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Sample records for dna biosensors fret

  1. Development of a Fluorescence Resonance Energy Transfer (FRET)-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense.

    Science.gov (United States)

    Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Abdul Halim; Hussein, Mohd Zobir

    2013-12-01

    An optical DNA biosensor based on fluorescence resonance energy transfer (FRET) utilizing synthesized quantum dot (QD) has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA) via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10(-9) M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

  2. Development of a Fluorescence Resonance Energy Transfer (FRET-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

    Directory of Open Access Journals (Sweden)

    Noremylia Mohd Bakhori

    2013-12-01

    Full Text Available An optical DNA biosensor based on fluorescence resonance energy transfer (FRET utilizing synthesized quantum dot (QD has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

  3. A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection.

    Directory of Open Access Journals (Sweden)

    Smarajit Chakraborty

    2015-04-01

    Full Text Available In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein and a response regulator (usually a DNA binding protein. The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor ("I-switch" to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2. Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a

  4. Development of FRET biosensors for mammalian and plant systems

    NARCIS (Netherlands)

    Hamers, D.; van Voorst Vader, L.; Borst, J.W.; Goedhart, J.

    2014-01-01

    Genetically encoded biosensors are increasingly used in visualising signalling processes in different organisms. Sensors based on green fluorescent protein technology are providing a great opportunity for using Forster resonance energy transfer (FRET) as a tool that allows for monitoring dynamic

  5. NIR FRET Fluorophores for Use as an Implantable Glucose Biosensor

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    Majed DWEIK

    2008-12-01

    Full Text Available Development of an in vivo optical sensor requires the utilization of Near Infra Red (NIR fluorophores due to their ability to operate within the biological tissue window. Alexa Fluor 750 (AF750 and Alexa Fluor 680 (AF680 were examined as potential NIR fluorophores for an in vivo fluorescence resonance energy transfer (FRET glucose biosensor. AF680 and AF750 found to be a FRET pair and percent energy transfer was calculated. Next, the tested dye pair was utilized in a competitive binding assay in order to detect glucose. Concanavalin A (Con A and dextran have binding affinity, but in the presence of glucose, glucose displaces dextran due to its higher affinity to Con A than dextran. Finally, the percent signal transfer through porcine skin was examined. The results showed with approximately 4.0 mm porcine skin thickness, 1.98 % of the fluorescence was transmitted and captured by the detector.

  6. Mining the Sinorhizobium meliloti transportome to develop FRET biosensors for sugars, dicarboxylates and cyclic polyols.

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    Alexandre Bourdès

    Full Text Available Förster resonance energy transfer (FRET biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens. To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.Two new vectors were developed for cloning genes for solute-binding proteins (SBPs between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2 and red fluorescent protein (mKate2 FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose, D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate. To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP transport systems.FRET based on orange (mOrange2 and red fluorescent protein (mKate2 partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i cyclic polyols, (ii L-deoxy sugars, (iii β-linked disaccharides and (iv C4-dicarboxylates could be developed to study metabolism in vivo.

  7. DNA nanotechnology-enabled biosensors.

    Science.gov (United States)

    Chao, Jie; Zhu, Dan; Zhang, Yinan; Wang, Lianhui; Fan, Chunhai

    2016-02-15

    Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. DNA-based biosensors, as a sub-field to biosensor, utilize DNA strands with short oligonucleotides as probes for target recognition. Although DNA-based biosensors have offered a promising alternative for fast, simple and cheap detection of target molecules, there still exist key challenges including poor stability and reproducibility that hinder their competition with the current gold standard for DNA assays. By exploiting the self-recognition properties of DNA molecules, researchers have dedicated to make versatile DNA nanostructures in a highly rigid, controllable and functionalized manner, which offers unprecedented opportunities for developing DNA-based biosensors. In this review, we will briefly introduce the recent advances on design and fabrication of static and dynamic DNA nanostructures, and summarize their applications for fabrication and functionalization of DNA-based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells.

    Science.gov (United States)

    Elliott, Amicia D; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A; Davidson, Michael W; Tkaczyk, Tomasz; Piston, David W

    2017-01-01

    Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

  9. Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

    Science.gov (United States)

    Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

    2018-04-19

    Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Biosensors for DNA sequence detection

    Science.gov (United States)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  11. A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

    Directory of Open Access Journals (Sweden)

    Victoria Steffen

    2016-09-01

    Full Text Available Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP, Citrine. Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

  12. Studying DNA looping by single-molecule FRET.

    Science.gov (United States)

    Le, Tung T; Kim, Harold D

    2014-06-28

    Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

  13. Electroacoustic miniaturized DNA-biosensor.

    Science.gov (United States)

    Gamby, Jean; Lazerges, Mathieu; Pernelle, Christine; Perrot, Hubert; Girault, Hubert H; Tribollet, Bernard

    2007-11-01

    A micrometer-sized electroacoustic DNA-biosensor was developed. The device included a thin semi-crystalline polyethylene terephthalate (PET) dielectric layer with two Ag microband electrodes on one side and a DNA thiol-labeled monolayer adsorbed on a gold surface on the other. A resonance wave was observed at 29 MHz with a network analyzer, upon AC voltage application between the two Ag electrodes, corresponding to electromechanical coupling induced by molecular dipoles of the PET polymer chain in the dielectric layer. It was found that the device size and geometry were well adapted to detect DNA hybridization, by measuring the capacity of the resonance response evolution: hybridization induced polarization of the dielectric material that affected the electromechanical coupling established in the dielectric layer. The 0.2 mm(2) sensor sensitive area allows detection in small volumes and still has higher detection levels for bioanalytical applications, the non-contact configuration adopted avoids electric faradic reactions that may damage biosensor sensitive layers, and finally, PET is a costless raw material, easy to process and well adapted for large scale production. The well-balanced technological and economic advantages of this kind of device make it a good candidate for biochip integration.

  14. SH2 Domain-Based FRET Biosensor for Measuring BCR-ABL Activity in Living CML Cells.

    Science.gov (United States)

    Fujioka, Mari; Asano, Yumi; Nakada, Shigeyuki; Ohba, Yusuke

    2017-01-01

    Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.

  15. A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water-Soluble Blue Light Emitting Polymer

    Directory of Open Access Journals (Sweden)

    Nidhi Mathur

    2008-01-01

    Full Text Available A reliable, fast, and low-cost biosensor for medical diagnostics using DNA sequence detection has been developed and tested for the detection of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium hexylfluorenyl-2, 7-diyl-alt-co- (1,4-phenylene] dibromide salt (PFP has been taken as cationic conjugated polymer (CCP and PNA attached with fluorescein dye (PNAC∗ as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA having complementary sequence, Forster resonance energy transfer (FRET may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.

  16. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

    Directory of Open Access Journals (Sweden)

    Potzkei Janko

    2012-03-01

    Full Text Available Abstract Background Molecular oxygen (O2 is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results We developed a genetically encoded Förster resonance energy transfer (FRET-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen consists of the yellow fluorescent protein (YFP that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP. Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (pathophysiological processes in living cells.

  17. Polyfluorophore Excimers and Exciplexes as FRET Donors in DNA

    Science.gov (United States)

    Teo, Yin Nah; Kool, Eric T.

    2009-01-01

    We describe studies aimed at testing whether oligomeric exciplex- and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Förster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3’ end of a 14mer DNA probe sequence. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Förster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single

  18. FRET-based biosensors for the detection and quantification of AI-2 class of quorum sensing compounds.

    Science.gov (United States)

    Rajamani, Sathish; Sayre, Richard

    2011-01-01

    Intercellular small molecular weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum sensing molecules. These molecules mediate a variety of population-dependent responses, including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules has important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. A set of ligand-insensitive LuxP-mutant FRET protein sensor was also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of

  19. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    Science.gov (United States)

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

  20. New Catalytic DNA Biosensors for Radionuclides and Metal ions

    International Nuclear Information System (INIS)

    Lu, Yi

    2003-01-01

    The goals of the project are to develop new catalytic DNA biosensors for simultaneous detection and quantification of bioavailable radionuclides and metal ions, and apply the sensors for on-site, real-time assessment of concentration, speciation and stability of the individual contaminants during and after bioremediation. A negative selection strategy was tested and validated. In vitro selection was shown to yield highly active and specific transition metal ion-dependent catalytic DNA/RNA. A fluorescence resonance energy transfer (FRET) study of in vitro selected DNA demonstrated that the trifluorophore labeled system is a simple and powerful tool in studying complex biomolecules structure and dynamics, and is capable of revealing new sophisticated structural changes. New fluorophore/quenchers in a single fluorosensor yielded improved signal to noise ratio in detection, identification and quantification of metal contaminants. Catalytic DNA fluorescent and colorimetric sensors were shown useful in sensing lead in lake water and in leaded paint. Project results were described in two papers and two patents, and won an international prize

  1. FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

    Science.gov (United States)

    This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The...

  2. Disposable electrochemical DNA biosensor for environmental ...

    Indian Academy of Sciences (India)

    been used due to its rapid, easy handling and cost effective responses for the toxicity assessment in real water ... in the application of DNA as biosensors as it is found ... used as a preclinical safety assessment tool to screen ... out the work.

  3. Recent Development of Nano-Materials Used in DNA Biosensors

    Directory of Open Access Journals (Sweden)

    Yibin Ying

    2009-07-01

    Full Text Available As knowledge of the structure and function of nucleic acid molecules has increased, sequence-specific DNA detection has gained increased importance. DNA biosensors based on nucleic acid hybridization have been actively developed because of their specificity, speed, portability, and low cost. Recently, there has been considerable interest in using nano-materials for DNA biosensors. Because of their high surface-to-volume ratios and excellent biological compatibilities, nano-materials could be used to increase the amount of DNA immobilization; moreover, DNA bound to nano-materials can maintain its biological activity. Alternatively, signal amplification by labeling a targeted analyte with nano-materials has also been reported for DNA biosensors in many papers. This review summarizes the applications of various nano-materials for DNA biosensors during past five years. We found that nano-materials of small sizes were advantageous as substrates for DNA attachment or as labels for signal amplification; and use of two or more types of nano-materials in the biosensors could improve their overall quality and to overcome the deficiencies of the individual nano-components. Most current DNA biosensors require the use of polymerase chain reaction (PCR in their protocols. However, further development of nano-materials with smaller size and/or with improved biological and chemical properties would substantially enhance the accuracy, selectivity and sensitivity of DNA biosensors. Thus, DNA biosensors without PCR amplification may become a reality in the foreseeable future.

  4. Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET

    NARCIS (Netherlands)

    Hohlbein, J.; Kapanidis, A.N.

    2016-01-01

    Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open

  5. DNA Nanotechnology-Enabled Interfacial Engineering for Biosensor Development.

    Science.gov (United States)

    Ye, Dekai; Zuo, Xiaolei; Fan, Chunhai

    2018-06-12

    Biosensors represent biomimetic analytical tools for addressing increasing needs in medical diagnosis, environmental monitoring, security, and biodefense. Nevertheless, widespread real-world applications of biosensors remain challenging due to limitations of performance, including sensitivity, specificity, speed, and reproducibility. In this review, we present a DNA nanotechnology-enabled interfacial engineering approach for improving the performance of biosensors. We first introduce the main challenges of the biosensing interfaces, especially under the context of controlling the DNA interfacial assembly. We then summarize recent progress in DNA nanotechnology and efforts to harness DNA nanostructures to engineer various biological interfaces, with a particular focus on the use of framework nucleic acids. We also discuss the implementation of biosensors to detect physiologically relevant nucleic acids, proteins, small molecules, ions, and other biomarkers. This review highlights promising applications of DNA nanotechnology in interfacial engineering for biosensors and related areas.

  6. Sub-ensemble monitoring of DNA strand displacement using multiparameter single-molecule FRET

    OpenAIRE

    Baltierra Jasso, Laura; Morten, Michael; Magennis, Steven William

    2018-01-01

    Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constan...

  7. Innovative configurations of electrochemical DNA biosensors (a review)

    OpenAIRE

    Girousi, Stella; Karastogianni, Sofia; Serpi, Constantina

    2011-01-01

    In the field of electrochemical biosensing, transition metal complexes achieved a significant importance as hybridization indicators or electroactive markers of DNA. Their incorporation in electro-chemical DNA biosensors enables to offer a promising perspective in understanding of the biological activity of some chemical compounds. In this context, the development of innovative configurations of electrochemical DNA biosensors applied to life sciences during the last years were reviewed ...

  8. Branched DNA nanostructures efficiently stabilised and monitored by novel pyrene-perylene 2'-α-l-amino-LNA FRET pairs

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Santhosh Kumar, T; Campbell, Meghan A

    2013-01-01

    Novel pyrene-perylene α-l-LNA FRET pairs described herein effectively detect assembly of 2- and 3-way branched DNA nanostructures prepared by postsynthetic microwave-assisted CuAAC click chemistry. The fluorescent signalling of assembly by internally positioned FRET pairs is achieved with low...

  9. Decoding spatial and temporal features of neuronal cAMP/PKA signaling with FRET biosensors.

    Science.gov (United States)

    Castro, Liliana R V; Guiot, Elvire; Polito, Marina; Paupardin-Tritsch, Daniéle; Vincent, Pierre

    2014-02-01

    Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Indicator Based and Indicator - Free Electrochemical DNA Biosensors

    National Research Council Canada - National Science Library

    Kerman, Kagan

    2001-01-01

    The utility and advantages of an indicator free and MB based sequence specific DNA hybridization biosensor based on guanine and adenine oxidation signals and MB reduction signals have been demonstrated...

  11. Studying DNA Looping by Single-Molecule FRET

    OpenAIRE

    Le, Tung T.; Kim, Harold D.

    2014-01-01

    Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can als...

  12. Electroporation and microinjection successfully deliver single-stranded and duplex DNA into live cells as detected by FRET measurements.

    Directory of Open Access Journals (Sweden)

    Rosemary A Bamford

    Full Text Available Förster resonance energy transfer (FRET technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5 complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

  13. DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

    Science.gov (United States)

    Wang, Mengke; Lin, Zihan; Liu, Qing; Jiang, Shan; Liu, Hua; Su, Xingguang

    2018-07-05

    A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL -1 with a detection limit (LOD) of 0.039 U mL -1 . Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts

    Directory of Open Access Journals (Sweden)

    Max Nobis

    2017-10-01

    Full Text Available The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

  15. Ratiometric FRET-based detection of DNA and micro-RNA in solution

    International Nuclear Information System (INIS)

    Matveeva, Evgenia G.; Gryczynski, Zygmunt; Stewart, Donald R.; Gryczynski, Ignacy

    2009-01-01

    A ratiometric method for detecting DNA oligomers in bulk solution based on Foerster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).

  16. FRET enhancement close to gold nanoparticles positioned in DNA origami constructs.

    Science.gov (United States)

    Aissaoui, Nesrine; Moth-Poulsen, Kasper; Käll, Mikael; Johansson, Peter; Wilhelmsson, L Marcus; Albinsson, Bo

    2017-01-05

    Here we investigate the energy transfer rates of a Förster resonance energy transfer (FRET) pair positioned in close proximity to a 5 nm gold nanoparticle (AuNP) on a DNA origami construct. We study the distance dependence of the FRET rate by varying the location of the donor molecule, D, relative to the AuNP while maintaining a fixed location of the acceptor molecule, A. The presence of the AuNP induces an alteration in the spontaneous emission of the donor (including radiative and non-radiative rates) which is strongly dependent on the distance between the donor and AuNP surface. Simultaneously, the energy transfer rates are enhanced at shorter D-A (and D-AuNP) distances. Overall, in addition to the direct influence of the acceptor and AuNP on the donor decay there is also a significant increase in decay rate not explained by the sum of the two interactions. This leads to enhanced energy transfer between donor and acceptor in the presence of a 5 nm AuNP. We also demonstrate that the transfer rate in the three "particle" geometry (D + A + AuNP) depends approximately linearly on the transfer rate in the donor-AuNP system, suggesting the possibility to control FRET process with electric field induced by 5 nm AuNPs close to the donor fluorophore. It is concluded that DNA origami is a very versatile platform for studying interactions between molecules and plasmonic nanoparticles in general and FRET enhancement in particular.

  17. FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca(2+)/PKA crosstalk.

    Science.gov (United States)

    Schott, Micah B; Gonowolo, Faith; Maliske, Benjamin; Grove, Bryon

    2016-04-01

    Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca(2+)]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium "switch" that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca(2+) and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca(2+)/PKA crosstalk was not observed in cells expressing a gravin mutant that resisted calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca(2+) and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may

  18. FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

    Science.gov (United States)

    Schott, Micah; Gonowolo, Faith; Maliske, Ben; Grove, Bryon

    2016-01-01

    Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca2+]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium “switch” that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca2+ and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca2+/PKA crosstalk was not observed in cells expressing a gravin mutant that resists calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream β-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca2+ and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may

  19. A fluorescence resonance energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) for the detection of mecA gene sequence of Staphylococcus aureus.

    Science.gov (United States)

    Shi, Jingyu; Chan, Chunyu; Pang, Yukting; Ye, Weiwei; Tian, Feng; Lyu, Jing; Zhang, Yu; Yang, Mo

    2015-05-15

    In this work, a novel fluorescence resonance energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) pairs was developed for Staphylococcus aureus specific gene sequence detection. This FRET biosensor platform was realized by immobilization of capture probes on GQDs and conjugation of reporter probes on AuNPs. Target oligos then co-hybridized with capture probes and reporter probes to form a sandwich structure which brought GQDs and AuNPs to close proximity to trigger FRET effect. The fluorescence signals before and after addition of targets were measured and the fluorescence quenching efficiency could reach around 87% with 100 nM target oligo. The limit of detection (LOD) of this FRET biosensor was around 1 nM for S.aureus gene detection. Experiments with both single-base mismatched oligos and double-base mismatched oligos demonstrated the good sequence selectivity of this FRET biosensor. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

    Science.gov (United States)

    Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

    2018-03-05

    Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Development of an electrochemical DNA biosensor for detection of ...

    Indian Academy of Sciences (India)

    2.4 million of deaths.1,2 Southern hybridization tech- niques, radiographic .... Electrochemical DNA sensors can be greatly affected .... 3.5 Diagnostic performance of the biosensor ... Silva M M S, Cavalcanti I T, Barroso M F, Sales M G F.

  2. DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

    Science.gov (United States)

    A rapid and sensitive fiber optic biosensor assay for radiation-induced DNA damage is reported. For this assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed...

  3. Biosensors and environmental health

    National Research Council Canada - National Science Library

    Preedy, Victor R; Patel, Vinood B

    2012-01-01

    ..., bacterial biosensors, antibody-based biosensors, enzymatic, amperometric and electrochemical aspects, quorum sensing, DNA-biosensors, cantilever biosensors, bioluminescence and other methods and applications...

