free and highly selective electrochemical immunosensor was developed for the detection of m6A. The method is established on that the anti-m6A-Ab can recognize both m6A-RNA and m6A-DNA. An analogous modified DNA probe (L1) serves as a signal molecule, by competing with m6A-RNA for binding to Abs to broaden the linear range. The detection of m6A-RNA by this method is unaffected by the lengths and base sequences of RNA. Under optimal conditions, the proposed immunosensor presented a wide linear range from 0.05 to 200 nM with a detection limit as low as 0.016 nM (S/N = 3). The specificity and reproducibility of the method are satisfactory. Furthermore, the developed immunosensor was validated for m6A determination in human cell lines. Thus, the immunosensor provides a promising platform for m6A-RNA detection with simplicity, high specificity and sensitivity. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of Different Disinfectants on the Performance of an On-Meter Dosed Amperometric Glucose-Oxidase-Based Glucose Meter

PubMed Central

Sarmaga, Don; DuBois, Jeffrey A; Lyon, Martha E

2011-01-01

Background Off-meter dosed photometric glucose-oxidase-based glucose meters have been reported to be susceptible to interference by hydrogen-peroxide-based disinfecting agents. The objective of this study was to determine if a single application of hydrogen-peroxide-containing Accel® wipe to disinfect an on-meter dosed amperometric glucose-oxidase-based glucose meter will influence its performance. Method The performance of five on-meter dosed amperometric glucose-oxidase-based glucose meters was determined before and after disinfecting the devices with a single application of either CaviWipes® (14.3% isopropanol and 0.23% diisobutyl-phenoxy-ethoxyethyl dimethyl benzyl ammonium chloride) or Accel (0.5% hydrogen peroxide) wipes. Replicate glucose measurements were conducted before disinfecting the devices, immediately after disinfecting, and then 1 and 2 min postdisinfecting, with measurements in triplicate. Analysis was sequentially completed for five different meters. Results were analyzed by a two-way analysis of variance (Analyze-it software). Results No clinical (<0.3 mmol/liter) or statistical differences (p > .05) in glucose concentration were detected when the on-meter dosed amperometric glucose-oxidase-based glucose meters were disinfected with either CaviWipes or Accel wipes and measured immediately or 1 or 2 min postdisinfecting. No clinically significant difference in glucose concentration was detected between meters (<0.3 mmol/liter). Conclusion The on-meter dosed glucose oxidase amperometric-based glucose meters are not analytically susceptible to interference by a single application of hydrogen-peroxide-containing Accel disinfectant wipes. PMID:22226263

Evaluation of different disinfectants on the performance of an on-meter dosed amperometric glucose-oxidase-based glucose meter.

PubMed

Sarmaga, Don; Dubois, Jeffrey A; Lyon, Martha E

2011-11-01

Off-meter dosed photometric glucose-oxidase-based glucose meters have been reported to be susceptible to interference by hydrogen-peroxide-based disinfecting agents. The objective of this study was to determine if a single application of hydrogen-peroxide-containing Accel® wipe to disinfect an on-meter dosed amperometric glucose-oxidase-based glucose meter will influence its performance. The performance of five on-meter dosed amperometric glucose-oxidase-based glucose meters was determined before and after disinfecting the devices with a single application of either CaviWipes® (14.3% isopropanol and 0.23% diisobutyl-phenoxy-ethoxyethyl dimethyl benzyl ammonium chloride) or Accel (0.5% hydrogen peroxide) wipes. Replicate glucose measurements were conducted before disinfecting the devices, immediately after disinfecting, and then 1 and 2 min postdisinfecting, with measurements in triplicate. Analysis was sequentially completed for five different meters. Results were analyzed by a two-way analysis of variance (Analyze-it software). No clinical (<0.3 mmol/liter) or statistical differences (p > .05) in glucose concentration were detected when the on-meter dosed amperometric glucose-oxidase-based glucose meters were disinfected with either CaviWipes or Accel wipes and measured immediately or 1 or 2 min postdisinfecting. No clinically significant difference in glucose concentration was detected between meters (<0.3 mmol/liter). The on-meter dosed glucose oxidase amperometric-based glucose meters are not analytically susceptible to interference by a single application of hydrogen-peroxide-containing Accel disinfectant wipes. © 2011 Diabetes Technology Society.