  4. Multicolor fluorescent biosensor for multiplexed detection of DNA.

    Science.gov (United States)

    Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Huan, Shuang-Yan; Wu, Cuichen; Fu, Ting; Tan, Weihong

    2014-05-20

    Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

  5. Building an aptamer/graphene oxide FRET biosensor for one-step detection of bisphenol A.

    Science.gov (United States)

    Zhu, Yingyue; Cai, Yilin; Xu, Liguang; Zheng, Lixue; Wang, Limei; Qi, Bin; Xu, Chuanlai

    2015-04-15

    Bisphenol A (BPA) is an important industrial chemical for polycarbonate (PC) and epoxy resins in paper and plastic industries. In our work, a kind of new method for detection of BPA was designed based on graphene oxide and anti-BPA aptamer. The graphene oxide can specifically adsorb and quench the fluorescence of fluorescently modified ssDNA probes. Meanwhile, the BPA can combine with anti-BPA optamer and switch its configuration to prevent the aptamer from adsorbing on the surface of graphene oxide (GO). Under different concentrations of BPA, based on the target-induced conformational change of anti-BPA aptamer and the interactions between the fluorescently modified anti-BPA aptamer (FAM-ssDNA) and GO, the experimental results show that the intensity of the fluorescence signal was changed. A low limit of detection of 0.05 ng/mL was obtained in the range 0.1-10 ng/mL. In addition, the specificity was outstanding among analogues of BPA. The recovery rate in actual water samples spiked with BPA can be 96.0% to 104.5%. The developed method was successfully used to determine BPA in actual water samples.

  6. Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

    International Nuclear Information System (INIS)

    Liu, P.; Hun, X.; Qing, H.

    2011-01-01

    We report on a highly sensitive chemiluminescent (CL) biosensor for the sequence-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H 2 O 2 and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L -1 of target DNA. (author)

  7. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina

    2017-01-01

    specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection...

  8. Electrochemical DNA biosensors based on platinum nanoparticles combined carbon nanotubes

    International Nuclear Information System (INIS)

    Zhu Ningning; Chang Zhu; He Pingang; Fang Yuzhi

    2005-01-01

    Platinum nanoparticles were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced electrochemical DNA biosensor. Multi-walled carbon nanotubes and platinum nanoparticles were dispersed in Nafion, which were used to fabricate the modification of the glassy carbon electrode (GCE) surface. Oligonucleotides with amino groups at the 5' end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated daunomycin. Due to the ability of carbon nanotubes to promote electron-transfer reactions, the high catalytic activities of platinum nanoparticles for chemical reactions, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.0 x 10 -11 mol l -1

  9. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET

    Directory of Open Access Journals (Sweden)

    Mengyi Yang

    2018-01-01

    Full Text Available Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing

  10. DNA biosensor by self-assembly of carbon nanotubes and DNA to detect riboflavin

    Energy Technology Data Exchange (ETDEWEB)

    Li Jing [College of Chemistry and Chemical Engineering. Chongqing University, ChongQing, 400044 (China); Zhang Yunhuai, E-mail: xp2031@163.com [College of Chemistry and Chemical Engineering. Chongqing University, ChongQing, 400044 (China); Yang Tongyi [School of Life Science. NanJing University, Nanjing, 210093 (China); Zhang Huai [Liming Research Institute of Chemical Industry, LuoYang, 471001 (China); Yang Yixuan [State Key Laboratory of Chemical Resource Engineering. Beijing University of Chemical Technology, Beijing 100029 (China); Xiao Peng [College of Mathematics and Physics, Chongqing University, Chongqing 400044 (China)

    2009-10-15

    The fabrication of biosensors via self-assembly of single-walled carbon nanotubes (SWNTs) and DNA on a platinum electrode was presented in this paper. The carboxylic SWNTs were assembled on an amine-modified platinum electrode surface and followed by the assembly of NH{sub 2}-DNA with the carboxyl-amine coupling. The decorated surface was characterized by Field Emission Electron Microscopy (FEG-SEM) and electrochemical experiments, which showed that the reaction of DNA-SWNTs biosensor was quasi-reversible. The mechanism of DNA and riboflavin (VB{sub 2}) was studied by cyclic voltammetry and UV-Vis spectroscopy. The fabricated SWNTs-reinforced biosensor exhibits high sensitivity and low detection limit for the tested VB{sub 2} compared to the reported methods.

  11. A graphene oxide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin A (BoNT/A) enzymatic activity.

    Science.gov (United States)

    Shi, Jingyu; Guo, Jiubiao; Bai, Gongxun; Chan, Chunyu; Liu, Xuan; Ye, Weiwei; Hao, Jianhua; Chen, Sheng; Yang, Mo

    2015-03-15

    Botulinum neurotoxins (BoNTs) are among the most potent toxic bacterial proteins for humans, which make them potential agents for bioterrorism. Therefore, an ultrasensitive detection of BoNTs and their active states is in great need as field-deployable systems for anti-terrorism applications. We report the construction of a novel graphene oxide (GO)-peptide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of the BoNT serotype A light chain (BoNT-LcA) protease activity. A green fluorescence protein (GFP) modified SNAP-25 peptide substrate (SNAP-25-GFP) was optimally designed and synthesized with the centralized recognition/cleavage sites. This FRET platform was constructed by covalent immobilization of peptide substrate on GO with BSA passivation which have advantages of low non-specific adsorption and high stability in protein abundant solution. BoNT-LcA can specifically cleave SNAP-25-GFP substrate covalently immobilized on GO to release the fragment with GFP. Based on fluorescence signal recovery measurement, the target BoNT-LcA was detected sensitively and selectively with the linear detection range from 1fg/mL to 1pg/mL. The limit of detection (LOD) for BoNT-LcA is around 1fg/mL. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Effect of DNA type on response of DNA biosensor for carcinogens

    Science.gov (United States)

    Sani, Nor Diyana bt. Md.; Heng, Lee Yook; Surif, Salmijah; Lazim, Azwani Mat

    2013-11-01

    Carcinogens are cancer causing chemicals that can bind to DNA and cause damage to the DNA. These chemicals are available everywhere including in water, air, soil and food. Therefore, a sensor that can detect the presence of these chemicals will be a very useful tool. Since carcinogens bind to DNA, DNA can be used as the biological element in a biosensor. This study has utilized different types of DNA in a biosensor for carcinogen detection. The DNAs include double stranded calf thymus DNA, single stranded calf thymus DNA and guanine rich single stranded DNA. The modified SPE was exposed to a carcinogen followed by interaction with methylene blue which acts as the electroactive indicator. The SPE was then analysed using differential pulse voltammetry (DPV). Optimization studies were conducted for MB concentration and accumulation time, DNA concentration, as well as effect of buffer concentration, buffer pH and ionic strength. The performance of the biosensor was tested on a group 1 carcinogen, formaldehyde. The results indicated that the usage of guanine rich single stranded DNA also gives higher response as carcinogens prefer to bind with guanine compared to other bases.

  13. Application of Gold Nanoparticles for Electrochemical DNA Biosensor

    Directory of Open Access Journals (Sweden)

    Ahmed Mishaal Mohammed

    2014-01-01

    Full Text Available An electrochemical DNA biosensor was successfully fabricated by using (3-aminopropyltriethoxysilane (APTES as a linker molecule combined with the gold nanoparticles (GNPs on thermally oxidized SiO2 thin films. The SiO2 thin films surface was chemically modified with a mixture of APTES and GNPs for DNA detection in different time periods of 30 min, 1 hour, 2 hours, and 4 hours, respectively. The DNA immobilization and hybridization were conducted by measuring the differences of the capacitance value within the frequency range of 1 Hz to 1 MHz. The capacitance values for DNA immobilization were 160 μF, 77.8 μF, 70 μF, and 64.6 μF, respectively, with the period of time from 30 min to 4 hours. Meanwhile the capacitance values for DNA hybridization were 44 μF, 54 μF, 55 μF, and 61.5 μF, respectively. The capacitance value of bare SiO2 thin film was 0.42 μF, which was set as a base line for a reference in DNA detection. The differences of the capacitance value between the DNA immobilization and hybridization revealed that the modified SiO2 thin films using APTES and GNPs were successfully developed for DNA detection.

  14. Biosensors.

    Science.gov (United States)

    Rechnitz, Garry A.

    1988-01-01

    Describes theory and principles behind biosensors that incorporate biological components as part of a sensor or probe. Projects major applications in medicine and veterinary medicine, biotechnology, food and agriculture, environmental studies, and the military. Surveys current use of biosensors. (ML)

  15. Electrochemical DNA biosensor based on the BDD nanograss array electrode.

    Science.gov (United States)

    Jin, Huali; Wei, Min; Wang, Jinshui

    2013-04-10

    The development of DNA biosensor has attracted considerable attention due to their potential applications, including gene analysis, clinical diagnostics, forensic study and more medical applications. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry in this study. Electrochemical DNA biosensor was developed based on the BDD film electrode (fBDD) and BDD nanograss array electrode (nBDD). In comparison with fBDD and AuNPs/CA/fBDD electrode, the lower semicircle diameter of electrochemical impedance spectroscopy obtained on nBDD and AuNPs/CA/nBDD electrode indicated that the presence of nanograss array improved the reactive site, reduced the interfacial resistance, and made the electron transfer easier. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry. The experimental results demonstrated that the prepared AuNPs/CA/nBDD electrode was suitable for DNA hybridization with favorable performance of faster response, higher sensitivity, lower detection limit and satisfactory selectivity, reproducibility and stability.

  16. A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

    Science.gov (United States)

    Cox, Dianne; Hodgson, Louis

    2014-01-01

    Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

  17. Nanostructured ZnO-based biosensor: DNA immobilization and hybridization

    Directory of Open Access Journals (Sweden)

    Ahmed Mishaal Mohammed

    2017-09-01

    Full Text Available An electrochemical DNA biosensor was successfully fabricated by using (3-aminopropyl triethoxysilane (APTES with zinc oxide (ZnO nanorods synthesized using microwave-assisted chemical bath deposition method on thermally oxidized SiO2 thin films. The structural quality and morphology of the ZnO nanorods were determined by employing scanning electron microscopy (SEM and X-ray diffraction (XRD, which show a hexagonal wurtzite structure with a preferred orientation along the (101 direction. The surface of the SiO2 thin films was chemically modified with ZnO. Label-free detection DNA immobilization and hybridization were performed using potassium hexacyanoferrate with cyclic voltammetry (CV measurements. The capacitance, permittivity, and conductivity profiles of the fabricated sensor clearly indicate DNA immobilization and hybridization. Results show that the capacitance values of bare, ZnO- modified surface immobilization, and target DNA hybridization were 46×10−12F, 47×10−8F, 27μF, and 17μF, respectively, at 1Hz. The permittivity measurement increased from 3.94×103 to 251×103 and 165×103 at the frequency range of approximately 200 to 1Hz for bare and DNA immobilization and hybridization, respectively. The measured conductivity values for the bare, ZnO, immobilized, and hybridization device were 2.4×10−9, 10×10−8, 1.6×10−7, and 1.3×10−7Scm−1, respectively. Keywords: Zinc oxide, Biosensor, Capacitance, Permittivity, Conductivity

  18. Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

    Science.gov (United States)

    Chung, Da-Jung; Kim, Ki-Chul; Choi, Seong-Ho

    2011-09-01

    An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin-biotin conjugation of a biotinylated probe DNA, 5'-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3', and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5'-TTC GAC CTC GGT TAG AAG ACT CAT-3' and two-base mismatched DNA, 5'-TTC GAC AGC GGT TAT AAG ACT CAT-3'.

  19. Fluorescent carbon nanoparticle-based lateral flow biosensor for ultrasensitive detection of DNA.

    Science.gov (United States)

    Takalkar, Sunitha; Baryeh, Kwaku; Liu, Guodong

    2017-12-15

    We report a fluorescent carbon nanoparticle (FCN)-based lateral flow biosensor for ultrasensitive detection of DNA. Fluorescent carbon nanoparticle with a diameter of around 15nm was used as a tag to label a detection DNA probe, which was complementary with the part of target DNA. A capture DNA probe was immobilized on the test zone of the lateral flow biosensor. Sandwich-type hybridization reactions among the FCN-labeled DNA probe, target DNA and capture DNA probe were performed on the lateral flow biosensor. In the presence of target DNA, FCNs were captured on the test zone of the biosensor and the fluorescent intensity of the captured FCNs was measured with a portable fluorescent reader. After systematic optimizations of experimental parameters (the components of running buffers, the concentration of detection DNA probe used in the preparation of FCN-DNA conjugates, the amount of FCN-DNA dispensed on the conjugate pad and the dispensing cycles of the capture DNA probes on the test-zone), the biosensor could detect a minimum concentration of 0.4 fM DNA. This study provides a rapid and low-cost approach for DNA detection with high sensitivity, showing great promise for clinical application and biomedical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Electrochemical DNA biosensor based on grafting-to mode of terminal deoxynucleoside transferase-mediated extension.

    Science.gov (United States)

    Chen, Jinyuan; Liu, Zhoujie; Peng, Huaping; Zheng, Yanjie; Lin, Zhen; Liu, Ailin; Chen, Wei; Lin, Xinhua

    2017-12-15

    Previously reported electrochemical DNA biosensors based on in-situ polymerization approach reveal that terminal deoxynucleoside transferase (TdTase) has good amplifying performance and promising application in the design of electrochemical DNA biosensor. However, this method, in which the background is significantly affected by the amount of TdTase, suffers from being easy to produce false positive result and poor stability. Herein, we firstly present a novel electrochemical DNA biosensor based on grafting-to mode of TdTase-mediated extension, in which DNA targets are polymerized in homogeneous solution and then hybridized with DNA probes on BSA-based DNA carrier platform. It is surprising to find that the background in the grafting-to mode of TdTase-based electrochemical DNA biosensor have little interference from the employed TdTase. Most importantly, the proposed electrochemical DNA biosensor shows greatly improved detection performance over the in-situ polymerization approach-based electrochemical DNA biosensor. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Hall effect biosensors with ultraclean graphene film for improved sensitivity of label-free DNA detection

    KAUST Repository

    Loan, Phan Thi Kim; Wu, Dongqin; Ye, Chen; Li, Xiaoqing; Tra, Vu Thanh; Wei, Qiuping; Fu, Li; Yu, Aimin; Li, Lain-Jong; Lin, Cheng-Te

    2017-01-01

    The quality of graphene strongly affects the performance of graphene-based biosensors which are highly demanded for the sensitive and selective detection of biomolecules, such as DNA. This work reported a novel transfer process for preparing a

  2. Voltammetric Detection of Damage to DNA by Arsenic Compounds at a DNA Biosensor

    Directory of Open Access Journals (Sweden)

    R. Wennrich

    2005-11-01

    Full Text Available DNA biosensor can serve as a powerfull tool for simple in vitro tests of chemicaltoxicity. In this paper, damage to DNA attached to the surface of screen-printed carbonelectrode by arsenic compounds in solution is described. Using the Co(III complex with1,10-phenanthroline, [Co(phen3]3+ , as an electrochemical DNA marker and the Ru(IIcomplex with bipyridyne, [Ru(bipy3]2+ , as a DNA oxidation catalyst, the portion of originaldsDNA which survives an incubation of the biosensor in the cleavage medium was evaluated.The model cleavage mixture was composed of an arsenic compound at 10-3 mol/Lconcentration corresponding to real contaminated water, 2x10-4 mol/L Fe(II or Cu(II ions asthe redox catalyst, and 1.5x10-2 mol/L hydrogen peroxide. DNA damage by arsenite,dimethylarsinic acid as the metabolic product of inorganic arsenic and widely used herbicide,as well as phenylarsonic acid and p-arsanilic acid as the representatives of feed additives wasfound in difference to arsenate.

  3. One-step synthesis of DNA functionalized cadmium-free quantum dots and its application in FRET-based protein sensing

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Cuiling, E-mail: clzhang@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Ding, Caiping [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Zhou, Guohua [School of Chemistry and Chemical Engineering, Lingnan Normal University, Zhanjiang, 524048 (China); Xue, Qin [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China); Xian, Yuezhong, E-mail: yzxian@chem.ecnu.edu.cn [Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 (China)

    2017-03-08

    DNA functionalized quantum dots (QDs) are promising nanoprobes for the fluorescence resonance energy transfer (FRET)-based biosensing. Herein, cadmium-free DNA functionalized Mn-doped ZnS (DNA-ZnS:Mn{sup 2+}) QDs were successfully synthesized by one-step route. As-synthesized QDs show excellent photo-stability with the help of PAA and DNA. Then, we constructed a novel FRET model based on the QDs and WS{sub 2} nanosheets as the energy donor-acceptor pairs, which was successfully applied for the protein detection through the terminal protection of small molecule-linked DNA assay. This work not only explores the potential bioapplication of the DNA-ZnS:Mn{sup 2+} QDs, but also provides a platform for the investigation of small molecule-protein interaction. - Highlights: • The stable and cadmium-free DNA functionalized ZnS:Mn{sup 2+} QDs were successfully synthesized through a facile one-step route. • We constructed a novel FRET system based on one-step synthesized DNA-ZnS:Mn{sup 2+} QDs (donor) and WS{sub 2} nanosheets (acceptor). • The FRET-based strategy was applied for the detection of streptavidin and folate receptor by combining TPSMLD and Exo III.

  4. Label-free DNA biosensor based on resistance change of platinum nanoparticles assemblies.

    Science.gov (United States)

    Skotadis, Evangelos; Voutyras, Konstantinos; Chatzipetrou, Marianneza; Tsekenis, Georgios; Patsiouras, Lampros; Madianos, Leonidas; Chatzandroulis, Stavros; Zergioti, Ioanna; Tsoukalas, Dimitris

    2016-07-15

    A novel nanoparticle based biosensor for the fast and simple detection of DNA hybridization events is presented. The sensor utilizes hybridized DNA's charge transport properties, combining them with metallic nanoparticle networks that act as nano-gapped electrodes. The DNA hybridization events can be detected by a significant reduction in the sensor's resistance due to the conductive bridging offered by hybridized DNA. By modifying the nanoparticle surface coverage, which can be controlled experimentally being a function of deposition time, and the structural properties of the electrodes, an optimized biosensor for the in situ detection of DNA hybridization events is ultimately fabricated. The fabricated biosensor exhibits a wide response range, covering four orders of magnitude, a limit of detection of 1nM and can detect a single base pair mismatch between probe and complementary DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Determination of cDNA encoding BCR/ABL fusion gene in patients with chronic myelogenous leukemia using a novel FRET-based quantum dots-DNA nanosensor.

    Science.gov (United States)

    Shamsipur, Mojtaba; Nasirian, Vahid; Barati, Ali; Mansouri, Kamran; Vaisi-Raygani, Asad; Kashanian, Soheila

    2017-05-08

    In the present study, we developed a sensitive method based on fluorescence resonance energy transfer (FRET) for the determination of the BCR/ABL fusion gene, which is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). For this purpose, CdTe quantum dots (QDs) were conjugated to amino-modified 18-mer oligonucleotide ((N)DNA) to form the QDs-(N)DNA nanosensor. In the presence of methylene blue (MB) as an intercalator, the hybridization of QDs-(N)DNA with the target BCR/ABL fusion gene (complementary DNA), brings the MB (acceptor) at close proximity of the QDs (donor), leading to FRET upon photoexcitation of the QDs. The enhancement in the emission intensity of MB was used to follow up the hybridization, which was linearly proportional to concentration of the target complementary DNA in a range from 1.0 × 10 -9 to 1.25 × 10 -7  M. The detection limit of the proposed method was obtained to be 1.5 × 10 -10  M. Finally, the feasibility and selectivity of the proposed nanosensor was evaluated by the analysis of derived nucleotides from both mismatched sequences and clinical samples of patients with leukemia as real samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Ratiometric FRET-based detection of DNA and micro-RNA on the surface using TIRF detection

    International Nuclear Information System (INIS)

    Matveeva, Evgenia G.; Gryczynski, Zygmunt; Stewart, Donald R.; Gryczynski, Ignacy

    2010-01-01

    A new FRET-based method for the ratiometric detection of DNA oligomers on a surface using TIRF detection mode is reported. The dual-labeled system consisting of two hybridized oligomers, Cy3oligoY:Cy5oligoX was immobilized on the surface, and the total internal reflection fluorescence (TIRF) was used to detect emission signals from the surface. Two signals, green and red, which originated from the green donor Cy3 and the red acceptor Cy5, have been simultaneously detected. When the target single-stranded complimentary oligomer was present in the solution, this oligomer replaced the Cy3oligoY in the donor:acceptor complex on the surface and the ratio of red-to-green signal was dramatically changed. This detection scheme is generally applicable to the detection of DNA or RNA on a surface.

  7. A Graphene-Based Biosensing Platform Based on Regulated Release of an Aptameric DNA Biosensor.

    Science.gov (United States)

    Mao, Yu; Chen, Yongli; Li, Song; Lin, Shuo; Jiang, Yuyang

    2015-11-09

    A novel biosensing platform was developed by integrating an aptamer-based DNA biosensor with graphene oxide (GO) for rapid and facile detection of adenosine triphosphate (ATP, as a model target). The DNA biosensor, which is locked by GO, is designed to contain two sensing modules that include recognition site for ATP and self-replication track that yields the nicking domain for Nt.BbvCI. By taking advantage of the different binding affinity of single-stranded DNA, double-stranded DNA and aptamer-target complex toward GO, the DNA biosensor could be efficiently released from GO in the presence of target with the help of a complementary DNA strand (CPDNA) that partially hybridizes to the DNA biosensor. Then, the polymerization/nicking enzyme synergetic isothermal amplification could be triggered, leading to the synthesis of massive DNA amplicons, thus achieving an enhanced sensitivity with a wide linear dynamic response range of four orders of magnitude and good selectivity. This biosensing strategy expands the applications of GO-DNA nanobiointerfaces in biological sensing, showing great potential in fundamental research and biomedical diagnosis.

  8. Hall effect biosensors with ultraclean graphene film for improved sensitivity of label-free DNA detection

    KAUST Repository

    Loan, Phan Thi Kim

    2017-07-19

    The quality of graphene strongly affects the performance of graphene-based biosensors which are highly demanded for the sensitive and selective detection of biomolecules, such as DNA. This work reported a novel transfer process for preparing a residue-free graphene film using a thin gold supporting layer. A Hall effect device made of this gold-transferred graphene was demonstrated to significantly enhance the sensitivity (≈ 5 times) for hybridization detection, with a linear detection range of 1 pM – 100nM for DNA target. Our findings provide an efficient method to boost the sensitivity of graphene-based biosensors for DNA recognition.

  9. Electrochemical behavior of antioxidants: Part 3. Electrochemical studies of caffeic Acid–DNA interaction and DNA/carbon nanotube biosensor for DNA damage and protection

    Directory of Open Access Journals (Sweden)

    Refat Abdel-Hamid

    2016-05-01

    Full Text Available Multi-walled carbon nanotubes-modified glassy carbon electrode biosensor was used for electrochemical studies of caffeic acid–dsDNA interaction in phosphate buffer solution at pH 2.12. Caffeic acid, CAF, shows a well-defined cyclic voltammetric wave. Its anodic peak current decreases and the peak potential shifts positively on the addition of dsDNA. This behavior was ascribed to an interaction of CAF with dsDNA giving CAF–dsDNA complex by intercalative binding mode. The apparent binding constant of CAF–dsDNA complex was determined using amperometric titrations. The oxidative damage caused to DNA was detected using the biosensor. The damage caused by the reactive oxygen species, hydroxyl radical (·−OH generated by the Fenton system on the DNA-biosensor was detected. It was found that CAF has the capability of scavenging the hydroxide radical and protecting the DNA immobilized on the GCE surface.

  10. Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

    Czech Academy of Sciences Publication Activity Database

    Fessl, Tomáš; Adamec, František; Polívka, Tomáš; Foldynová-Trantírková, Silvie; Vácha, František; Trantírek, L.

    2012-01-01

    Roč. 40, č. 16 (2012), s. 10 ISSN 0305-1048 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z60220518 Keywords : in-cell FRET * fluorescence * DNA * nucleic acid * ATTO * in vivo Subject RIV: BO - Biophysics Impact factor: 8.278, year: 2012

  11. Droplet-based microscale colorimetric biosensor for multiplexed DNA analysis via a graphene nanoprobe

    International Nuclear Information System (INIS)

    Xiang Xia; Luo Ming; Shi Liyang; Ji Xinghu; He Zhike

    2012-01-01

    Graphical abstract: With a microvalve manipulate technique combined with droplet platform, a microscale fluorescence-based colorimetric sensor for multiplexed DNA analysis is developed via a graphene nanoprobe. Highlights: ► A quantitative detection for multiplexed DNA is first realized on droplet platform. ► The DNA detection is relied on a simple fluorescence-based colorimetric method. ► GO is served as a quencher for two different DNA fluorescent probes. ► This present work provides a rapid, sensitive, visual and convenient detection tool for droplet biosensor. - Abstract: The development of simple and inexpensive DNA detection strategy is very significant for droplet-based microfluidic system. Here, a droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device by using fluorescence-based colorimetric method and a graphene nanoprobe. With the aid of droplet manipulation technique, droplet size adjustment, droplet fusion and droplet trap are realized accurately and precisely. Due to the high quenching efficiency of graphene oxide (GO), in the absence of target DNAs, the droplet containing two single-stranded DNA probes and GO shows dark color, in which the DNA probes are labeled carboxy fluorescein (FAM) and 6-carboxy-X-rhodamine (ROX), respectively. The droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of 9.46 and 9.67 × 10 −8 M, respectively. It is also demonstrated that this biosensor platform can become a promising detection tool in high throughput applications with low consumption of reagents. Moreover, the incorporation of graphene nanoprobe and droplet technique can drive the biosensor field one more step to some extent.

  12. An ultrasensitive hollow-silica-based biosensor for pathogenic Escherichia coli DNA detection.

    Science.gov (United States)

    Ariffin, Eda Yuhana; Lee, Yook Heng; Futra, Dedi; Tan, Ling Ling; Karim, Nurul Huda Abd; Ibrahim, Nik Nuraznida Nik; Ahmad, Asmat

    2018-03-01

    A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer-Emmett-Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10 -12 -1.0×10 -2 μM, with a low detection limit of 8.17×10 -14 μM (R 2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstract Step-by-step DNA biosensor fabrication based on aminated hollow silica spheres.

  13. Preparation of DNA biosensor application from fuel oil waste by functionalization and characterization of MWCNT

    Directory of Open Access Journals (Sweden)

    Ahmed Mishaal Mohammed

    2017-11-01

    Full Text Available The potential of using a multi-wall carbon nanotube (MWCNT synthesized from a fuel oil waste of power plants has discovered for the first time for DNA biosensors application. The MWCNT surface morphologies were examined by field emission scanning electron microscopy (FE-SEM and atomic force microscopy (AFM. The thickness of the MWCNT was found 203nm and confirmed by FESEM. The electrochemical DNA biosensor was successfully developed using a MWCNT modified on SiO2 thin films. The capacitance measurements were performed to detect the sensitivity of DNA detection. The change in capacitance before and after immobilization of the DNA was measured in the frequency range of 1Hz to 1MHz. The results indicate that bare device exhibited the lowest capacitance value, which was 32.7μF. The capacitance value of the DNA immobilization increase to 52μF. The permittivity and conductivity also were examined to study the effect of the DNA immobilization toward the MWCNT modified surface. This present demonstrated that the MWCNT modified SiO2 a thin film was successfully fabricated for DNA biosensor detection. Keywords: Carbon nanotubes, Sensors, Thin films, Electrochemical DNA

  14. Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

    Science.gov (United States)

    Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

    2007-05-15

    The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

  15. A DNA biosensor for molecular diagnosis of Aeromonas hydrophila using zinc sulfide nanospheres

    Directory of Open Access Journals (Sweden)

    M. Negahdary

    2017-07-01

    Full Text Available Today, identification of pathogenic bacteria using modern and accurate methods is inevitable. Integration in electrochemical measurements with nanotechnology has led to the design of efficient and sensitive DNA biosensors against bacterial agents. Here, efforts were made to detect Aeromonas hydrophila using aptamers as probes and zinc sulfide (ZnS nanospheres as signal enhancers and electron transfer facilitators. After modification of the working electrode area (in a screen-printed electrode with ZnS nanospheres through electrodeposition, the coated surface of a modified electrode with ZnS nanospheres was investigated through scanning electron microscopy (SEM. The size of synthesized ZnS nanospheres was estimated at about 20–50 nm and their shape was in the form of porous plates in microscopic observations. All electrochemical measurements were performed using cyclic voltammetry (CV, electrochemical impedance spectroscopy (EIS, and constant potential amperometry (CPA techniques. The designed DNA biosensor was able to detect deoxyribonucleic acid (DNA of Aeromonas hydrophila in the range 1.0  ×  10−4 to 1.0  ×  10−9 mol L−1; the limit of detection (LOD in this study was 1  ×  10−13 mol L−1. This DNA biosensor showed satisfactory thermal and pH stability. Reproducibility for this DNA biosensor was measured and the relative standard deviation (RSD of the performance of this DNA biosensor was calculated as 5 % during 42 days.

  16. The effect of pH and DNA concentration on organic thin-film transistor biosensors

    KAUST Repository

    Khan, Hadayat Ullah

    2012-03-01

    Organic electronics are beginning to attract more interest for biosensor technology as they provide an amenable interface between biology and electronics. Stable biosensor based on electronic detection platform would represent a significant advancement in technology as costs and analysis time would decrease immensely. Organic materials provide a route toward that goal due to their compatibility with electronic applications and biological molecules. In this report, we detail the effects of experimental parameters, such as pH and concentration, toward the selective detection of DNA via surface-bound peptide nucleic acid (PNA) sequences on organic transistor biosensors. The OTFT biosensors are fabricated with thin-films of the organic semiconductor, 5,5′-bis-(7-dodecyl-9H-fluoren-2-yl)-2,2′-bithiophene (DDFTTF), in which they exhibit a stable mobility of 0.2 cm 2 V -1 s -1 in buffer solutions (phosphate-buffer saline, pH 7.4 or sodium acetate, pH 7). Device performance were optimized to minimize the deleterious effects of pH on gate-bias stress such that the sensitivity toward DNA detection can be improved. In titration experiments, the surface-bound PNA probes were saturated with 50 nM of complementary target DNA, which required a 10-fold increase in concentration of single-base mismatched target DNA to achieve a similar surface saturation. The binding constant of DNA on the surface-bound PNA probes was determined from the concentration-dependent response (titration measurements) of our organic transistor biosensors. © 2011 Elsevier B.V. All rights reserved.

  17. The effect of pH and DNA concentration on organic thin-film transistor biosensors

    KAUST Repository

    Khan, Hadayat Ullah; Roberts, Mark E.; Johnson, Olasupo B.; Knoll, Wolfgang; Bao, Zhenan

    2012-01-01

    Organic electronics are beginning to attract more interest for biosensor technology as they provide an amenable interface between biology and electronics. Stable biosensor based on electronic detection platform would represent a significant advancement in technology as costs and analysis time would decrease immensely. Organic materials provide a route toward that goal due to their compatibility with electronic applications and biological molecules. In this report, we detail the effects of experimental parameters, such as pH and concentration, toward the selective detection of DNA via surface-bound peptide nucleic acid (PNA) sequences on organic transistor biosensors. The OTFT biosensors are fabricated with thin-films of the organic semiconductor, 5,5′-bis-(7-dodecyl-9H-fluoren-2-yl)-2,2′-bithiophene (DDFTTF), in which they exhibit a stable mobility of 0.2 cm 2 V -1 s -1 in buffer solutions (phosphate-buffer saline, pH 7.4 or sodium acetate, pH 7). Device performance were optimized to minimize the deleterious effects of pH on gate-bias stress such that the sensitivity toward DNA detection can be improved. In titration experiments, the surface-bound PNA probes were saturated with 50 nM of complementary target DNA, which required a 10-fold increase in concentration of single-base mismatched target DNA to achieve a similar surface saturation. The binding constant of DNA on the surface-bound PNA probes was determined from the concentration-dependent response (titration measurements) of our organic transistor biosensors. © 2011 Elsevier B.V. All rights reserved.

  18. Hall effect biosensors with ultraclean graphene film for improved sensitivity of label-free DNA detection.

    Science.gov (United States)

    Loan, Phan Thi Kim; Wu, Dongqin; Ye, Chen; Li, Xiaoqing; Tra, Vu Thanh; Wei, Qiuping; Fu, Li; Yu, Aimin; Li, Lain-Jong; Lin, Cheng-Te

    2018-01-15

    The quality of graphene strongly affects the performance of graphene-based biosensors which are highly demanded for the sensitive and selective detection of biomolecules, such as DNA. This work reported a novel transfer process for preparing a residue-free graphene film using a thin gold supporting layer. A Hall effect device made of this gold-transferred graphene was demonstrated to significantly enhance the sensitivity (≈ 5 times) for hybridization detection, with a linear detection range of 1pM to 100nM for DNA target. Our findings provide an efficient method to boost the sensitivity of graphene-based biosensors for DNA recognition. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. A DNA biosensor based on the electrocatalytic oxidation of amine by a threading intercalator

    International Nuclear Information System (INIS)

    Gao Zhiqiang; Tansil, Natalia

    2009-01-01

    An electrochemical biosensor for the detection of DNA based a peptide nucleic acid (PNA) capture probe (CP) modified indium tin oxide electrode (ITO) is described in this report. After hybridization, a threading intercalator, N,N'-bis[(3-propyl)-imidazole]-1,4,5,8-naphthalene diimide (PIND) imidazole complexed with Ru(bpy) 2 Cl (PIND-Ru, bpy = 2,2'-bipyridine), was introduced to the biosensor. PIND-Ru selectively intercalated to double-stranded DNA (ds-DNA) and became immobilized on the biosensor surface. Voltammetric tests showed highly stable and reversible electrochemical oxidation/reduction processes and the peak currents can directly be utilized for DNA quantification. When the tests were conducted in an amine-containing medium, Tris-HCl buffer for example, a remarkable improvement in the voltammetric response and noticeable enhancements of voltammetric and amperometric sensitivities were observed due to the electrocatalytic activity of the [Ru(bpy) 2 Cl] redox moieties. Electrocatalytic current was observed when as little as 3.0 attomoles of DNA was present in the sample solution

  20. A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization

    International Nuclear Information System (INIS)

    Wang, Qingxiang; Ding, Yingtao; Gao, Feng; Jiang, Shulian; Zhang, Bin; Ni, Jiancong; Gao, Fei

    2013-01-01

    Graphical abstract: A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction between probe DNA and the sulfonic dye of 1-amino-2-naphthol-4-sulfonic acid that electrodeposited on a glassy carbon electrode. -- Highlights: •A versatile sulfonic dye of ANS was electrodeposited on a GCE. •A DNA biosensor was fabricated based on a facile sulfamide coupling reaction. •High probe DNA density of 3.18 × 10 13 strands cm −2 was determined. •A wide linear range and a low detection limit were obtained. -- Abstract: A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO 3 − ) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO 3 − layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO 3 − -AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO 3 − . The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH 3 ) 6 3+ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 10 13 strands cm −2 and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen) 3 3+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen) 3 3+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10 −13 M to 1.0 × 10 −8 M with

  1. Fabrication of Ultrasensitive Field-Effect Transistor DNA Biosensors by a Directional Transfer Technique Based on CVD-Grown Graphene.

    Science.gov (United States)

    Zheng, Chao; Huang, Le; Zhang, Hong; Sun, Zhongyue; Zhang, Zhiyong; Zhang, Guo-Jun

    2015-08-12

    Most graphene field-effect transistor (G-FET) biosensors are fabricated through a routine process, in which graphene is transferred onto a Si/SiO2 substrate and then devices are subsequently produced by micromanufacture processes. However, such a fabrication approach can introduce contamination onto the graphene surface during the lithographic process, resulting in interference for the subsequent biosensing. In this work, we have developed a novel directional transfer technique to fabricate G-FET biosensors based on chemical-vapor-deposition- (CVD-) grown single-layer graphene (SLG) and applied this biosensor for the sensitive detection of DNA. A FET device with six individual array sensors was first fabricated, and SLG obtained by the CVD-growth method was transferred onto the sensor surface in a directional manner. Afterward, peptide nucleic acid (PNA) was covalently immobilized on the graphene surface, and DNA detection was realized by applying specific target DNA to the PNA-functionalized G-FET biosensor. The developed G-FET biosensor was able to detect target DNA at concentrations as low as 10 fM, which is 1 order of magnitude lower than those reported in a previous work. In addition, the biosensor was capable of distinguishing the complementary DNA from one-base-mismatched DNA and noncomplementary DNA. The directional transfer technique for the fabrication of G-FET biosensors is simple, and the as-constructed G-FET DNA biosensor shows ultrasensitivity and high specificity, indicating its potential application in disease diagnostics as a point-of-care tool.

  2. pH-dependence of the optical bio-sensor based on DNA-carbon nanotube

    International Nuclear Information System (INIS)

    Vu Thuy Huong; Quach Kha Quang; Tran Thanh Thuy; Phan Duc Anh; Ngo Van Thanh; Nguyen Ai Viet

    2010-01-01

    In 2006, Daniel A. Heller et al. [1] demonstrated that carbon nanotubes (CNNTs) wrapped with DNA can be placed inside living cells and detect trace amounts of harmful contaminants using near infrared light. This discovery could lead to new types of optical sensors and biomarkers at the sub cellular level. The working principle of this optical bio-sensor from DNA and CNNTs can be explained by a simple theoretical model which was introduced in [3]. In this paper, the pH-dependence of DNA and the pH-dependence of solution around CNNTs are shown by using data analysis method. By substituting them into the same model, the pH-dependence of DNA-wrapped CNNTs was elicited in this paper. The range of parameters for workable conditions of this bio-sensor was indicated that the solution should have pH from 6 to 9 and the concentration of ions should be more than a critical value. These results are according to the experimental data and the deduction about pH and salt concentration in solution. They are very useful as using such a new bio-sensor like this in living environment. (author)

  3. A Highly Sensitive Electrochemical DNA Biosensor from Acrylic-Gold Nano-composite for the Determination of Arowana Fish Gender

    Science.gov (United States)

    Rahman, Mahbubur; Heng, Lee Yook; Futra, Dedi; Chiang, Chew Poh; Rashid, Zulkafli A.; Ling, Tan Ling

    2017-08-01

    The present research describes a simple method for the identification of the gender of arowana fish ( Scleropages formosus). The DNA biosensor was able to detect specific DNA sequence at extremely low level down to atto M regimes. An electrochemical DNA biosensor based on acrylic microsphere-gold nanoparticle (AcMP-AuNP) hybrid composite was fabricated. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesised with a facile and well-established one-step photopolymerization procedure and physically adsorbed on the AuNPs at the surface of a carbon screen printed electrode (SPE). The DNA biosensor was constructed simply by grafting an aminated DNA probe on the succinimide functionalised AcMPs via a strong covalent attachment. DNA hybridisation response was determined by differential pulse voltammetry (DPV) technique using anthraquinone monosulphonic acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10-18 M with a wide linear calibration range of 1.0 × 10-18 to 1.0 × 10-8 M ( R 2 = 0.99) can be achieved by the proposed DNA biosensor under optimal conditions. Electrochemical detection of arowana DNA can be completed within 1 hour. Due to its small size and light weight, the developed DNA biosensor holds high promise for the development of functional kit for fish culture usage.