Free-Labeling Facial Expressions and Emotional Situations in Children Aged 3-7 Years: Developmental Trajectory and a Face Inferiority Effect

ERIC Educational Resources Information Center

Wang, Zhenhong; Lü, Wei; Zhang, Hui; Surina, Alyssa

2014-01-01

Chinese children (N = 185, aged 3-7 years) were assessed on their abilities to freely label facial expressions and emotional situations. Results indicated that the overall accuracy of free-labeling facial expressions increased relatively quickly in children aged 3-5 years, but slowed down in children aged 5-7 years. In contrast, the overall…

Prototype amperometric biosensor for sialic acid determination.

PubMed

Marzouk, Sayed A M; Ashraf, S S; Tayyari, Khawla A Al

2007-02-15

This paper describes the first report on the development, characterization, and applications of a prototype amperometric biosensor for free sialic acid (SA). The sensor was constructed by the coimmobilization of two enzymes, i.e., N-acetylneuraminic acid aldolase and pyruvate oxidase, on a polyester microporous membrane, which was then mounted on top of a platinum disk electrode. The SA biosensor operation was based on the sequential action of the two enzymes to ultimately produce hydrogen peroxide, which was then detected by anodic amperometry at the platinum electrode. The surface of the platinum electrode was coated with an electropolymeric layer to enhance the biosensor selectivity in the presence of interfering oxidizable species. Optimization of the enzyme layer composition resulted in a fast and steady current response in phosphate buffer pH 7.2 at 37 degrees C. The limit of detection was 10 microM, and the response was linear to 3.5 mM (r = 0.9987). The prepared SA biosensors retained approximately 85% of their initial sensitivity after 8 days and showed excellent response reproducibility (CV = 2.3%). Utilization of a third enzyme, sialidase, expanded the scope of the present SA biosensor to determine bound sialic acid as well. The merits of the described biosensor allowed its successful application in determining SA in biological and pharmaceutical samples. The obtained results indicated that the presented SA biosensor should be a useful bioanalytical tool in several biological and clinical applications such as screening of SA as a nonspecific tumor marker as well as monitoring of tumor therapy.

Old tree with new shoots: silver nanoparticles for label-free and colorimetric mercury ions detection

NASA Astrophysics Data System (ADS)

Gao, Shuyan; Jia, Xiaoxia; Chen, Yanli

2013-01-01

Mercury in the environment from global mercury emissions as well as various forms of contamination poses severe threats to both human health and the environment. Long-term exposure to high levels of Hg-based toxins results in serious and irreversible damage of the central nervous system and other organs. Therefore, the development of effective sensing systems for mercury detection becomes an increasing demand. In this article, a yogurt-mediated silver nanostructure is reported to be unprecedentedly used in the naked-eye and label-free detection of mercury. The method relies on the redox reaction resulting from the electrode potential difference between Ag+/Ag (0.7996 V) and Hg2+/Hg2 2+ (0.920 V) that makes colorless Hg2+ ions which oxidize colored silver nanoparticle (AgNP) to colorless Ag+. The labor-intensive modification of AgNPs and expensive labeling are avoided, and the traditional AuNPs are substituted by AgNPs in this Hg2+ ions sensing platform, which makes it facile, low-cost, and particularly useful for home, clinic, or field applications as well as resource-limited conditions. This sensing system achieves a detection limit as low as 10 nM, lower than the toxicity level of Hg2+ ions in drinking water (30 nM) defined by World Health Organization, and exhibits excellent selectivity, largely free from the matrix effect of the real water samples. This visual label-free Hg2+ ions sensing motif shows great promise for sensing Hg2+ ions in terms of sensitivity, selectivity, cost, and maneuverability. It is also a good example for the organic combination of green chemistry and functional materials, which may trigger interest in furthering biosystems for environmental science applications.

Label-free biodetection using a smartphone.

PubMed

Gallegos, Dustin; Long, Kenneth D; Yu, Hojeong; Clark, Peter P; Lin, Yixiao; George, Sherine; Nath, Pabitra; Cunningham, Brian T

2013-06-07

Utilizing its integrated camera as a spectrometer, we demonstrate the use of a smartphone as the detection instrument for a label-free photonic crystal biosensor. A custom-designed cradle holds the smartphone in fixed alignment with optical components, allowing for accurate and repeatable measurements of shifts in the resonant wavelength of the sensor. Externally provided broadband light incident upon an entrance pinhole is subsequently collimated and linearly polarized before passing through the biosensor, which resonantly reflects only a narrow band of wavelengths. A diffraction grating spreads the remaining wavelengths over the camera's pixels to display a high resolution transmission spectrum. The photonic crystal biosensor is fabricated on a plastic substrate and attached to a standard glass microscope slide that can easily be removed and replaced within the optical path. A custom software app was developed to convert the camera images into the photonic crystal transmission spectrum in the visible wavelength range, including curve-fitting analysis that computes the photonic crystal resonant wavelength with 0.009 nm accuracy. We demonstrate the functionality of the system through detection of an immobilized protein monolayer, and selective detection of concentration-dependent antibody binding to a functionalized photonic crystal. We envision the capability for an inexpensive, handheld biosensor instrument with web connectivity to enable point-of-care sensing in environments that have not been practical previously.

Development of cadmium-free quantum dot for intracellular labelling through electroporation or lipid-calcium-phosphate

NASA Astrophysics Data System (ADS)

Liu, Ying-Feng; Hung, Wei-Ling; Hou, Tzh-Yin; Huang, Hsiu-Ying; Lin, Cheng-An J.

2016-04-01

Traditional fluorescent labelling techniques has severe photo-bleaching problem such as organic dyes and fluorescent protein. Quantum dots made up of traditional semiconductor (CdSe/ZnS) material has sort of biological toxicity. This research has developed novel Cd-free quantum dots divided into semiconductor (Indium phosphide, InP) and noble metal (Gold). Former has lower toxicity compared to traditional quantum dots. Latter consisting of gold (III) chloride (AuCl3) and toluene utilizes sonochemical preparation and different stimulus to regulate fluorescent wavelength. Amphoteric macromolecule surface technology and ligand Exchange in self-Assembled are involved to develop hydrophilic nanomaterials which can regulate the number of grafts per molecule of surface functional groups. Calcium phosphate (CaP) nanoparticle (NP) with an asymmetric lipid bilayer coating technology developed for intracellular delivery and labelling has synthesized Cd-free quantum dots possessing high brightness and multi-fluorescence successfully. Then, polymer coating and ligand exchange transfer to water-soluble materials to produce liposome nanomaterials as fluorescent probes and enhancing medical applications of nanotechnology.

A highly-sensitive label-free biosensor based on two dimensional photonic crystals with negative refraction

NASA Astrophysics Data System (ADS)

Malmir, Narges; Fasihi, Kiazand

2017-11-01

In this work, we present a novel high-sensitive optical label-free biosensor based on a two-dimensional photonic crystal (2D PC). The suggested structure is composed of a negative refraction structure in a hexagonal lattice PC, along with a positive refraction structure which is arranged in a square lattice PC. The frequency shift of the transmission peak is measured respect to the changes of refractive indices of the studied materials (the blood plasma, water, dry air and normal air). The studied materials are filled into a W1 line-defect waveguide which is located in the PC structure with positive refraction (the microfluidic nanochannel). Our numerical simulations, which are based on finite-difference time-domain (FDTD) method, show that in the proposed structure, a sensitivity about 1100 nm/RIU and a transmission efficiency more than 75% can be achieved. With this design, to the best of our knowledge, the obtained sensitivity and the transmission efficiency are one of the highest values in the reported PC label-free biosensors.

Photocatalytic silver enhancement reaction for gravimetric immunosensors.

PubMed

Seo, Hyejung; Joo, Jinmyoung; Ko, Wooree; Jung, Namchul; Jeon, Sangmin

2010-12-17

A novel microgravimetric immunosensor has been developed using TiO(2) nanoparticle-modified immunoassay and silver enhancement reaction. An antibody-conjugated TiO(2) nanoparticle is bound to the AFP antigen immobilized on a quartz resonator. When the nanoparticles are exposed to UV light in a silver nitrate solution, the photocatalytic reduction of silver ions results in the formation of metallic silver onto the nanoparticles and induces a decrease in the resonance frequency. The frequency change by this photocatalytic reduction reaction is three orders of magnitude larger than the change by antigen binding alone. The efficiency of the photocatalytic reaction has been found to increase with the fraction of anatase crystallites in the nanoparticles and the concentration of the AgNO(3) solution. The results highlight the potential of the photocatalytic nanoparticles for the detection of low concentrations of target molecules using gravimetric sensors.

Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

PubMed

Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

2017-10-03

Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

Quantum Cascade Laser-Based Infrared Microscopy for Label-Free and Automated Cancer Classification in Tissue Sections.

PubMed

Kuepper, Claus; Kallenbach-Thieltges, Angela; Juette, Hendrik; Tannapfel, Andrea; Großerueschkamp, Frederik; Gerwert, Klaus

2018-05-16

A feasibility study using a quantum cascade laser-based infrared microscope for the rapid and label-free classification of colorectal cancer tissues is presented. Infrared imaging is a reliable, robust, automated, and operator-independent tissue classification method that has been used for differential classification of tissue thin sections identifying tumorous regions. However, long acquisition time by the so far used FT-IR-based microscopes hampered the clinical translation of this technique. Here, the used quantum cascade laser-based microscope provides now infrared images for precise tissue classification within few minutes. We analyzed 110 patients with UICC-Stage II and III colorectal cancer, showing 96% sensitivity and 100% specificity of this label-free method as compared to histopathology, the gold standard in routine clinical diagnostics. The main hurdle for the clinical translation of IR-Imaging is overcome now by the short acquisition time for high quality diagnostic images, which is in the same time range as frozen sections by pathologists.

An Electrochemical Immunosensor for Detection of Staphylococcus aureus Bacteria Based on Immobilization of Antibodies on Self-Assembled Monolayers-Functionalized Gold Electrode

PubMed Central

Braiek, Mohamed; Rokbani, Karima Bekir; Chrouda, Amani; Mrabet, Béchir; Bakhrouf, Amina; Maaref, Abderrazak; Jaffrezic-Renault, Nicole

2012-01-01

The detection of pathogenic bacteria remains a challenge for the struggle against biological weapons, nosocomial diseases, and for food safety. In this research, our aim was to develop an easy-to-use electrochemical immunosensor for the detection of pathogenic Staphylococcus aureus ATCC25923. The biosensor was elaborated by the immobilization of anti-S. aureus antibodies using a self-assembled monolayer (SAMs) of 3-Mercaptopropionic acid (MPA). These molecular assemblies were spontaneously formed by the immersion of the substrate in an organic solvent containing the SAMs that can covalently bond to the gold surface. The functionalization of the immunosensor was characterized using two electrochemical techniques: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Here, the analysis was performed in phosphate buffer with ferro/ferricyanide as the redox probe. The EIS technique was used for affinity assays: antibody-cell binding. A linear relationship between the increment in the electron transfer resistance (RCT) and the logarithmic value of S. aureus concentration was observed between 10 and 106 CFU/mL. The limit of detection (LOD) was observed at 10 CFU/mL, and the reproducibility was calculated to 8%. Finally, a good selectivity versus E. coli and S. epidermidis was obtained for our developed immunosensor demonstrating its specificity towards only S. aureus. PMID:25586032

An Electrochemical Immunosensor for Detection of Staphylococcus aureus Bacteria Based on Immobilization of Antibodies on Self-Assembled Monolayers-Functionalized Gold Electrode.

PubMed

Braiek, Mohamed; Rokbani, Karima Bekir; Chrouda, Amani; Mrabet, Béchir; Bakhrouf, Amina; Maaref, Abderrazak; Jaffrezic-Renault, Nicole

2012-10-16

The detection of pathogenic bacteria remains a challenge for the struggle against biological weapons, nosocomial diseases, and for food safety. In this research, our aim was to develop an easy-to-use electrochemical immunosensor for the detection of pathogenic Staphylococcus aureus ATCC25923. The biosensor was elaborated by the immobilization of anti-S. aureus antibodies using a self-assembled monolayer (SAMs) of 3-Mercaptopropionic acid (MPA). These molecular assemblies were spontaneously formed by the immersion of the substrate in an organic solvent containing the SAMs that can covalently bond to the gold surface. The functionalization of the immunosensor was characterized using two electrochemical techniques: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Here, the analysis was performed in phosphate buffer with ferro/ferricyanide as the redox probe. The EIS technique was used for affinity assays: antibody-cell binding. A linear relationship between the increment in the electron transfer resistance (RCT) and the logarithmic value of S. aureus concentration was observed between 10 and 106 CFU/mL. The limit of detection (LOD) was observed at 10 CFU/mL, and the reproducibility was calculated to 8%. Finally, a good selectivity versus E. coli and S. epidermidis was obtained for our developed immunosensor demonstrating its specificity towards only S. aureus.