  4. Development of Piezoelectric DNA-Based Biosensor for Direct Detection of Mycobacterium Tuberculosis in Clinical Specimens

    Directory of Open Access Journals (Sweden)

    Thongchai KAEWPHINIT

    2010-02-01

    Full Text Available This study was focused on establishment of piezoelectric biosensor for direct detection of Mycobacterium tuberculosis (MTB in clinical specimens. The quartz crystal immobilized via 3-mercaptopropionic acid (MPA/avidin/DNA biotinylated probe on gold surface and hybridization of the DNA target to DNA biotinylated probe. The optimal concentration of MPA, avidin and 5’-biotinylated DNA probe for immobilization of specific DNA probe on gold surface were 15 mM, 0.1 mg/ml and 1.5 μM, respectively. The detection of genomic DNA digestion in the range from 0.5 to 30 μg/ml. The fabricated biosensor was evaluated through an examination of 200 samples. No cross hybridization were observed against M. avium complex (MAC and other microorganism. This target DNA preparation without amplification will reduce time consuming, costs, and the tedious step of amplification. This study can be extended to develop the new method which is high sensitivity, specificity, cheap, easy to use, and rapid for detection of MTB in many fields.

  5. Hydrogen peroxide biosensor based on DNA-Hb modified gold electrode

    International Nuclear Information System (INIS)

    Kafi, A.K.M.; Fan Yin; Shin, Hoon-Kyu; Kwon, Young-Soo

    2006-01-01

    A hydrogen peroxide (H 2 O 2 ) biosensor based on DNA-hemoglobin (Hb) modified electrode is described in this paper. The sensor was designed by DNA and hemoglobin dropletting onto gold electrode surface layer by layer. The sensor based on the direct electron transfer of iron of hemoglobin showed a well electrocatalytic response to the reduction of the H 2 O 2 . This sensor offered an excellent electrochemical response for H 2 O 2 concentration below micromole level with high sensitivity and selectivity and short response time. Experimental conditions influencing the biosensor performance such as, pH, potential were optimized and assessed. The levels of the RSD's ( 2 O 2 was observed from 10 to 120 μM with the detection limit of 0.4 μM (based on the S/N = 3)

  6. A single-surface electrochemical biosensor for the detection of DNA triplet repeat expansion

    Czech Academy of Sciences Publication Activity Database

    Fojta, Miroslav; Horáková Brázdilová, Petra; Cahová, Kateřina; Pečinka, Petr

    2006-01-01

    Roč. 18, č. 2 (2006), s. 141-151 ISSN 1040-0397 R&D Projects: GA MPO(CZ) 1H-PK/42; GA AV ČR(CZ) IAA4004402 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA hybridization * electrochemical biosensor * enzyme-linked assay Subject RIV: BO - Biophysics Impact factor: 2.444, year: 2006

  7. A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

    DEFF Research Database (Denmark)

    Schneider, Uffe V; Severinsen, Jette K; Géci, Imrich

    2010-01-01

    BACKGROUND: Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acid...

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications. (paper)

  9. An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite

    International Nuclear Information System (INIS)

    Chen, Mei; Hou, Changjun; Huo, Danqun; Yang, Mei; Fa, Huanbao

    2016-01-01

    Graphical abstract: A novel and sensitive electrochemical biosensor based on hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized single-walled carbon nanotubes (SWCNTs-COOH) was first developed for the detection of the specific-sequence target DNA. This schematic represents the fabrication procedure of our DNA biosensor. - Highlights: • An ultrasensitive DNA electrochemical biosensor was developed. • CuO NWs entangled with the SWCNTs formed a mesh structure with good conductivity. • It is the first time use of CuONWs-SWCNTs hybrid nanocomposite for DNA detection. • The biosensor is simple, selective, stable, and sensitive. • The biosensor has great potential for use in analysis of real samples. - Abstract: Here, we developed a novel and sensitive electrochemical biosensor to detect specific-sequence target DNA. The biosensor was based on a hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized single-walled carbon nanotubes (SWCNTs-COOH). The resulting CuO NWs/SWCNTs layers exhibited a good differential pulse voltammetry (DPV) current response for the target DNA sequences, which we attributed to the properties of CuO NWs and SWCNTs. CuO NWs and SWCNTs hybrid composites with highly conductive and biocompatible nanostructure were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and cyclic voltammetry (CV). Immobilization of the probe DNA on the electrode surface was largely improved due to the unique synergetic effect of CuO NWs and SWCNTs. DPV was applied to monitor the DNA hybridization event, using adriamycin as an electrochemical indicator. Under optimal conditions, the peak currents of adriamycin were linear with the logarithm of target DNA concentrations (ranging from 1.0 × 10"−"1"4 to 1.0 × 10"−"8 M), with a detection limit of 3.5 × 10"−"1"5 M (signal/noise ratio of 3). The biosensor also showed high selectivity to

  10. An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Mei [Key Laboratory of Biorheology Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Hou, Changjun, E-mail: houcj@cqu.edu.cn [Key Laboratory of Biorheology Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); National Key Laboratory of Fundamental Science of Micro/Nano-Device and System Technology, Chongqing University, Chongqing 400044 (China); Huo, Danqun [Key Laboratory of Biorheology Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); National Key Laboratory of Fundamental Science of Micro/Nano-Device and System Technology, Chongqing University, Chongqing 400044 (China); Yang, Mei [Key Laboratory of Biorheology Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Fa, Huanbao [College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China)

    2016-02-28

    Graphical abstract: A novel and sensitive electrochemical biosensor based on hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized single-walled carbon nanotubes (SWCNTs-COOH) was first developed for the detection of the specific-sequence target DNA. This schematic represents the fabrication procedure of our DNA biosensor. - Highlights: • An ultrasensitive DNA electrochemical biosensor was developed. • CuO NWs entangled with the SWCNTs formed a mesh structure with good conductivity. • It is the first time use of CuONWs-SWCNTs hybrid nanocomposite for DNA detection. • The biosensor is simple, selective, stable, and sensitive. • The biosensor has great potential for use in analysis of real samples. - Abstract: Here, we developed a novel and sensitive electrochemical biosensor to detect specific-sequence target DNA. The biosensor was based on a hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized single-walled carbon nanotubes (SWCNTs-COOH). The resulting CuO NWs/SWCNTs layers exhibited a good differential pulse voltammetry (DPV) current response for the target DNA sequences, which we attributed to the properties of CuO NWs and SWCNTs. CuO NWs and SWCNTs hybrid composites with highly conductive and biocompatible nanostructure were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and cyclic voltammetry (CV). Immobilization of the probe DNA on the electrode surface was largely improved due to the unique synergetic effect of CuO NWs and SWCNTs. DPV was applied to monitor the DNA hybridization event, using adriamycin as an electrochemical indicator. Under optimal conditions, the peak currents of adriamycin were linear with the logarithm of target DNA concentrations (ranging from 1.0 × 10{sup −14} to 1.0 × 10{sup −8} M), with a detection limit of 3.5 × 10{sup −15} M (signal/noise ratio of 3). The biosensor also showed high

  11. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

    Science.gov (United States)

    Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

    2018-01-09

    Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Conducting polymer based DNA biosensor for the detection of the Bacillus cereus group species

    Science.gov (United States)

    Velusamy, Vijayalakshmi; Arshak, Khalil; Korostynska, Olga; Oliwa, Kamila; Adley, Catherine

    2009-05-01

    Biosensor designs are emerging at a significant rate and play an increasingly important role in foodborne pathogen detection. Conducting polymers are excellent tools for the fabrication of biosensors and polypyrrole has been used in the detection of biomolecules due to its unique properties. The prime intention of this paper was to pioneer the design and fabrication of a single-strand (ss) DNA biosensor for the detection of the Bacillus cereus (B.cereus) group species. Growth of B. cereus, results in production of several highly active toxins. Therefore, consumption of food containing >106 bacteria/gm may results in emetic and diarrhoeal syndromes. The most common source of this bacterium is found in liquid food products, milk powder, mixed food products and is of particular concern in the baby formula industry. The electrochemical deposition technique, such as cyclic voltammetry, was used to develop and test a model DNA-based biosensor on a gold electrode electropolymerized with polypyrrole. The electrically conducting polymer, polypyrrole is used as a platform for immobilizing DNA (1μg) on the gold electrode surface, since it can be more easily deposited from neutral pH aqueous solutions of pyrrolemonomers. The average current peak during the electrodeposition event is 288μA. There is a clear change in the current after hybridization of the complementary oligonucleotide (6.35μA) and for the noncomplementary oligonucleotide (5.77μA). The drop in current after each event was clearly noticeable and it proved to be effective.

  13. Detection of DNA and poly-l-lysine using CVD graphene-channel FET biosensors

    International Nuclear Information System (INIS)

    Kakatkar, Aniket; Craighead, H G; Abhilash, T S; Alba, R De; Parpia, J M

    2015-01-01

    A graphene channel field-effect biosensor is demonstrated for detecting the binding of double-stranded DNA and poly-l-lysine. Sensors consist of chemical vapor deposition graphene transferred using a clean, etchant-free transfer method. The presence of DNA and poly-l-lysine are detected by the conductance change of the graphene transistor. A readily measured shift in the Dirac voltage (the voltage at which the graphene’s resistance peaks) is observed after the graphene channel is exposed to solutions containing DNA or poly-l-lysine. The ‘Dirac voltage shift’ is attributed to the binding/unbinding of charged molecules on the graphene surface. The polarity of the response changes to positive direction with poly-l-lysine and negative direction with DNA. This response results in detection limits of 8 pM for 48.5 kbp DNA and 11 pM for poly-l-lysine. The biosensors are easy to fabricate, reusable and are promising as sensors of a wide variety of charged biomolecules. (paper)

  14. AFFINITY BIOSENSOR BASED ON SCREEN-PRINTED ELECTRODE MODIFIED WITH DNA FOR GENOTOXIC COMPOUNDS DETECTION

    Directory of Open Access Journals (Sweden)

    Bambang Kuswandi

    2010-06-01

    Full Text Available An electrochemical method for the detection of the genotoxic compounds using a DNA-modified electrode was developed. This electrode was successfully used for the electrochemical detection of genotoxic compounds in water samples. The electrochemical results clearly demonstrated that, the development is related to the molecular interaction between the surface-linked DNA obtained from calf thymus and the target compounds, such as pollutants, in order to develop a simple device for rapid screening of genotoxic compounds in environmental samples. The detection of such compounds was measured by their effect on the oxidation signal of the guanine peak of the DNA immobilised on the surface of carbon based Screen-Printed Electrode (SPE in disposable mode, and monitored by square-wave voltametric analysis. The DNA biosensor is able to detect known intercalating and groove-binding genotoxic compounds such as Dioxin, Bisphenol A, PCBs, and Phtalates. Application to real water samples is discussed and reported.   Keywords: electrochemical, screen-printed electrode, DNA biosensor, genotoxic compounds

  15. A fractal analysis of protein to DNA binding kinetics using biosensors.

    Science.gov (United States)

    Sadana, Ajit

    2003-08-01

    A fractal analysis of a confirmative nature only is presented for the binding of estrogen receptor (ER) in solution to its corresponding DNA (estrogen response element, ERE) immobilized on a sensor chip surface [J. Biol. Chem. 272 (1997) 11384], and for the cooperative binding of human 1,25-dihydroxyvitamin D(3) receptor (VDR) to DNA with the 9-cis-retinoic acid receptor (RXR) [Biochemistry 35 (1996) 3309]. Ligands were also used to modulate the first reaction. Data taken from the literature may be modeled by using a single- or a dual-fractal analysis. Relationships are presented for the binding rate coefficient as a function of either the analyte concentration in solution or the fractal dimension that exists on the biosensor surface. The binding rate expressions developed exhibit a wide range of dependence on the degree of heterogeneity that exists on the surface, ranging from sensitive (order of dependence equal to 1.202) to very sensitive (order of dependence equal to 12.239). In general, the binding rate coefficient increases as the degree of heterogeneity or the fractal dimension of the surface increases. The predictive relationships presented provide further physical insights into the reactions occurring on the biosensor surface. Even though these reactions are occurring on the biosensor surface, the relationships presented should assist in understanding and in possibly manipulating the reactions occurring on cellular surfaces.

  16. Spiky gold shells on magnetic particles for DNA biosensors.

    Science.gov (United States)

    Bedford, Erin E; Boujday, Souhir; Pradier, Claire-Marie; Gu, Frank X

    2018-05-15

    Combined separation and detection of biomolecules has the potential to speed up and improve the sensitivity of disease detection, environmental testing, and biomolecular analysis. In this work, we synthesized magnetic particles coated with spiky nanostructured gold shells and used them to magnetically separate out and detect oligonucleotides using SERS. The distance dependence of the SERS signal was then harnessed to detect DNA hybridization using a Raman label bound to a hairpin probe. The distance of the Raman label from the surface increased upon complementary DNA hybridization, leading to a decrease in signal intensity. This work demonstrates the use of the particles for combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Modified surface of titanium dioxide nanoparticles-based biosensor for DNA detection

    Science.gov (United States)

    Nadzirah, Sh.; Hashim, U.; Rusop, M.

    2018-05-01

    A new technique was used to develop a simple and selective picoammeter DNA biosensor for identification of E. coli O157:H7. This biosensor was fabricated from titanium dioxide nanoparticles that was synthesized by sol-gel method and spin-coated on silicon dioxide substrate via spinner. 3-Aminopropyl triethoxy silane (APTES) was used to modify the surface of TiO2. Simple surface modification approach has been applied; which is single dropping of APTES onto the TiO2 nanoparticles surface. Carboxyl modified probe DNA has been bind onto the surface of APTES/TiO2 without any amplifier element. Electrical signal has been used as the indicator to differentiate each step (surface modification of TiO2 and probe DNA immobilization). The I-V measurements indicate extremely low current (pico-ampere) flow through the device which is 2.8138E-10 A for pure TiO2 nanoparticles, 2.8124E-10 A after APTES modification and 3.5949E-10 A after probe DNA immobilization.

  18. Electrochemical DNA biosensor based on MNAzyme-mediated signal amplification

    International Nuclear Information System (INIS)

    Diao, Wei; Tang, Min; Ding, Xiaojuan; Zhang, Ye; Yang, Jianru; Cheng, Wenbin; Mo, Fei; Wen, Bo; Xu, Lulu; Yan, Yurong

    2016-01-01

    The authors describe an electrochemical sensing strategy for highly sensitive and specific detection of target (analyte) DNA based on an amplification scheme mediated by a multicomponent nucleic acid enzyme (MNAzyme). MNAzymes were formed by multicomponent complexes which produce amplified “output” signals in response to specific “input” signal. In the presence of target nucleic acid, multiple partial enzymes (partzymes) oligonucleotides are assembled to form active MNAzymes. These can cleave H0 substrate into two pieces, thereby releasing the activated MNAzyme to undergo an additional cycle of amplification. Here, the two pieces contain a biotin-tagged sequence and a byproduct. The biotin-tagged sequences are specifically captured by the detection probes immobilized on the gold electrode. By employing streptavidinylated alkaline phosphatase as an enzyme label, an electrochemical signal is obtained. The electrode, if operated at a working potential of 0.25 V (vs. Ag/AgCl) in solution of pH 7.5, covers the 100 pM to 0.25 μM DNA concentration range, with a 79 pM detection limit. In our perception, the strategy introduced here has a wider potential in that it may be applied to molecular diagnostics and pathogen detection. (author)

  19. A sensitive DNA biosensor fabricated from gold nanoparticles, carbon nanotubes, and zinc oxide nanowires on a glassy carbon electrode

    International Nuclear Information System (INIS)

    Wang Jie; Li Shuping; Zhang Yuzhong

    2010-01-01

    We outline here the fabrication of a sensitive electrochemical DNA biosensor for the detection of sequence-specific target DNA. Zinc oxide nanowires (ZnONWs) were first immobilized on the surface of a glassy carbon electrode. Multi-walled carbon nanotubes (MWCNTs) with carboxyl groups were then dropped onto the surface of the ZnONWs. Gold nanoparticles (AuNPs) were subsequently introduced to the surface of the MWNTs/ZnONWs by electrochemical deposition. A single-stranded DNA probe with a thiol group at the end (HS-ssDNA) was covalently immobilized on the surface of the AuNPs by forming an Au-S bond. Scanning electron microscopy (SEM) and cyclic voltammetry (CV) were used to investigate the film assembly process. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of [Ru(NH 3 ) 6 ] 3+ bounding to double-stranded DNA (dsDNA). The incorporation of ZnONWs and MWCNTs in this sensor design significantly enhances the sensitivity and the selectivity. This DNA biosensor can detect the target DNA quantitatively in the range of 1.0 x 10 -13 to 1.0 x 10 -7 M, with a detection limit of 3.5 x 10 -14 M (S/N = 3). In addition, the DNA biosensor exhibits excellent selectivity, even for single-mismatched DNA detection.

  20. Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components

    Directory of Open Access Journals (Sweden)

    Marian Filipiak

    2008-03-01

    Full Text Available An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5’-phosphate end to amino group of cysteamine self-assembled monolayer (SAM on gold electrode surface with the use of activating reagents – water soluble 1-ethyl-3(3’- dimethylaminopropyl-carbodiimide (EDC and N-hydroxy-sulfosuccinimide (NHS. The hybridization reaction on the electrode surface was detected via methylene blue (MB presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs give a broad perspectives for analytical application of the biosensor.

  1. DNA tetrahedral scaffolds-based platform for the construction of electrochemiluminescence biosensor.

    Science.gov (United States)

    Feng, Qiu-Mei; Zhou, Zhen; Li, Mei-Xing; Zhao, Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-04-15

    Proximal metallic nanoparticles (NPs) could quench the electrochemiluminescence (ECL) emission of semiconductor quantum dots (QDs) due to Förster energy transfer (FRET), but at a certain distance, the coupling of light-emission with surface plasmon resonance (SPR) result in enhanced ECL. Thus, the modification strategies and distances control between QDs and metallic NPs are critical for the ECL intensity of QDs. In this strategy, a SPR enhanced ECL sensor based on DNA tetrahedral scaffolds modified platform was reported for the detection of telomerase activity. Due to the rigid three-dimensional structure, DNA tetrahedral scaffolds grafting on the electrode surface could accurately modulate the distance between CdS QDs and luminol labelled gold nanoparticles (L-Au NPs), meanwhile provide an enhanced spatial dimension and accessibility for the assembly of multiple L-Au NPs. The ECL intensities of both CdS QDs (-1.25V vs. SCE) and luminol (+0.33V vs. SCE) gradually increased along with the formation of multiple L-Au NPs at the vertex of DNA tetrahedral scaffolds induced by telomerase, bringing in a dual-potential ECL analysis. The proposed method showed high sensitivity for the identification of telomerase and was successfully applied for the differentiation of cancer cells from normal cells. This work suggests that DNA tetrahedral scaffolds could serve as an excellent choice for the construction of SPR-ECL system. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Biosensor for label-free DNA quantification based on functionalized LPGs.