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

PubMed

Sjödin, Marcus O D; Wetterhall, Magnus; Kultima, Kim; Artemenko, Konstantin

2013-06-01

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar. Copyright © 2013 Elsevier B.V. All rights reserved.

Amperometric biosensor based on glassy carbon electrode modified with long-length carbon nanotube and enzyme

NASA Astrophysics Data System (ADS)

Furutaka, Hajime; Nemoto, Kentaro; Inoue, Yuki; Hidaka, Hiroki; Muguruma, Hitoshi; Inoue, Hitoshi; Ohsawa, Tatsuya

2016-05-01

An amperometric biosensor based on a glassy carbon electrode modified with long-length multiwalled carbon nanotubes (MWCNTs) and enzyme nicotinamide-adenine-dinucleotide-dependent glucose dehydrogenase (GDH) is presented. We demonstrate the effect of the MWCNT length on the amperometric response of the enzyme biosensor. The long length of MWCNT is 200 µm (average), whereas the normal length of MWCNT is 1 µm (average). The response of the long MWCNT-GDH electrode is 2 times more sensitive than that of the normal-length MWCNT-GDH electrode in the concentration range from 0.25-35 mM. The result of electrochemical impedance spectroscopy measurements suggest that the long-length MWCNT-GDH electrode formed a better electron transfer network than the normal-length one.

Label-free characterization of degenerative changes in articular cartilage by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Oshima, Yusuke; Akehi, Mayu; Kiyomatsu, Hiroshi; Miura, Hiromasa

2017-04-01

Osteoarthritis (OA) is very common joint disease in the aging population. Main symptom of OA is accompanied by degenerative changes of articular cartilage. Raman spectroscopy is a label-free technique which enables to analyze molecular composition in degenerative cartilage. We generated an animal OA model surgically induced by knee joint instability and performed Raman spectroscopic analysis for the articular cartilage. In the result, Raman spectral data of the articular cartilage showed drastic changes in comparison between OA and control side. The relative intensity of phosphate band increases in the degenerative cartilage.

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

PubMed

Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

2017-01-01

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

An Ultrasensitive Electrochemical Immunosensor for HIV p24 Based on Fe3O4@SiO2 Nanomagnetic Probes and Nanogold Colloid-Labeled Enzyme–Antibody Copolymer as Signal Tag

PubMed Central

Gan, Ning; Du, Xiaowen; Cao, Yuting; Hu, Futao; Li, Tianhua; Jiang, Qianli

2013-01-01

An ultrasensitive portable electrochemical immunosensor for human immunodeficiency virus p24 (HIV p24) antigen detection has been developed, whereby the detection sensitivity was 1000 times higher than that of the ELISA method. Firstly, a novel HRP enzyme–antibody copolymer (EV-p24 Ab2) was synthesized through an EnVision regent (EV, a dextrin amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of anti IgG), then incubated in the secondary antibody of p24. Secondly, the copolymer was immobilized on the gold nanocolloids (AuNPs) to fabricate a novel signal tag (AuNPs/EV-p24 Ab2). Subsequently, a sandwich-type immunoreaction would take place between the capture probe (silicon dioxide-coated magnetic Fe3O4 nanoparticles (MNPs) labeled with the primary p24 antibody (MNPs-p24 Ab1)), p24 (different concentrations) and the signal tag [AuNPs/EV-p24 Ab2)] to form the immunocomplex. Finally, the immunocomplex was absorbed on the surface of screen printed carbon electrode (SPCE) by a magnet and immersed in the o-hydroxyl phenol (HQ) and H2O2. The large amounts of HRP on the signal tag can catalyze the oxidation of HQ by H2O2, which can induce an amplified reductive current. Moreover, the capture probe could improve the accumulation ability of p24 and facilitate its separation from the substrate through the magnet. Under optimal conditions, the proposed immunoassay exhibited good sensitivity to p24 within a certain concentration range from 0.001 to 10.00 ng/mL, with a detection limit of 0.5 pg/mL (S/N = 3). The proposed method can be used for real-time and early detection of HIV-infected people. PMID:28809208

Label-free optical detection of action potential in mammalian neurons (Conference Presentation)

NASA Astrophysics Data System (ADS)

Batabyal, Subrata; Satpathy, Sarmishtha; Bui, Loan; Kim, Young-Tae; Mohanty, Samarendra K.; Davé, Digant P.