    Science.gov (United States)

    Gonçalves, Helena M R; Moreira, Luis; Pereira, Leonor; Jorge, Pedro; Gouveia, Carlos; Martins-Lopes, Paula; Fernandes, José R A

    2016-10-15

    A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62±2nM and the limit of quantification was 209±7nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Development of swine-specific DNA markers for biosensor-based halal authentication.

    Science.gov (United States)

    Ali, M E; Hashim, U; Kashif, M; Mustafa, S; Che Man, Y B; Abd Hamid, S B

    2012-06-29

    The pig (Sus scrofa) mitochondrial genome was targeted to design short (15-30 nucleotides) DNA markers that would be suitable for biosensor-based hybridization detection of target DNA. Short DNA markers are reported to survive harsh conditions in which longer ones are degraded into smaller fragments. The whole swine mitochondrial-genome was in silico digested with AluI restriction enzyme. Among 66 AluI fragments, five were selected as potential markers because of their convenient lengths, high degree of interspecies polymorphism and intraspecies conservatism. These were confirmed by NCBI blast analysis and ClustalW alignment analysis with 11 different meat-providing animal and fish species. Finally, we integrated a tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in deliberately contaminated autoclaved pork-beef binary mixtures and no cross-species detection was recorded, demonstrating the feasibility of this type of probe for biosensor-based detection of pork adulteration of halal and kosher foods.

  4. SiPM as miniaturised optical biosensor for DNA-microarray applications

    Directory of Open Access Journals (Sweden)

    M.F. Santangelo

    2015-12-01

    Full Text Available A miniaturized optical biosensor for low-level fluorescence emitted by DNA strands labelled with CY5 is showed. Aim of this work is to demonstrate that a Si-based photodetector, having a low noise and a high sensitivity, can replace traditional detection systems in DNA-microarray applications. The photodetector used is a photomultiplier (SiPM, with 25 pixels. It exhibits a higher sensitivity than commercial optical readers and we experimentally found a detection limit for spotted dried samples of ∼1 nM. We measured the fluorescence signal in different operating conditions (angle of analysis, fluorophores concentrations, solution volumes and support. Once fixed the angle of analysis, for samples spotted on Al-TEOS slide dried, the system is proportional to the concentration of the analyte in the sample and is linear in the range 1 nM–1 μM. For solutions, the range of linearity ranges from 100 fM to 10 nM. The system potentialities and the device low costs suggest it as basic component for the design and fabrication of a cheap, easy and portable optical system. Keywords: Optical Biosensor, SiPM, DNA microarray, Fluorophore detection

  5. Amperometric biosensor for hydrogen peroxide based on Hemoglobin/DNA/Poly-2,6-pyridinediamine modified gold electrode

    International Nuclear Information System (INIS)

    Tong Zhongqiang; Yuan Ruo; Chai Yaqin; Chen Shihong; Xie Yi

    2007-01-01

    An amperometric biosensor for hydrogen peroxide (H 2 O 2 ) was fabricated based on immobilization of hemoglobin (Hb) on DNA/Poly-2,6-pyridinediamine (PPD) modified Au electrode. PPD thin films were firstly electro-deposited on Au electrode surface which provide a template to attach negatively charged DNA molecules by electrostatic attraction. The adsorbed DNA network provides a good microenvironment for the immobilization of biomolecules and promotes electron transfer between the immobilized Hb and the electrode surface. The fabrication process of the biosensor was characterized by electrochemical impedance spectroscopy. Experimental conditions influencing the biosensor performance such as pH, potential and temperature were assessed and optimized. The proposed biosensor displayed a good electrocatalytic response to the reduction of H 2 O 2 , its linear range is 1.7 μM to 3 mM with a detection limit of 1.0 μM based on the signal-to-noise ratio of 3 (S/N = 3) under the optimized conditions. The Michaelis-Menten constant K m app of Hb immobilized on the electrode surface was found to be 0.8 mM. The biosensor shows high sensitivity and stability. Importantly, this deposition methodology could be further developed for the immobilization of other proteins and biocompounds

  6. Efficient Fluorescence Resonance Energy Transfer between Quantum Dots and Gold Nanoparticles Based on Porous Silicon Photonic Crystal for DNA Detection.

    Science.gov (United States)

    Zhang, Hongyan; Lv, Jie; Jia, Zhenhong

    2017-05-10

    A novel assembled biosensor was prepared for detecting 16S rRNA, a small-size persistent specific for Actinobacteria. The mechanism of the porous silicon (PS) photonic crystal biosensor is based on the fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and gold nanoparticles (AuNPs) through DNA hybridization, where QDs act as an emission donor and AuNPs serve as a fluorescence quencher. Results showed that the photoluminescence (PL) intensity of PS photonic crystal was drastically increased when the QDs-conjugated probe DNA was adhered to the PS layer by surface modification using a standard cross-link chemistry method. The PL intensity of QDs was decreased when the addition of AuNPs-conjugated complementary 16S rRNA was dropped onto QDs-conjugated PS. Based on the analysis of different target DNA concentration, it was found that the decrease of the PL intensity showed a good linear relationship with complementary DNA concentration in a range from 0.25 to 10 μM, and the detection limit was 328.7 nM. Such an optical FRET biosensor functions on PS-based photonic crystal for DNA detection that differs from the traditional FRET, which is used only in liquid. This method will benefit the development of a new optical FRET label-free biosensor on Si substrate and has great potential in biochips based on integrated optical devices.

  7. Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

    DEFF Research Database (Denmark)

    Jensen, Pia Wrensted; Falconi, Mattia; Kristoffersen, Emil Laust

    2013-01-01

    structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only “signal amplification” the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1......Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very...... simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples...

  8. The field effect transistor DNA biosensor based on ITO nanowires in label-free hepatitis B virus detecting compatible with CMOS technology.

    Science.gov (United States)

    Shariati, Mohsen

    2018-05-15

    In this paper the field-effect transistor DNA biosensor for detecting hepatitis B virus (HBV) based on indium tin oxide nanowires (ITO NWs) in label free approach has been fabricated. Because of ITO nanowires intensive conductance and functional modified surface, the probe immobilization and target hybridization were increased strongly. The high resolution transmission electron microscopy (HRTEM) measurement showed that ITO nanowires were crystalline and less than 50nm in diameter. The single-stranded hepatitis B virus DNA (SS-DNA) was immobilized as probe on the Au-modified nanowires. The DNA targets were measured in a linear concentration range from 1fM to 10µM. The detection limit of the DNA biosensor was about 1fM. The time of the hybridization process for defined single strand was 90min. The switching ratio of the biosensor between "on" and "off" state was ~ 1.1 × 10 5 . For sensing the specificity of the biosensor, non-complementary, mismatch and complementary DNA oligonucleotide sequences were clearly discriminated. The HBV biosensor confirmed the highly satisfied specificity for differentiating complementary sequences from non-complementary and the mismatch oligonucleotides. The response time of the DNA sensor was 37s with a high reproducibility. The stability and repeatability of the DNA biosensor showed that the peak current of the biosensor retained 98% and 96% of its initial response for measurements after three and five weeks, respectively. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

    OpenAIRE

    Bajar, Bryce T.; Wang, Emily S.; Lam, Amy J.; Kim, Bongjae B.; Jacobs, Conor L.; Howe, Elizabeth S.; Davidson, Michael W.; Lin, Michael Z.; Chu, Jun

    2016-01-01

    Many genetically encoded biosensors use F?rster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intra...

  10. DNA Nanobiosensors: An Outlook on Signal Readout Strategies

    Directory of Open Access Journals (Sweden)

    Arun Richard Chandrasekaran

    2017-01-01

    Full Text Available A suite of functionalities and structural versatility makes DNA an apt material for biosensing applications. DNA-based biosensors are cost-effective and sensitive and have the potential to be used as point-of-care diagnostic tools. Along with robustness and biocompatibility, these sensors also provide multiple readout strategies. Depending on the functionality of DNA-based biosensors, a variety of output strategies have been reported: fluorescence- and FRET-based readout, nanoparticle-based colorimetry, spectroscopy-based techniques, electrochemical signaling, gel electrophoresis, and atomic force microscopy.

  11. Impedimetric DNA Biosensor Based on a Nanoporous Alumina Membrane for the Detection of the Specific Oligonucleotide Sequence of Dengue Virus

    Directory of Open Access Journals (Sweden)

    Chee-Seng Toh

    2013-06-01

    Full Text Available A novel and integrated membrane sensing platform for DNA detection is developed based on an anodic aluminum oxide (AAO membrane. Platinum electrodes (~50–100 nm thick are coated directly on both sides of the alumina membrane to eliminate the solution resistance outside the nanopores. The electrochemical impedance technique is employed to monitor the impedance changes within the nanopores upon DNA binding. Pore resistance (Rp linearly increases in response towards the increasing concentration of the target DNA in the range of 1 × 10−12 to 1 × 10−6 M. Moreover, the biosensor selectively differentiates the complementary sequence from single base mismatched (MM-1 strands and non-complementary strands. This study reveals a simple, selective and sensitive method to fabricate a label-free DNA biosensor.

  12. Impedimetric DNA biosensor based on a nanoporous alumina membrane for the detection of the specific oligonucleotide sequence of dengue virus.

    Science.gov (United States)

    Deng, Jiajia; Toh, Chee-Seng

    2013-06-17

    A novel and integrated membrane sensing platform for DNA detection is developed based on an anodic aluminum oxide (AAO) membrane. Platinum electrodes (~50-100 nm thick) are coated directly on both sides of the alumina membrane to eliminate the solution resistance outside the nanopores. The electrochemical impedance technique is employed to monitor the impedance changes within the nanopores upon DNA binding. Pore resistance (Rp) linearly increases in response towards the increasing concentration of the target DNA in the range of 1 × 10⁻¹² to 1 × 10⁻⁶ M. Moreover, the biosensor selectively differentiates the complementary sequence from single base mismatched (MM-1) strands and non-complementary strands. This study reveals a simple, selective and sensitive method to fabricate a label-free DNA biosensor.

  13. In Vitro Evaluation of Fluorescence Glucose Biosensor Response

    OpenAIRE

    Aloraefy, Mamdouh; Pfefer, T. Joshua; Ramella-Roman, Jessica C.; Sapsford, Kim E.

    2014-01-01

    Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensor...

  14. Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

    Science.gov (United States)

    Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

    2014-10-07

    We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

  15. An Electrochemical DNA Biosensor Developed on a Nanocomposite Platform of Gold and Poly(propyleneimine Dendrimer

    Directory of Open Access Journals (Sweden)

    Omotayo Arotiba

    2008-11-01

    Full Text Available An electrochemical DNA nanobiosensor was prepared by immobilization of a 20mer thiolated probe DNA on electro-deposited generation 4 (G4 poly(propyleneimine dendrimer (PPI doped with gold nanoparticles (AuNP as platform, on a glassy carbon electrode (GCE. Field emission scanning electron microscopy results confirmed the codeposition of PPI (which was linked to the carbon electrode surface by C-N covalent bonds and AuNP ca 60 nm. Voltammetric interrogations showed that the platform (GCE/PPI-AuNP was conducting and exhibited reversible electrochemistry (E°′ = 235 mV in pH 7.2 phosphate buffer saline solution (PBS due to the PPI component. The redox chemistry of PPI was pH dependent and involves a two electron, one proton process, as interpreted from a 28 mV/pH value obtained from pH studies. The charge transfer resistance (Rct from the electrochemical impedance spectroscopy (EIS profiles of GCE/PPI-AuNP monitored with ferro/ferricyanide (Fe(CN63-/4- redox probe, decreased by 81% compared to bare GCE. The conductivity (in PBS and reduced Rct (in Fe(CN63-/4- values confirmed PPI-AuNP as a suitable electron transfer mediator platform for voltammetric and impedimetric DNA biosensor. The DNA probe was effectively wired onto the GCE/PPI-AuNP via Au-S linkage and electrostatic interactions. The nanobiosensor responses to target DNA which gave a dynamic linear range of 0.01 - 5 nM in PBS was based on the changes in Rct values using Fe(CN63-/4- redox probe.

  16. Comparison of impedimetric detection of DNA hybridization on the various biosensors based on modified glassy carbon electrodes with PANHS and nanomaterials of RGO and MWCNTs.

    Science.gov (United States)

    Benvidi, Ali; Tezerjani, Marzieh Dehghan; Jahanbani, Shahriar; Mazloum Ardakani, Mohammad; Moshtaghioun, Seyed Mohammad

    2016-01-15

    In this research, we have developed lable free DNA biosensors based on modified glassy carbon electrodes (GCE) with reduced graphene oxide (RGO) and carbon nanotubes (MWCNTs) for detection of DNA sequences. This paper compares the detection of BRCA1 5382insC mutation using independent glassy carbon electrodes (GCE) modified with RGO and MWCNTs. A probe (BRCA1 5382insC mutation detection (ssDNA)) was then immobilized on the modified electrodes for a specific time. The immobilization of the probe and its hybridization with the target DNA (Complementary DNA) were performed under optimum conditions using different electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The proposed biosensors were used for determination of complementary DNA sequences. The non-modified DNA biosensor (1-pyrenebutyric acid-N- hydroxysuccinimide ester (PANHS)/GCE), revealed a linear relationship between ∆Rct and logarithm of the complementary target DNA concentration ranging from 1.0×10(-16)molL(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.992, for DNA biosensors modified with multi-wall carbon nanotubes (MWCNTs) and reduced graphene oxide (RGO) wider linear range and lower detection limit were obtained. For ssDNA/PANHS/MWCNTs/GCE a linear range 1.0×10(-17)mol L(-1)-1.0×10(-10)mol L(-1) with a correlation coefficient of 0.993 and for ssDNA/PANHS/RGO/GCE a linear range from 1.0×10(-18)mol L(-1) to 1.0×10(-10)mol L(-1) with a correlation coefficient of 0.985 were obtained. In addition, the mentioned biosensors were satisfactorily applied for discriminating of complementary sequences from noncomplementary sequences, so the mentioned biosensors can be used for the detection of BRCA1-associated breast cancer. Copyright © 2015. Published by Elsevier B.V.

  17. Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

    Energy Technology Data Exchange (ETDEWEB)

    Jafari, Safiye [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Faridbod, Farnoush, E-mail: faridbodf@khayam.ut.ac.ir [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Biosensor Research Center, Endocrinology & Metabolism Molecular and Cellular Research Institute, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Norouzi, Parviz [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Biosensor Research Center, Endocrinology & Metabolism Molecular and Cellular Research Institute, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Dezfuli, Amin Shiralizadeh [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Ajloo, Davood [School of Chemistry, Damghan University, Damghan (Iran, Islamic Republic of); Mohammadipanah, Fatemeh [Department of Microbial Biotechnology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, 14155-6455 Tehran (Iran, Islamic Republic of); Ganjali, Mohammad Reza [Center of Excellence in Electrochemistry, University of Tehran, Tehran (Iran, Islamic Republic of); Biosensor Research Center, Endocrinology & Metabolism Molecular and Cellular Research Institute, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2015-10-01

    A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO{sub 2}NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy){sub 3}]{sup 2+/3+} redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy){sub 3}]{sup 2+/3+} FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10{sup −15} to 1 × 10{sup −8} mol L{sup −1}. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL{sup −1} with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy){sub 3}]{sup 2+/3+} interaction with ssDNA before and after hybridization. - Highlights: • New DNA biosensor is designed for sub-femtomolar detection of Aeromonas hydrophila DNA sequence. • Reduced graphene oxide decorated Ceria nanoparticles was used as a new immobilization platform. • Biosensor was successfully used to detect A. hydrophila DNA sequence in fish pond water.

  18. Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

    International Nuclear Information System (INIS)

    Jafari, Safiye; Faridbod, Farnoush; Norouzi, Parviz; Dezfuli, Amin Shiralizadeh; Ajloo, Davood; Mohammadipanah, Fatemeh; Ganjali, Mohammad Reza

    2015-01-01

    A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO_2NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)_3]"2"+"/"3"+ redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)_3]"2"+"/"3"+ FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10"−"1"5 to 1 × 10"−"8 mol L"−"1. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL"−"1 with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)_3]"2"+"/"3"+ interaction with ssDNA before and after hybridization. - Highlights: • New DNA biosensor is designed for sub-femtomolar detection of Aeromonas hydrophila DNA sequence. • Reduced graphene oxide decorated Ceria nanoparticles was used as a new immobilization platform. • Biosensor was successfully used to detect A. hydrophila DNA sequence in fish pond water.

  19. cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors

    DEFF Research Database (Denmark)

    Mathiesen, Jesper Mosolff; Vedel, Line; Bräuner-Osborne, Hans

    2013-01-01

    end-point assays for quantifying GPCR-mediated changes in intracellular cAMP levels exist. More recently, fluorescence resonance energy transfer (FRET)-based cAMP biosensors that can quantify intracellular cAMP levels in real time have been developed. These FRET-based cAMP biosensors have been used...... primarily in single cell FRET microscopy to monitor and visualize changes in cAMP upon GPCR activation. Here, a similar cAMP biosensor with a more efficient mCerulean/mCitrine FRET pair is described for use in the 384-well plate format. After cloning and expression in HEK293 cells, the biosensor...... is characterized in the 384-well plate format and used for measuring the signaling of the G(s)-coupled ß(2)-adrenergic receptor. The procedures described may be applied for other FRET-based biosensors in terms of characterization and conversion to the 384-well plate format....

  20. Enhanced sensing of dengue virus DNA detection using O{sub 2} plasma treated-silicon nanowire based electrical biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, S.F.A., E-mail: siti_fatimah0410@yahoo.com [Institute of Advanced Technology, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor (Malaysia); Yusof, N.A., E-mail: azahy@upm.edu.my [Institute of Advanced Technology, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor (Malaysia); Chemistry Department, Faculty of Science, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor (Malaysia); Hashim, U. [Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000, Kangar, Perlis (Malaysia); Hushiarian, R. [La Trobe Institute for Molecular Science, La Trobe University, Victoria, 3086 (Australia); Nuzaihan, M.N.M. [Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000, Kangar, Perlis (Malaysia); Hamidon, M.N. [Institute of Advanced Technology, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor (Malaysia); Zawawi, R.M. [Chemistry Department, Faculty of Science, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor (Malaysia); Fathil, M.F.M. [Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000, Kangar, Perlis (Malaysia)

    2016-10-26

    Dengue Virus (DENV) has become one of the most serious arthropod-borne viral diseases, causing death globally. The existing methods for DENV detection suffer from the late stage treatment due to antibodies-based detection which is feasible only after five days following the onset of the illness. Here, we demonstrated the highly effective molecular electronic based detection utilizing silicon nanowire (SiNW) integrated with standard complementary metal-oxide-semiconductor (CMOS) process as a sensing device for detecting deoxyribonucleic acid (DNA) related to DENV in an early stage diagnosis. To transform the fabricated devices as a functional sensing element, three-step procedure consist of SiNW surface modification, DNA immobilization and DNA hybridization were employed. The detection principle works by detecting the changes in current of SiNW which bridge the source and drain terminal to sense the immobilization of probe DNA and their hybridization with target DNA. The oxygen (O{sub 2}) plasma was proposed as an effective strategy for increasing the binding amounts of target DNA by modified the SiNW surface. It was found that the detection limit of the optimized O{sub 2} plasma treated-SiNW device could be reduced to 1.985 × 10{sup −14} M with a linear detection range of the sequence-specific DNA from 1.0 × 10{sup −9} M to 1.0 × 10{sup −13} M. In addition, the developed biosensor device was able to discriminate between complementary, single mismatch and non-complementary DNA sequences. This highly sensitive assay was then applied to the detection of reverse transcription-polymerase chain reaction (RT-PCR) product of DENV-DNA, making it as a potential method for disease diagnosis through electrical biosensor. - Highlights: • Molecular electronic detection of Dengue Virus (DENV) DNA using SiNW biosensor is presented. • Oxygen plasma surface treatment as an enhancer technique for device sensitivity is highlighted. • The limit of detection (Lo

  1. Enhanced sensing of dengue virus DNA detection using O_2 plasma treated-silicon nanowire based electrical biosensor

    International Nuclear Information System (INIS)

    Rahman, S.F.A.; Yusof, N.A.; Hashim, U.; Hushiarian, R.; Nuzaihan, M.N.M.; Hamidon, M.N.; Zawawi, R.M.; Fathil, M.F.M.

    2016-01-01

    Dengue Virus (DENV) has become one of the most serious arthropod-borne viral diseases, causing death globally. The existing methods for DENV detection suffer from the late stage treatment due to antibodies-based detection which is feasible only after five days following the onset of the illness. Here, we demonstrated the highly effective molecular electronic based detection utilizing silicon nanowire (SiNW) integrated with standard complementary metal-oxide-semiconductor (CMOS) process as a sensing device for detecting deoxyribonucleic acid (DNA) related to DENV in an early stage diagnosis. To transform the fabricated devices as a functional sensing element, three-step procedure consist of SiNW surface modification, DNA immobilization and DNA hybridization were employed. The detection principle works by detecting the changes in current of SiNW which bridge the source and drain terminal to sense the immobilization of probe DNA and their hybridization with target DNA. The oxygen (O_2) plasma was proposed as an effective strategy for increasing the binding amounts of target DNA by modified the SiNW surface. It was found that the detection limit of the optimized O_2 plasma treated-SiNW device could be reduced to 1.985 × 10"−"1"4 M with a linear detection range of the sequence-specific DNA from 1.0 × 10"−"9 M to 1.0 × 10"−"1"3 M. In addition, the developed biosensor device was able to discriminate between complementary, single mismatch and non-complementary DNA sequences. This highly sensitive assay was then applied to the detection of reverse transcription-polymerase chain reaction (RT-PCR) product of DENV-DNA, making it as a potential method for disease diagnosis through electrical biosensor. - Highlights: • Molecular electronic detection of Dengue Virus (DENV) DNA using SiNW biosensor is presented. • Oxygen plasma surface treatment as an enhancer technique for device sensitivity is highlighted. • The limit of detection (LoD) as low as 1.985

  2. A novel GMO biosensor for rapid ultrasensitive and simultaneous detection of multiple DNA components in GMO products.

    Science.gov (United States)

    Huang, Lin; Zheng, Lei; Chen, Yinji; Xue, Feng; Cheng, Lin; Adeloju, Samuel B; Chen, Wei

    2015-04-15

    Since the introduction of genetically modified organisms (GMOs), there has been on-going and continuous concern and debates on the commercialization of products derived from GMOs. There is an urgent need for development of highly efficient analytical methods for rapid and high throughput screening of GMOs components, as required for appropriate labeling of GMO-derived foods, as well as for on-site inspection and import/export quarantine. In this study, we describe, for the first time, a multi-labeling based electrochemical biosensor for simultaneous detection of multiple DNA components of GMO products on the same sensing interface. Two-round signal amplification was applied by using both an exonuclease enzyme catalytic reaction and gold nanoparticle-based bio-barcode related strategies, respectively. Simultaneous multiple detections of different DNA components of GMOs were successfully achieved with satisfied sensitivity using this electrochemical biosensor. Furthermore, the robustness and effectiveness of the proposed approach was successfully demonstrated by application to various GMO products, including locally obtained and confirmed commercial GMO seeds and transgenetic plants. The proposed electrochemical biosensor demonstrated unique merits that promise to gain more interest in its use for rapid and on-site simultaneous multiple screening of different components of GMO products. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Molecular beacon based biosensor for the sequence-specific detection of DNA using DNA-capped gold nanoparticles-streptavidin conjugates for signal amplification

    International Nuclear Information System (INIS)

    Fang, Xian; Jiang, Wei; Han, Xiaowei; Zhang, Yuzhong

    2013-01-01

    We describe a highly sensitive and selective molecular beacon-based electrochemical impedance biosensor for the sequence-specific detection of DNA. DNA-capped conjugates between gold nanoparticles (Au-NPs) and streptavidin are used for signal amplification. The molecular beacon was labeled with a thiol at its 5′ end and with biotin at its 3′ end, and then immobilized on the surface of a bare gold electrode through the formation of Au-S bonds. Initially, the molecular beacon is present in the “closed” state, and this shields the biotin from being approached by streptavidin due to steric hindrance. In the presence of the target DNA, the target DNA molecules hybridize with the loop and cause a conformational change that moves the biotin away from the surface of the electrode. The biotin thereby becomes accessible for the reporter (the DNA-streptavidin capped Au-NPs), and this results in a distinct increase in electron transfer resistance. Under optimal conditions, the increase in resistance is linearly related to the logarithm of the concentration of complementary target DNA in the range from 1.0 fM to 0.1 μM, with a detection limit of 0.35 fM (at an S/N of 3). This biosensor exhibits good selectivity, and acceptable stability and reproducibility. (author)

  4. Sensitive DNA impedance biosensor for detection of cancer, chronic lymphocytic leukemia, based on gold nanoparticles/gold modified electrode

    International Nuclear Information System (INIS)

    Ensafi, Ali A.; Taei, M.; Rahmani, H.R.; Khayamian, T.

    2011-01-01

    Highlights: → Chronic lymphocytic leukemia causes an increase in the number of white blood cells. → We introduced a highly sensitive biosensor for the detection of chronic lymphocytic leukemia. → A suitable 25-mer ssDNA probe was immobilized on the surface of the gold nanoparticles. → We used electrochemical impedance spectroscopy as a suitable tool for the detection. → Detection of chronic lymphocytic leukemia in blood sample was checked using the sensor. - Abstract: A simple and sensitive DNA impedance sensor was prepared for the detection of chronic lymphocytic leukemia. The DNA electrochemical biosensor is worked based on the electrochemical impedance spectroscopic (EIS) detection of the sequence-specific DNA related to chronic lymphocytic leukemia. The ssDNA probe was immobilized on the surface of the gold nanoparticles. Compared to the bare gold electrode, the gold nanoparticles-modified electrode could improve the density of the probe DNA attachment and hence the sensitivity of the DNA sensor greatly. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy were performed in a solution containing 1.0 mmol L -1 K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] and 50 mmol L -1 phosphate buffer saline pH 6.87 plus 50 mmol L -1 KCl. In the CV studied, the potential was cycled from 0.0 to +0.65 V with a scan rate of 50 mV s -1 . Using EIS, the difference of the electron transfer resistance (ΔR et ) was linear with the logarithm of the complementary oligonucleotides sequence concentrations in the range of 7.0 x 10 -12 -2.0 x 10 -7 mol L -1 , with a detection limit of 1.0 x 10 -12 mol L -1 . In addition, the DNA sensor showed a good reproducibility and stability during repeated regeneration and hybridization cycles.

  5. Roughness Effects on Fretting Fatigue

    Science.gov (United States)

    Yue, Tongyan; Abdel Wahab, Magd

    2017-05-01

    Fretting is a small oscillatory relative motion between two normal loaded contact surfaces. It may cause fretting fatigue, fretting wear and/or fretting corrosion damage depending on various fretting couples and working conditions. Fretting fatigue usually occurs at partial slip condition, and results in catastrophic failure at the stress levels below the fatigue limit of the material. Many parameters may affect fretting behaviour, including the applied normal load and displacement, material properties, roughness of the contact surfaces, frequency, etc. Since fretting damage is undesirable due to contacting, the effect of rough contact surfaces on fretting damage has been studied by many researchers. Experimental method on this topic is usually focusing on rough surface effects by finishing treatment and random rough surface effects in order to increase fretting fatigue life. However, most of numerical models on roughness are based on random surface. This paper reviewed both experimental and numerical methodology on the rough surface effects on fretting fatigue.

  6. An Electrochemical DNA Biosensor for the Detection of Salmonella Using Polymeric Films and Electrochemical Labels

    Science.gov (United States)

    Diaz Serrano, Madeline

    Waterborne and foodborne diseases are one of the principal public health problems worldwide. Microorganisms are the major agents of foodborne illness: pathogens such as Salmonella, Campylobacter jejuni and Escherichia coli, and parasites such as cryptosporidium. The most popular methods to detect Salmonella are based on culture and colony counting methods, ELISA, Gel electrophoresis and the polymerase chain reaction. Conventional detection methods are laborious and time-consuming, allowing for portions of the food to be distributed, marketed, sold and eaten before the analysis is done and the problem even detected. By these reasons, the rapid, easy and portable detection of foodborne organisms will facilitate the disease treatment. Our particular interest is to develop a nucleic acid biosensor (NAB) for the detection of pathogenic microorganisms in food and water samples. In this research, we report on the development of a NAB prototype using a polymer modified electrode surface together with sequences of different lengths for the OmpC gene from Salmonella as probes and Ferrocene-labeled target (Fc-ssDNA), Ferrocene-labeled tri(ethylene glycol) (Fc-PEG) and Ruthenium-Ferrocene (Ru-Fe) bimetallic complex as an electrochemical labels. We have optimized several PS films and anchored nucleic acid sequences with different lengths at gold and carbon surfaces. Non contact mode AFM and XPS were used to monitor each step of the NAB preparation, from polymer modification to oligos hybridization (conventional design). The hybridization reaction was followed electrochemically using a Fc-ssDNA and Fc-PEG in solution taking advantage of the morphological changes generated upon hybridization. We observed a small current at the potential for the Fe oxidation without signal amplification at +296 mV vs. Ag/AgCl for the Fc-ssDNA strategy and a small current at +524 mV for the Fc-PEG strategy. The immobilization, hybridization and signal amplification of Biotin- OmpC Salmonella genes

  7. A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection.

    Science.gov (United States)

    Chi, Chun-Wei; Lao, Yeh-Hsing; Li, Yi-Shan; Chen, Lin-Chi

    2011-03-15

    A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

    Energy Technology Data Exchange (ETDEWEB)

    Lucarelli, Fausto; Marrazza, Giovanna; Palchetti, Ilaria; Cesaretti, S.; Mascini, Marco

    2002-09-26

    This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence. The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised. The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.

  9. Facilitating the indirect detection of genomic DNA in an electrochemical DNA biosensor using magnetic nanoparticles and DNA ligase

    Directory of Open Access Journals (Sweden)

    Roozbeh Hushiarian

    2015-12-01

    This technique was found to be reliably repeatable. The indirect detection of genomic DNA using this method is significantly improved and showed high efficiency in small amounts of samples with the detection limit of 5.37 × 10−14 M.

  10. Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox

    Energy Technology Data Exchange (ETDEWEB)

    Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Heng, Lee Yook; Karim, Nurul Huda Abd [School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan (Malaysia); Ahmad, Haslina; Harun, Siti Norain [Chemistry Department, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor (Malaysia)

    2014-09-03

    A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy){sub 2}(PIP)]{sup 2+}, (bpy = 2,2′bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy){sub 2}(PIP)]{sup 2+} was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy){sub 2}(PIP)]{sup 2+} with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.

  11. A DNA biosensor based on gold nanoparticle decorated on carboxylated multi-walled carbon nanotubes for gender determination of Arowana fish.

    Science.gov (United States)

    Saeedfar, Kasra; Heng, Lee Yook; Chiang, Chew Poh

    2017-12-01

    Multi-wall carbon nanotubes (MWCNTs) were modified to design a new DNA biosensor. Functionalized MWCNTs were equipped with gold nanoparticles (GNPs) (~15nm) (GNP-MWCNTCOOH) to construct DNA biosensors based on carbon-paste screen-printed (SPE) electrodes. GNP attachment onto functionalized MWCNTs was carried out by microwave irradiation and was confirmed by spectroscopic studies and surface analysis. DNA biosensors based on differential pulse voltammetry (DPV) were constructed by immobilizing thiolated single-stranded DNA probes onto GNP-MWCNTCOOH. Ruthenium (III) chloride hexaammoniate [Ru(NH 3 ) 6 ,2Cl - ] (RuHex) was used as hybridization redox indicator. RuHex and MWCNT interaction was low in compared to other organic redox hybridization indicators. The linear response range for DNA determination was 1×10 -21 to 1×10 -9 M with a lower detection limit of 1.55×10 -21 M. Thus, the attachment of GNPs onto functionalized MWCNTs yielded sensitive DNA biosensor with low detection limit and stability more than 30days. Constructed electrode was used to determine gender of arowana fish. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Electrochemical study of quinone redox cycling: A novel application of DNA-based biosensors for monitoring biochemical reactions.

    Science.gov (United States)

    Ensafi, Ali A; Jamei, Hamid Reza; Heydari-Bafrooei, Esmaeil; Rezaei, B

    2016-10-01

    This paper presents the results of an experimental investigation of voltammetric and impedimetric DNA-based biosensors for monitoring biological and chemical redox cycling reactions involving free radical intermediates. The concept is based on associating the amounts of radicals generated with the electrochemical signals produced, using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). For this purpose, a pencil graphite electrode (PGE) modified with multiwall carbon nanotubes and poly-diallydimethlammonium chloride decorated with double stranded fish sperm DNA was prepared to detect DNA damage induced by the radicals generated from a redox cycling quinone (i.e., menadione (MD; 2-methyl-1,4-naphthoquinone)). Menadione was employed as a model compound to study the redox cycling of quinones. A direct relationship was found between free radical production and DNA damage. The relationship between MD-induced DNA damage and free radical generation was investigated in an attempt to identify the possible mechanism(s) involved in the action of MD. Results showed that DPV and EIS were appropriate, simple and inexpensive techniques for the quantitative and qualitative comparisons of different reducing reagents. These techniques may be recommended for monitoring DNA damages and investigating the mechanisms involved in the production of redox cycling compounds. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

    Science.gov (United States)

    Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

    2015-06-16

    A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

  14. Electrochemical DNA biosensor for the detection of Trichoderma harzianum based on a gold electrode modified with a composite membrane made from an ionic liquid, ZnO nanoparticles and chitosan, and by using acridine orange as a redox indicator

    International Nuclear Information System (INIS)

    Siddiquee, S.; Yusof, N.A.; Salleh, A.B.; Tan, S.G.; Bakar, F.A.

    2011-01-01

    An electrochemical DNA biosensor was developed that is based on a gold electrode modified with a nanocomposite membrane made from an ionic liquid, ZnO nanoparticles and chitosan. A single-stranded DNA probe was immobilized on this electrode. Acridine orange was used as the hybridization probe for monitoring the hybridization of the target DNA. The biosensor was capable of detecting target DNA in the concentration range from 1.0 x 10 -14 to 1.8 x 10 -4 mol L -1 , with a detection limit of 1.0 x 10 -15 mol L -1 . The approach towards constructing a DNA biosensor allows studies on the hybridization even with crude DNA fragments and also to analyze sample obtained from real samples. The results show that the DNA biosensor has the potential for sensitive detection of a specific sequence of the Trichoderma harzianum gene and provides a quick, sensitive and convenient method for the study of microorganisms. (author)

  15. In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

    International Nuclear Information System (INIS)

    Hsu, Y.-Y.; Liu, Y.-N.; Wang Wenyen; Kao, Fu-Jen; Kung, S.-H.

    2007-01-01

    An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP 2 )-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A pro ) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A pro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research

  16. DNA directed protein immobilization on mixed ssDNA/oligo /ethylene glycol/ self-assembled monolayers for sensitive biosensors

    Czech Academy of Sciences Publication Activity Database

    Boozer, C.; Ladd, J.; Chen, S.; Yu, Q.; Homola, Jiří; Jiang, S. Y.

    2004-01-01

    Roč. 76, č. 23 (2004), s. 6967-6972 ISSN 0003-2700 Grant - others:US FDA(US) FD-U-002250 Institutional research plan: CEZ:AV0Z2067918 Keywords : arrays * biosensors * surface plasmon resonance * gold Subject RIV: JB - Sensors, Measurment, Regulation Impact factor: 5.450, year: 2004

  17. Investigation of cleaning and regeneration methods for reliable construction of DNA cantilever biosensors

    DEFF Research Database (Denmark)

    Quan, Xueling; Yi, Sun; Heiskanen, Arto

    to clean and regenerate the sensing surface of cantilever biosensors. Perchloric acid potential sweep, potassium hydroxide-hydrogen peroxide, and piranha cleaning are investigated here. Peak-current potential differences from cyclic voltammetry, X-ray photo-electron spectroscopy and fluorescence detection...

  18. Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy

    Science.gov (United States)

    Zhao, Ming; Huang, Run; Peng, Leilei

    2012-01-01

    Förster resonant energy transfer (FRET) is extensively used to probe macromolecular interactions and conformation changes. The established FRET lifetime analysis method measures the FRET process through its effect on the donor lifetime. In this paper we present a method that directly probes the time-resolved FRET signal with frequency domain Fourier lifetime excitation-emission matrix (FLEEM) measurements. FLEEM separates fluorescent signals by their different phonon energy pathways from excitation to emission. The FRET process generates a unique signal channel that is initiated by donor excitation but ends with acceptor emission. Time-resolved analysis of the FRET EEM channel allows direct measurements on the FRET process, unaffected by free fluorophores that might be present in the sample. Together with time-resolved analysis on non-FRET channels, i.e. donor and acceptor EEM channels, time resolved EEM analysis allows precise quantification of FRET in the presence of free fluorophores. The method is extended to three-color FRET processes, where quantification with traditional methods remains challenging because of the significantly increased complexity in the three-way FRET interactions. We demonstrate the time-resolved EEM analysis method with quantification of three-color FRET in incompletely hybridized triple-labeled DNA oligonucleotides. Quantitative measurements of the three-color FRET process in triple-labeled dsDNA are obtained in the presence of free single-labeled ssDNA and double-labeled dsDNA. The results establish a quantification method for studying multi-color FRET between multiple macromolecules in biochemical equilibrium. PMID:23187535

  19. Titanium Dioxide Nanoparticle-Based Interdigitated Electrodes: A Novel Current to Voltage DNA Biosensor Recognizes E. coli O157:H7.