2017-02-01

Electrophysiology techniques are the gold standard in neuroscience for studying functionality of a single neuron to a complex neuronal network. However, electrophysiology techniques are not flawless, they are invasive nature, procedures are cumbersome to implement with limited capability of being used as a high-throughput recording system. Also, long term studies of neuronal functionality with aid of electrophysiology is not feasible. Non-invasive stimulation and detection of neuronal electrical activity has been a long standing goal in neuroscience. Introduction of optogenetics has ushered in the era of non-invasive optical stimulation of neurons, which is revolutionizing neuroscience research. Optical detection of neuronal activity that is comparable to electro-physiology is still elusive. A number of optical techniques have been reported recording of neuronal electrical activity but none is capable of reliably measuring action potential spikes that is comparable to electro-physiology. Optical detection of action potential with voltage sensitive fluorescent reporters are potential alternatives to electrophysiology techniques. The heavily rely on secondary reporters, which are often toxic in nature with background fluorescence, with slow response and low SNR making them far from ideal. The detection of one shot (without averaging)-single action potential in a true label-free way has been elusive so far. In this report, we demonstrate the optical detection of single neuronal spike in a cultured mammalian neuronal network without using any exogenous labels. To the best of our knowledge, this is the first demonstration of label free optical detection of single action potentials in a mammalian neuronal network, which was achieved using a high-speed phase sensitive interferometer. We have carried out stimulation and inhibition of neuronal firing using Glutamate and Tetrodotoxin respectively to demonstrate the different outcome (stimulation and inhibition) revealed in

A facile route to glycated albumin detection.

PubMed

Bohli, Nadra; Meilhac, Olivier; Rondeau, Philippe; Gueffrache, Syrine; Mora, Laurence; Abdelghani, Adnane

2018-07-01

In this paper we propose an easy way to detect the glycated form of human serum albumin which is biomarker for several diseases such as diabetes and Alzheimer. The detection platform is a label free impedimetric immunosensor, in which we used a monoclonal human serum albumin antibody as a bioreceptor and electrochemical impedance as a transducing method. The antibody was deposited onto a gold surface by simple physisorption technique. Bovine serum albumin was used as a blocking agent for non-specific binding interactions. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of each layer. Human serum albumin was glycated at different levels with several concentrations of glucose ranging from 0 mM to 500 mM representing physiological, pathological (diabetic albumin) and suprapathological concentration of glucose. Through the calibration curves, we could clearly distinguish between two different areas related to physiological and pathological albumin glycation levels. The immunosensor displayed a linear range from 7.49% to 15.79% of glycated albumin to total albumin with a good sensitivity. Surface plasmon resonance imaging was also used to characterize the developed immunosensor. Copyright © 2018 Elsevier B.V. All rights reserved.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

NASA Astrophysics Data System (ADS)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

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

Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.

2013-02-01

Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred frommore » one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.« less

Mechanism of amperometric biosensor with electronic-type-controlled carbon nanotube

NASA Astrophysics Data System (ADS)

Hidaka, Hiroki; Nowaki, Kohei; Muguruma, Hitoshi

2016-03-01

An amperometric enzyme biosensor with electronic-type-controlled (metallic and semiconducting) single-walled carbon nanotubes (CNTs) is presented. In this research, we investigate how the electronic types of CNTs influence the amperometric response of enzyme biosensors and what their working mechanisms are. The biosensor of interest is for glucose detection using enzyme glucose oxidase (GOD). In the presence of oxygen, the response of a metallic CNT-GOD electrode was 2.5 times more sensitive than that of a semiconducting CNT-GOD electrode. In contrast, in the absence of oxygen, the response of the semiconducting CNT-GOD electrode was retained, whereas that of the metallic CNT-GOD electrode was significantly reduced. This indicates that direct electron transfer occurred with the semiconducting CNT-GOD electrode, whereas the metallic CNT-GOD electrode was dominated by a hydrogen peroxide pathway caused by an enzymatic reaction. Electrochemical impedance spectroscopy was used to show that the semiconducting CNT network has less resistance for electron transfer than the metallic CNT network. The optimized glucose biosensor revealed a sensitivity of 5.6 µA mM-1 cm-2 at +0.6 V vs Ag/AgCl, a linear dynamic range of 0.025-1.4 mM, and a response time of 8 s.