    Directory of Open Access Journals (Sweden)

    Sh Nadzirah

    Full Text Available Nanoparticle-mediated bio-sensing promoted the development of novel sensors in the front of medical diagnosis. In the present study, we have generated and examined the potential of titanium dioxide (TiO2 crystalline nanoparticles with aluminium interdigitated electrode biosensor to specifically detect single-stranded E.coli O157:H7 DNA. The performance of this novel DNA biosensor was measured the electrical current response using a picoammeter. The sensor surface was chemically functionalized with (3-aminopropyl triethoxysilane (APTES to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system's physical characteristics. The complement of the target DNA of E. coli O157:H7 to the carboxylate-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. The specificity experiments indicate that the biosensor can discriminate between the complementary sequences from the base-mismatched and the non-complementary sequences. After duplex formation, the complementary target sequence can be quantified over a wide range with a detection limit of 1.0 x 10(-13M. With target DNA from the lysed E. coli O157:H7, we could attain similar sensitivity. Stability of DNA immobilized surface was calculated with the relative standard deviation (4.6%, displayed the retaining with 99% of its original response current until 6 months. This high-performance interdigitated DNA biosensor with high sensitivity, stability and non-fouling on a novel sensing platform is suitable for a wide range of biomolecular interactive analyses.

  20. PNA-PEG modified silicon platforms as functional bio-interfaces for applications in DNA microarrays and biosensors.

    Science.gov (United States)

    Cattani-Scholz, Anna; Pedone, Daniel; Blobner, Florian; Abstreiter, Gerhard; Schwartz, Jeffrey; Tornow, Marc; Andruzzi, Luisa

    2009-03-09

    The synthesis and characterization of two types of silicon-based biofunctional interfaces are reported; each interface bonds a dense layer of poly(ethylene glycol) (PEG(n)) and peptide nucleic acid (PNA) probes. Phosphonate self-assembled monolayers were derivatized with PNA using a maleimido-terminated PEG(45). Similarly, siloxane monolayers were functionalized with PNA using a maleimido-terminated PEG(45) spacer and were subsequently modified with a shorter methoxy-terminated PEG(12) ("back-filling"). The long PEG(45) spacer was used to distance the PNA probe from the surface and to minimize undesirable nonspecific adsorption of DNA analyte. The short PEG(12) "back-filler" was used to provide additional passivation of the surface against nonspecific DNA adsorption. X-ray photoelectron spectroscopic (XPS) analysis near the C 1s and N 1s ionization edges was done to characterize chemical groups formed in the near-surface region, which confirmed binding of PEG and PNA to the phosphonate and silane films. XPS also indicated that additional PEG chains were tethered to the surface during the back-filling process. Fluorescence hybridization experiments were carried out with complementary and noncDNA strands; both phosphonate and siloxane biofunctional surfaces were effective for hybridization of cDNA strands and significantly reduced nonspecific adsorption of the analyte. Spatial patterns were prepared by polydimethylsiloxane (PDMS) micromolding on the PNA-functionalized surfaces; selective hybridization of fluorescently labeled DNA was shown at the PNA functionalized regions, and physisorption at the probe-less PEG-functionalized regions was dramatically reduced. These results show that PNA-PEG derivatized phosphonate monolayers hold promise for the smooth integration of device surface chemistry with semiconductor technology for the fabrication of DNA biosensors. In addition, our results confirm that PNA-PEG derivatized self-assembled carboxyalkylsiloxane films are

  1. Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

    Science.gov (United States)

    Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

    2014-09-02

    A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

  2. A ratiometric electrochemical biosensor for the exosomal microRNAs detection based on bipedal DNA walkers propelled by locked nucleic acid modified toehold mediate strand displacement reaction.

    Science.gov (United States)

    Zhang, Jing; Wang, Liang-Liang; Hou, Mei-Feng; Xia, Yao-Kun; He, Wen-Hui; Yan, An; Weng, Yun-Ping; Zeng, Lu-Peng; Chen, Jing-Hua

    2018-04-15

    Sensitive and selective detection of microRNAs (miRNAs) in cancer cells derived exosomes have attracted rapidly growing interest owing to their potential in diagnostic and prognostic applications. Here, we design a ratiometric electrochemical biosensor based on bipedal DNA walkers for the attomolar detection of exosomal miR-21. In the presence of miR-21, DNA walkers are activated to walk continuously along DNA tracks, resulting in conformational changes as well as considerable increases of the signal ratio produced by target-respond and target-independent reporters. With the signal cascade amplification of DNA walkers, the biosensor exhibits ultrahigh sensitivity with the limit of detection (LOD) down to 67 aM. Furthermore, owing to the background-correcting function of target-independent reporters termed as reference reporters, the biosensor is robust and stable enough to be applied in the detection of exosomal miR-21 extracted from breast cancer cell lines and serums. In addition, because locked nucleic acid (LNA) modified toehold mediate strand displacement reaction (TMSDR) has extraordinary discriminative ability, the biosensor displays excellent selectivity even against the single-base-mismatched target. It is worth mentioning that our sensor is regenerative and stable for at least 5 cycles without diminution in sensitivity. In brief, the high sensitivity, selectivity and reproducibility, together with cheap, make the proposed biosensor a promising approach for exosomal miRNAs detection, in conjunction with early point-of-care testing (POCT) of cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Ultrasensitive label-free detection of DNA hybridization by sapphire-based graphene field-effect transistor biosensor

    Science.gov (United States)

    Xu, Shicai; Jiang, Shouzhen; Zhang, Chao; Yue, Weiwei; Zou, Yan; Wang, Guiying; Liu, Huilan; Zhang, Xiumei; Li, Mingzhen; Zhu, Zhanshou; Wang, Jihua

    2018-01-01

    Graphene has attracted much attention in biosensing applications for its unique properties. Because of one-atom layer structure, every atom of graphene is exposed to the environment, making the electronic properties of graphene are very sensitive to charged analytes. Therefore, graphene is an ideal material for transistors in high-performance sensors. Chemical vapor deposition (CVD) method has been demonstrated the most successful method for fabricating large area graphene. However, the conventional CVD methods can only grow graphene on metallic substrate and the graphene has to be transferred to the insulating substrate for further device fabrication. The transfer process creates wrinkles, cracks, or tears on the graphene, which severely degrade electrical properties of graphene. These factors severely degrade the sensing performance of graphene. Here, we directly fabricated graphene on sapphire substrate by high temperature CVD without the use of metal catalysts. The sapphire-based graphene was patterned and make into a DNA biosensor in the configuration of field-effect transistor. The sensors show high performance and achieve the DNA detection sensitivity as low as 100 fM (10-13 M), which is at least 10 times lower than prior transferred CVD G-FET DNA sensors. The use of the sapphire-based G-FETs suggests a promising future for biosensing applications.

  4. Biosensor Architectures for High-Fidelity Reporting of Cellular Signaling

    Science.gov (United States)

    Dushek, Omer; Lellouch, Annemarie C.; Vaux, David J.; Shahrezaei, Vahid

    2014-01-01

    Understanding mechanisms of information processing in cellular signaling networks requires quantitative measurements of protein activities in living cells. Biosensors are molecular probes that have been developed to directly track the activity of specific signaling proteins and their use is revolutionizing our understanding of signal transduction. The use of biosensors relies on the assumption that their activity is linearly proportional to the activity of the signaling protein they have been engineered to track. We use mechanistic mathematical models of common biosensor architectures (single-chain FRET-based biosensors), which include both intramolecular and intermolecular reactions, to study the validity of the linearity assumption. As a result of the classic mechanism of zero-order ultrasensitivity, we find that biosensor activity can be highly nonlinear so that small changes in signaling protein activity can give rise to large changes in biosensor activity and vice versa. This nonlinearity is abolished in architectures that favor the formation of biosensor oligomers, but oligomeric biosensors produce complicated FRET states. Based on this finding, we show that high-fidelity reporting is possible when a single-chain intermolecular biosensor is used that cannot undergo intramolecular reactions and is restricted to forming dimers. We provide phase diagrams that compare various trade-offs, including observer effects, which further highlight the utility of biosensor architectures that favor intermolecular over intramolecular binding. We discuss challenges in calibrating and constructing biosensors and highlight the utility of mathematical models in designing novel probes for cellular signaling. PMID:25099816

  5. A duplex DNA-gold nanoparticle probe composed as a colorimetric biosensor for sequence-specific DNA-binding proteins.

    Science.gov (United States)

    Ahn, Junho; Choi, Yeonweon; Lee, Ae-Ree; Lee, Joon-Hwa; Jung, Jong Hwa

    2016-03-21

    Using duplex DNA-AuNP aggregates, a sequence-specific DNA-binding protein, SQUAMOSA Promoter-binding-Like protein 12 (SPL-12), was directly determined by SPL-12-duplex DNA interaction-based colorimetric actions of DNA-Au assemblies. In order to prepare duplex DNA-Au aggregates, thiol-modified DNA 1 and DNA 2 were attached onto the surface of AuNPs, respectively, by the salt-aging method and then the DNA-attached AuNPs were mixed. Duplex-DNA-Au aggregates having the average size of 160 nm diameter and the maximum absorption at 529 nm were able to recognize SPL-12 and reached the equivalent state by the addition of ∼30 equivalents of SPL-12 accompanying a color change from red to blue with a red shift of the maximum absorption at 570 nm. As a result, the aggregation size grew to about 247 nm. Also, at higher temperatures of the mixture of duplex-DNA-Au aggregate solution and SPL-12, the equivalent state was reached rapidly. On the contrary, in the control experiment using Bovine Serum Albumin (BSA), no absorption band shift of duplex-DNA-Au aggregates was observed.

  6. An electrochemical impedance biosensor for Hg2+ detection based on DNA hydrogel by coupling with DNAzyme-assisted target recycling and hybridization chain reaction.

    Science.gov (United States)

    Cai, Wei; Xie, Shunbi; Zhang, Jin; Tang, Dianyong; Tang, Ying

    2017-12-15

    In this work, an electrochemical impedance biosensor for high sensitive detection of Hg 2+ was presented by coupling with Hg 2+ -induced activation of Mg 2+ -specific DNAzyme (Mg 2+ -DNAzyme) for target cycling and hybridization chain reaction (HCR) assembled DNA hydrogel for signal amplification. Firstly, we synthesized two different copolymer chains P1 and P2 by modifying hairpin DNA H3 and H4 with acrylamide polymer, respectively. Subsequently, Hg 2+ was served as trigger to activate the Mg 2+ -DNAzyme for selectively cleavage ribonucleobase-modified substrate in the presence of Mg 2+ . The partial substrate strand could dissociate from DNAzyme structure, and hybridize with capture probe H1 to expose its concealed sequence for further hybridization. With the help of the exposed sequence, the HCR between hairpin DNA H3 and H4 in P1 and P2 was initiated, and assembled a layer of DNA cross-linked hydrogel on the electrode surface. The formed non-conductive DNA hydrogel film could greatly hinder the interfacial electronic transfer which provided a possibility for us to construct a high sensitive impedance biosensor for Hg 2+ detection. Under the optimal conditions, the impedance biosensor showed an excellent sensitivity and selectivity toward Hg 2+ in a concentration range of 0.1pM - 10nM with a detection limit of 0.042pM Moreover, the real sample analysis reveal that the proposed biosensor is capable of discriminating Hg 2+ ions in reliable and quantitative manners, indicating this method has a promising potential for preliminary application in routine tests. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. In vitro evaluation of fluorescence glucose biosensor response.

    Science.gov (United States)

    Aloraefy, Mamdouh; Pfefer, T Joshua; Ramella-Roman, Jessica C; Sapsford, Kim E

    2014-07-08

    Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET) for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor.

  8. In Vitro Evaluation of Fluorescence Glucose Biosensor Response

    Directory of Open Access Journals (Sweden)

    Mamdouh Aloraefy

    2014-07-01

    Full Text Available Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor.

  9. [Application of DNA-based electrochemical biosensor in rapid detection of Escherichia coli exist in licorice decoction].

    Science.gov (United States)

    Zhao, Yu-Wen; Wang, Hai-Xia; Bie, Song-Tao; Shao, Qian; Wang, Chun-Hua; Wang, Dong-Heng; Li, Zheng

    2018-03-01

    A new method for detection of Escherichia coli exist in licorice decoction was developed by using DNA-based electrochemical biosensor. The thiolated capture probe was immobilized on a gold electrode at first. Then the aptamer for Escherichia coli was combined with the capture probe by hybridization. Due to the stronger interaction between the aptamer and the E. coli, the aptamer can dissociate from the capture probe in the presence of E. coli in licorice decoction. The biotinylated detection probe was hybridized with the single-strand capture probe. As a result, the electrochemical response to Escherichia coli can be measured by using differential pulse voltammetric in the presence of α-naphthyl phosphate. The plot of peak current vs. the logarithm of concentration in the range from 2.7×10² to 2.7×10⁸ CFU·mL⁻¹ displayed a linear relationship with a detection limit of 50 CFU·mL⁻¹. The relative standard deviation of 3 successive scans was 2.5%,2.1%,4.6% for 2×10²,2×10⁴,2×106:⁶ CFU·mL⁻¹ E. coli, respectively. The proposed procedure showed better specificity to E. coli in comparison to Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. In the detection of the real extractum glycyrrhizae, the results between the proposed strategy and the GB assay showed high degree of agreement, demonstrating the designed biosensor could be utilized as a powerful tool for microbial examination for traditional Chinese medicine. Copyright© by the Chinese Pharmaceutical Association.

  10. Development of DNA biosensor based on TiO2 nanoparticles

    Science.gov (United States)

    Nadzirah, Sh.; Hashim, U.; Rusop, M.

    2018-05-01

    A novel technique of DNA hybridization on the TiO2 nanoparticles film was developed by dropping a single droplet of target DNA onto the surface of TiO2 for the study of various concentrations of target DNA. The surface of TiO2 nanoparticle film was functionalized with APTES and covalently immobilized with 1 µM probe DNA on the silanized TiO2 nanoparticles surface. The effect of silanization, immobilization and hybridization were quantitatively measured by the output current signal obtained using a picoammeter. The 1 µM target DNA was found to be the most effective target towards the 1 µM probe DNA as the output current signal was within range; while the output current signal of the 10 µM target DNA was observed to beyond the range of the probe DNA control due to the excessive concentration as compared to the probe DNA. This approach has several advantages such as rapid, simple, low cost, and sensitive current signal during detection of different target DNA concentrations.

  11. Intercalating dye as an acceptor in quantum-dot-mediated FRET

    International Nuclear Information System (INIS)

    Lim, Teck Chuan; Bailey, Vasudev J; Wang, T-H; Ho, Y-P

    2008-01-01

    Fluorescence resonance energy transfer (FRET) is a popular tool to study intermolecular distances and characterize structural or conformational changes of biological macromolecules. We investigate a novel inorganic/organic FRET pair with quantum dots (QDs) as donors and DNA intercalating dyes, BOBO-3, as acceptors by using DNA as a linker. Typically, FRET efficiency increases with the number of stained DNA linked to a QD. However, with the use of intercalating dyes, we demonstrate that FRET efficiency at a fixed DNA:QD ratio can be further enhanced by increasing the number of dyes stained to a DNA strand through the use of an increased staining dye/bp ratio. We exploit this flexibility in the staining ratio to maintain a high FRET efficiency of >0.90 despite a sixfold decrease in DNA concentration. Having characterized this new QD-mediated FRET system, we test this system in a cellular environment using nanocomplexes generated by encapsulating DNA with commercial non-viral gene carriers. Using this novel FRET pair, we are able to monitor the configuration changes and fate of the DNA nanocomplexes during intracellular delivery, thereby providing an insight into the mechanistic study of gene delivery

  12. Effective immobilization of DNA for development of polypyrrole nanowires based biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Thi Luyen; Chu, Thi Xuan, E-mail: xuan@itims.edu.vn; Huynh, Dang Chinh; Pham, Duc Thanh; Luu, Thi Hoai Thuong; Mai, Anh Tuan, E-mail: tuan.maianh@hust.edu.vn

    2014-09-30

    Highlights: • Effective technique to immobilize probe DNA to the conducting polymer Polypyrrole nanowires (PPy NWs). • The PPy-NWs were electrochemically synthesized on the surface of the Pt electrodes using gelatin as the soft mold. • The DNA probe sequences were immobilized easily on the PPy NWs/Pt electrode using the adsorption method. • The DNA sensor has a low detection limit. - Abstract: This paper reports an easy technique for immobilization of the DNA to the conducting polymer polypyrrole nanowires (PPy NWs). The nanowires were electrochemically synthesized on the surface of working electrode in the presence of gelatin as a soft mold. The structure of obtained PPy NWs was investigated by Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy and Surface Enhanced Raman Spectroscopy (SERS). The DNA strands were directly immobilized on the PPy NWs. The amino groups at the up-end of the PPy nanowires facilitate the linkage with the phosphate groups of the probe DNA. The DNA immobilization and hybridization were characterized by Electrochemical Impedance Spectroscopy (EIS). The initial results show that the sensor responses to 10 pM of DNA sequence in the solution.

  13. Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

    DEFF Research Database (Denmark)

    Zhan, Zongrui; Ma, Xingyi; Cao, Cuong

    2011-01-01

    In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target...

  14. Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Viviane Devauges

    Full Text Available We present a novel imaging system combining total internal reflection fluorescence (TIRF microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

  15. DNA biosensor for detection of Salmonella typhi from blood sample of typhoid fever patient using gold electrode modified by self-assembled monolayers of thiols

    Science.gov (United States)

    Suryapratiwi, Windha Novita; Paat, Vlagia Indira; Gaffar, Shabarni; Hartati, Yeni Wahyuni

    2017-05-01

    Electrochemical biosensors are currently being developed in order to handle various clinical problems in diagnosing infectious diseases caused by pathogenic bacteria, or viruses. On this research, voltammetric DNA biosensor using gold electrode modified by thiols with self-assembled monolayers had been developed to detect a certain sequence of Salmonella typhi DNA from blood sample of typhoid fever patient. Thiol groups of cysteamines (Cys) and aldehyde groups from glutaraldehydes (Glu) were used as a link to increase the performance of gold electrode in detecting guanine oxidation signal of hybridized S. typhi DNA and ssDNA probe. Standard calibration method was used to determine analytical parameters from the measurements. The result shown that, the detection of S. typhi DNA from blood sample of typhoid fever patient can be carried out by voltammetry using gold electrode modified by self-assembled monolayers of thiols. A characteristic oxidation potential of guanine using Au/Cys/Gluwas obtained at +0.17 until +0.20 V. Limit of detection and limit of quantification from this measurements were 1.91μg mL-1 and 6.35 μg mL-1. The concentration of complement DNA from sample was 6.96 μg mL-1.