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

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

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

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

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

Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

PubMed

Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

2014-09-01

A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging

PubMed Central

2014-01-01

The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054. PMID:25181601

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

On-line removal of redox-active interferents by a porous electrode before amperometric blood glucose determination.

PubMed

Deng, Chunyan; Peng, Yong; Su, Lei; Liu, You-Nian; Zhou, Feimeng

2012-03-16

A porous reticulated vitreous carbon (RVC) electrode and a disk electrode coupled in tandem in an electrochemical flow cell has been used for electrolytic removal of interferents before amperometric glucose detection. The electrolytic efficiency at the upstream RVC electrode is 100% at a flow rate of 0.1 mL min(-1) or lower. Potential interferents such as acetaminophen, ascorbic acid, and uric acid can be completely eliminated by electrolysis at the RVC electrode. A mixed monolayer comprising glucose oxidase (GOD) and ferrocenyl-1-undecanethiol preformed at the downstream gold disk electrode was used as a mediator-based amperometric glucose sensor. The dependence of the amperometric current on the glucose concentration exhibits good linearity across over three orders of magnitude. The glucose measurements were also found to be reproducible (RSD<3.5%) and accurate. Unlike the chemiluminescence method, this device obviates the use of carcinogenic substrates and the glucose sensor performance is independent of the oxygen present in sample. On the basis that the RVC electrode requires minimal cleanup and the GOD-modified electrode remains stable for a week, the electrochemical flow cell should be amenable for automated on-line removal of redox interferents for other types of enzyme-based biosensors. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel self-enhanced electrochemiluminescence immunosensor based on hollow Ru-SiO2@PEI nanoparticles for NSE analysis.

PubMed

Zhou, Limin; Huang, Jianshe; Yu, Bin; You, Tianyan

2016-02-26

Poly(ethylenimine) (PEI) and Ru(bpy)3(2+)-doped silica (Ru-SiO2) nanoparticles were simply mixed together to prepare a novel self-enhanced electrochemiluminescence (ECL) composite of Ru-SiO2@PEI. The hollow Ru-SiO2@PEI nanoparticles were used to build an ECL immunosensor for the analysis of neuron specific enolase (NSE). PEI not only assembled on the surface of Ru-SiO2 nanoparticles through the electrostatic interaction to act as co-reactant for Ru(bpy)3(2+) ECL, but also provided alkaline condition to etch the Ru-SiO2 nanoparticles to form the hollow Ru-SiO2@PEI nanoparticles with porous shell. The unique structure of the Ru-SiO2@PEI nanoparticles loaded both a large amount of Ru(bpy)3(2+) and its co-reactant PEI at the same time, which shortened the electron-transfer distance, thereby greatly enhanced the luminous efficiency and amplified the ECL signal. The developed immunosensor showed a wide linear range from 1.0 × 10(-11) to 1.0 × 10(-5) mg mL(-1) with a low detection limit of 1.0 × 10(-11) mg mL(-1) for NSE. When the immunosensor was used for the determination of NSE in clinical human serum, the results were comparable with those obtained by using enzyme-linked immunosorbent assay (ELISA) method. The proposed method provides a promising alternative for NSE analysis in clinical samples.

Amperometric Enzyme-Based Biosensors for Application in Food and Beverage Industry

NASA Astrophysics Data System (ADS)

Csöoregi, Elisabeth; Gáspñr, Szilveszter; Niculescu, Mihaela; Mattiasson, Bo; Schuhmann, Wolfgang

Continuous, sensitive, selective, and reliable monitoring of a large variety of different compounds in various food and beverage samples is of increasing importance to assure a high-quality and tracing of any possible source of contamination of food and beverages. Most of the presently used classical analytical methods are often requiring expensive instrumentation, long analysis times and well-trained staff. Amperometric enzyme-based biosensors on the other hand have emerged in the last decade from basic science to useful tools with very promising application possibilities in food and beverage industry. Amperometric biosensors are in general highly selective, sensitive, relatively cheap, and easy to integrate into continuous analysis systems. A successful application of such sensors for industrial purposes, however, requires a sensor design, which satisfies the specific needs of monitoring the targeted analyte in the particular application, Since each individual application needs different operational conditions and sensor characteristics, it is obvious that biosensors have to be tailored for the particular case. The characteristics of the biosensors are depending on the used biorecognition element (enzyme), nature of signal transducer (electrode material) and the communication between these two elements (electron-transfer pathway).