  16. The effect of microscopic attractive interactions on piezoelectric coefficients of nanoscale DNA films and its resultant mirocantilever-based biosensor signals

    International Nuclear Information System (INIS)

    Wu, Jun-Zheng; Zhang, Neng-Hui; Zhou, Mei-Hong

    2017-01-01

    The adsorption of charged biomolecules on a substrate will trigger a self-induced electric potential field that could deflect microcantilever biosensors in the nanometer regime. The paper is devoted to a multiscale characterization of the piezoelectric coefficient of double-stranded DNA (dsDNA) films with microscopic attractive interactions in multivalence salt solutions, which has a close relationship with biosensor signals. First, two different analytical models of cantilever deflections based on macroscopic piezoelectric theories or mesoscopic liquid crystal theories were combined in the sense of equivalent deformation in order to bridge the relation between the macroscopic piezoelectric coefficient of an adsorbate film and the sensitivity of its microstructure to surrounding conditions. Second, two interaction potentials of the free energy for repulsion-dominated DNA films in NaCl solution or attraction-repulsion-coexisted DNA films in multivalent salt solutions were used to compare the piezoelectric effect and the resultant cantilever deformation at various packing conditions, such as different packing density, various nucleotide numbers and two packing technologies, i.e. nano-grafting or self-assembling technology. The variational tendency of microcantilever deflections predicted by the present multiscale analytical model agrees well with the related DNA-mirocantilever experiments. Negative piezoelectric coefficient of dsDNA film exists in multivalent salt solutions, and its distinctive size effect with different packing densities and nucleotide numbers provides us with an opportunity to obtain a more sensitive microcantilever sensor by careful control of packing conditions. (paper)

  17. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    Directory of Open Access Journals (Sweden)

    Huarong Guo

    2012-09-01

    Full Text Available p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase and pRL-CMV-luc (CMV promoter linked to Renilla luciferase into marine flatfish flounder gill (FG cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation, but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl phthalate (DEHP, a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

  18. Rapid and label-free electrochemical DNA biosensor for detecting hepatitis A virus.

    Science.gov (United States)

    Manzano, Marisa; Viezzi, Sara; Mazerat, Sandra; Marks, Robert S; Vidic, Jasmina

    2018-02-15

    Diagnostic systems that can deliver highly specific and sensitive detection of hepatitis A virus (HAV) in food and water are of particular interest in many fields including food safety, biosecurity and control of outbreaks. Our aim was the development of an electrochemical method based on DNA hybridization to detect HAV. A ssDNA probe specific for HAV (capture probe) was designed and tested on DNAs from various viral and bacterial samples using Nested-Reverse Transcription Polymerase Chain Reaction (nRT-PCR). To develop the electrochemical device, a disposable gold electrode was functionalized with the specific capture probe and tested on complementary ssDNA and on HAV cDNA. The DNA hybridization on the electrode was measured through the monitoring of the oxidative peak potential of the indicator tripropylamine by cyclic voltammetry. To prevent non-specific binding the gold surface was treated with 3% BSA before detection. High resolution atomic force microscopy (AFM) confirmed the efficiency of electrode functionalization and on-electrode hybridization. The proposed device showed a limit of detection of 0.65pM for the complementary ssDNA and 6.94fg/µL for viral cDNA. For a comparison, nRT-PCR quantified the target HAV cDNA with a limit of detection of 6.4fg/µL. The DNA-sensor developed can be adapted to a portable format to be adopted as an easy-to- use and low cost method for screening HAV in contaminated food and water. In addition, it can be useful for rapid control of HAV infections as it takes only a few minutes to provide the results. Copyright © 2017. Published by Elsevier B.V.

  19. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

    Science.gov (United States)

    Cox, Annie M; Goodwin, Kelly D

    2013-08-15

    The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. Published by Elsevier Ltd.

  20. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors

    International Nuclear Information System (INIS)

    Cox, Annie M.; Goodwin, Kelly D.

    2013-01-01

    Highlights: • DNA extraction methods affected measured qPCR target recovery. • Recovery and variability differed, sometimes by more than an order of magnitude. • SCODA did not offer significant improvement with PCR-inhibited seawater. • Aggressive lysis did appear to improve target recovery. • Reliable and affordable correction methods are needed for quantitative PCR. -- Abstract: The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications

  1. Rapid DNA multi-analyte immunoassay on a magneto-resistance biosensor

    NARCIS (Netherlands)

    Koets, M.; Wijk, van der T.; Eemeren, van J.T.W.M.; Amerongen, van A.; Prins, M.W.J.

    2009-01-01

    We present the rapid and sensitive detection of amplified DNA on a giant magneto-resistance sensor using superparamagnetic particles as a detection label. The one-step assay is performed on an integrated and miniaturized detection platform suitable for application into point-of-care devices. A

  2. Optical biosensors.

    Science.gov (United States)

    Damborský, Pavel; Švitel, Juraj; Katrlík, Jaroslav

    2016-06-30

    Optical biosensors represent the most common type of biosensor. Here we provide a brief classification, a description of underlying principles of operation and their bioanalytical applications. The main focus is placed on the most widely used optical biosensors which are surface plasmon resonance (SPR)-based biosensors including SPR imaging and localized SPR. In addition, other optical biosensor systems are described, such as evanescent wave fluorescence and bioluminescent optical fibre biosensors, as well as interferometric, ellipsometric and reflectometric interference spectroscopy and surface-enhanced Raman scattering biosensors. The optical biosensors discussed here allow the sensitive and selective detection of a wide range of analytes including viruses, toxins, drugs, antibodies, tumour biomarkers and tumour cells. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  3. Characteristic of fretting damage in metal material

    Energy Technology Data Exchange (ETDEWEB)

    Li, D.; Zhi, F.

    1988-10-01

    The fretting fatigue experiment of LC4 high strength aluminum alloy is described. An SEM examination of the fractology and morphology of fretting damage is carried out as well as an EDAX analysis of the chemical composition of fretting particles. The results show that many loose oxide particles were produced and accumulated in the fretting damage region. 10 references.

  4. Biosensors based on gold nanostructures

    OpenAIRE

    Vidotti,Marcio; Carvalhal,Rafaela F.; Mendes,Renata K.; Ferreira,Danielle C. M.; Kubota,Lauro T.

    2011-01-01

    The present review discusses the latest advances in biosensor technology achieved by the assembly of biomolecules associated with gold nanoparticles in analytical devices. This review is divided in sections according to the biomolecule employed in the biosensor development: (i) immunocompounds; (ii) DNA/RNA and functional DNA/RNA; and (iii) enzymes and Heme proteins. In order to facilitate the comprehension each section was subdivided according to the transduction mode. Gold nanoparticles bas...

  5. Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

    NARCIS (Netherlands)

    Mastop, M.; Bindels, D.S.; Shaner, N.C.; Postma, M.; Gadella, T.W.J.; Goedhart, J.

    2017-01-01

    The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching

  6. Characterizing single-molecule FRET dynamics with probability distribution analysis.

    Science.gov (United States)

    Santoso, Yusdi; Torella, Joseph P; Kapanidis, Achillefs N

    2010-07-12

    Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single-molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two-state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis-testing models of conformational dynamics against experimental data.

  7. A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

    Directory of Open Access Journals (Sweden)

    Jakobus van Unen

    Full Text Available G-protein coupled receptors (GPCRs can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.

  8. The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

    Directory of Open Access Journals (Sweden)

    Shaoying Lu

    2008-07-01

    Full Text Available Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP experiments, we have developed a finite element (FE method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2/sec than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2/sec and outside: 0.18+/-0.02 microm(2/sec. The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

  9. DNA impedance biosensor for detection of cancer, TP53 gene mutation, based on gold nanoparticles/aligned carbon nanotubes modified electrode.

    Science.gov (United States)

    Fayazfar, H; Afshar, A; Dolati, M; Dolati, A

    2014-07-11

    For the first time, a new platform based on electrochemical growth of Au nanoparticles on aligned multi-walled carbon nanotubes (A-MWCNT) was developed for sensitive lable-free DNA detection of the TP53 gene mutation, one of the most popular genes in cancer research. Electrochemical impedance spectroscopy (EIS) was used to monitor the sequence-specific DNA hybridization events related to TP53 gene. Compared to the bare Ta or MWCNT/Ta electrodes, the synergistic interactions of vertically aligned MWCNT array and gold nanoparticles at modified electrode could improve the density of the probe DNA attachment and resulting the sensitivity of the DNA sensor greatly. Using EIS, over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship in respect to the logarithm of the complementary oligonucleotides sequence concentrations in the wide range of 1.0×10(-15)-1.0×10(-7)M, with a detection limit of 1.0×10(-17)M (S/N=3). The prepared sensor also showed good stability (14 days), reproducibility (RSD=2.1%) and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining gold nanoparticles with the on-site fabricated aligned MWCNT array represents a promising platform for achieving sensitive biosensor for fast mutation screening related to most human cancer types. Copyright © 2014. Published by Elsevier B.V.

  10. Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection.

    Science.gov (United States)

    Yao, B C; Wu, Y; Yu, C B; He, J R; Rao, Y J; Gong, Y; Fu, F; Chen, Y F; Li, Y R

    2016-03-24

    Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel 'FRET on Fiber' concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based 'FRET on fiber' configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated 'FRET on Fiber' sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response.

  11. Synthesis and Characterization of Polyaniline/Graphene Composite Nanofiber and Its Application as an Electrochemical DNA Biosensor for the Detection of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Fatimah Syahidah Mohamad

    2017-12-01

    Full Text Available This article describes chemically modified polyaniline and graphene (PANI/GP composite nanofibers prepared by self-assembly process using oxidative polymerization of aniline monomer and graphene in the presence of a solution containing poly(methyl vinyl ether-alt-maleic acid (PMVEA. Characterization of the composite nanofibers was carried out by Fourier transform infrared (FTIR and Raman spectroscopy, transmission electron microscopy (TEM and scanning electron microscopy (SEM. SEM images revealed the size of the PANI nanofibers ranged from 90 to 360 nm in diameter and was greatly influenced by the proportion of PMVEA and graphene. The composite nanofibers with an immobilized DNA probe were used for the detection of Mycobacterium tuberculosis by using an electrochemical technique. A photochemical indicator, methylene blue (MB was used to monitor the hybridization of target DNA by using differential pulse voltammetry (DPV method. The detection range of DNA biosensor was obtained from of 10−6–10−9 M with the detection limit of 7.853 × 10−7 M under optimum conditions. The results show that the composite nanofibers have a great potential in a range of applications for DNA sensors.

  12. Microbial biosensors

    International Nuclear Information System (INIS)

    Le Yu; Chen, Wilfred; Mulchandani, Ashok

    2006-01-01

    A microbial biosensor is an analytical device that couples microorganisms with a transducer to enable rapid, accurate and sensitive detection of target analytes in fields as diverse as medicine, environmental monitoring, defense, food processing and safety. The earlier microbial biosensors used the respiratory and metabolic functions of the microorganisms to detect a substance that is either a substrate or an inhibitor of these processes. Recently, genetically engineered microorganisms based on fusing of the lux, gfp or lacZ gene reporters to an inducible gene promoter have been widely applied to assay toxicity and bioavailability. This paper reviews the recent trends in the development and application of microbial biosensors. Current advances and prospective future direction in developing microbial biosensor have also been discussed

  13. Synthetic Electric Microbial Biosensors

    Science.gov (United States)

    2017-06-10

    domains and DNA-binding domains into a single protein for deregulation of down stream genes of have been favored [10]. Initially experiments with... Germany DISTRIBUTION A. Approved for public release: distribution unlimited.   Talk title: “Synthetic biology based microbial biosensors for the...toolbox” in Heidelberg, Germany Poster title: “Anaerobic whole cell microbial biosensors” Link: http://phdsymposium.embl.org/#home   September, 2014

  14. Multi-step surface functionalization of polyimide based evanescent wave photonic biosensors and application for DNA hybridization by Mach-Zehnder interferometer

    Energy Technology Data Exchange (ETDEWEB)

    Melnik, Eva [Health and Environment Department, Nano Systems, AIT Austrian Institute of Technology GmbH, Donau-City-Strasse 1, 1220 Vienna (Austria); Department of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna (Austria); Bruck, Roman [Health and Environment Department, Nano Systems, AIT Austrian Institute of Technology GmbH, Donau-City-Strasse 1, 1220 Vienna (Austria); Hainberger, Rainer, E-mail: rainer.hainberger@ait.ac.at [Health and Environment Department, Nano Systems, AIT Austrian Institute of Technology GmbH, Donau-City-Strasse 1, 1220 Vienna (Austria); Laemmerhofer, Michael, E-mail: michael.laemmerhofer@univie.ac.at [Department of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna (Austria)

    2011-08-12

    Highlights: {yields} We realize a biosensing platform for polyimide evanescent photonic wave sensors. {yields} We show that the surface functionalization via silanisation and biotinylation followed by streptavidin immobilization do not destroy or damage the thin polyimide film. {yields} A highly dense streptavidin layer enables the immobilisation of biotinylated ligands such as biotinylated ssDNA for the selective measurement of DNA hybridization. - Abstract: The process of surface functionalization involving silanization, biotinylation and streptavidin bonding as platform for biospecific ligand immobilization was optimized for thin film polyimide spin-coated silicon wafers, of which the polyimide film serves as a wave guiding layer in evanescent wave photonic biosensors. This type of optical sensors make great demands on the materials involved as well as on the layer properties, such as the optical quality, the layer thickness and the surface roughness. In this work we realized the binding of a 3-mercaptopropyl trimethoxysilane on an oxygen plasma activated polyimide surface followed by subsequent derivatization of the reactive thiol groups with maleimide-PEG{sub 2}-biotin and immobilization of streptavidin. The progress of the functionalization was monitored by using different fluorescence labels for optimization of the chemical derivatization steps. Further, X-ray photoelectron spectroscopy and atomic force microscopy were utilized for the characterization of the modified surface. These established analytical methods allowed to derive information like chemical composition of the surface, surface coverage with immobilized streptavidin, as well as parameters of the surface roughness. The proposed functionalization protocol furnished a surface density of 144 fmol mm{sup -2} streptavidin with good reproducibility (13.9% RSD, n = 10) and without inflicted damage to the surface. This surface modification was applied to polyimide based Mach-Zehnder interferometer

  15. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.

    Science.gov (United States)

    Bajar, Bryce T; Wang, Emily S; Lam, Amy J; Kim, Bongjae B; Jacobs, Conor L; Howe, Elizabeth S; Davidson, Michael W; Lin, Michael Z; Chu, Jun

    2016-02-16

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.

  16. Highly Sensitive DNA Sensor Based on Upconversion Nanoparticles and Graphene Oxide.

    Science.gov (United States)

    Alonso-Cristobal, P; Vilela, P; El-Sagheer, A; Lopez-Cabarcos, E; Brown, T; Muskens, O L; Rubio-Retama, J; Kanaras, A G

    2015-06-17

    In this work we demonstrate a DNA biosensor based on fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er nanoparticles and graphene oxide (GO). Monodisperse NaYF4:Yb,Er nanoparticles with a mean diameter of 29.1 ± 2.2 nm were synthesized and coated with a SiO2 shell of 11 nm, which allowed the attachment of single strands of DNA. When these DNA-functionalized NaYF4:Yb,Er@SiO2 nanoparticles were in the proximity of the GO surface, the π-π stacking interaction between the nucleobases of the DNA and the sp(2) carbons of the GO induced a FRET fluorescence quenching due to the overlap of the fluorescence emission of the NaYF4:Yb,Er@SiO2 and the absorption spectrum of GO. By contrast, in the presence of the complementary DNA strands, the hybridization leads to double-stranded DNA that does not interact with the GO surface, and thus the NaYF4:Yb,Er@SiO2 nanoparticles remain unquenched and fluorescent. The high sensitivity and specificity of this sensor introduces a new method for the detection of DNA with a detection limit of 5 pM.

  17. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    Science.gov (United States)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  18. EXPERIMENTAL INVESTIGTION OF THE FRETTING PHENOMENON

    Directory of Open Access Journals (Sweden)

    Ştefan GHIMISI

    2015-12-01

    Full Text Available Fretting is now fully identified as a small amplitude oscilatory motion which induces a harmonic tangential force between two surfaces in contact.It is related to three main loadings, i.e. fretting-wear, fretting-fatigue and fretting corrosion.Fretting regimes were first mapped by Vingsbo. In a similar way, three fretting regimes will be considered: stick regime,slip regime and mixed regime. The mixed regime was made up of initial gross slip followed by partial slip condition after a few hundred cycles. Obviously the partial slip transition develops the highest stress levels which can induce fatigue crack nucleation depending on the fatigue properties of the two contacting first bodies. Therefore prediction of the frontier between partial slip and gross slip is required.

  19. A novel electrochemical DNA biosensor based on a modified magnetic bar carbon paste electrode with Fe{sub 3}O{sub 4}NPs-reduced graphene oxide/PANHS nanocomposite

    Energy Technology Data Exchange (ETDEWEB)

    Jahanbani, Shahriar; Benvidi, Ali, E-mail: abenvidi@yazd.ac.ir

    2016-11-01

    In this study, we have designed a label free DNA biosensor based on a magnetic bar carbon paste electrode (MBCPE) modified with nanomaterial of Fe{sub 3}O{sub 4}/reduced graphene oxide (Fe{sub 3}O{sub 4}NP-RGO) as a composite and 1- pyrenebutyric acid-N- hydroxysuccinimide ester (PANHS) as a linker for detection of DNA sequences. Probe (BRCA1 5382 insC mutation detection) strands were immobilized on the MBCPE/Fe{sub 3}O{sub 4}-RGO/PANHS electrode for the exact incubation time. The characterization of the modified electrode was studied using different techniques such as scanning electron microscopy (SEM), infrared spectroscopy (IR), vibrating sample magnetometer (VSM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry methods. Some experimental parameters such as immobilization time of probe DNA, time and temperature of hybridization process were investigated. Under the optimum conditions, the immobilization of the probe and its hybridization with the target DNA (Complementary DNA) were tested. This DNA biosensor revealed a good linear relationship between ∆ R{sub ct} and logarithm of the complementary target DNA concentration ranging from 1.0 × 10{sup −18} mol L{sup −1} to 1.0 × 10{sup −8} mol L{sup −1} with a correlation coefficient of 0.9935 and a detection limit of 2.8 × 10{sup −19} mol L{sup −1}. In addition, the mentioned biosensor was satisfactorily applied for discriminating of complementary sequences from non-complementary sequences. The constructed biosensor (MBCPE/Fe{sub 3}O{sub 4}-RGO/PANHS/ssDNA) with high sensitivity, selectivity, stability, reproducibility and low cost can be used for detection of BRCA1 5382 insC mutation. - Highlights: • We have designed a MBCPE/Fe{sub 3}O{sub 4}-RGO/PANHS/ssDNA for determination of BRCA1 5382. • The magnetic bar was used for fabrication of CPE for completely adsorption of Fe3O4-RGO. • The proposed electrode showed a detection limit as low as 2.8 × 10{sup −19} M for target

  20. Classic maximum entropy recovery of the average joint distribution of apparent FRET efficiency and fluorescence photons for single-molecule burst measurements.

    Science.gov (United States)

    DeVore, Matthew S; Gull, Stephen F; Johnson, Carey K

    2012-04-05

    We describe a method for analysis of single-molecule Förster resonance energy transfer (FRET) burst measurements using classic maximum entropy. Classic maximum entropy determines the Bayesian inference for the joint probability describing the total fluorescence photons and the apparent FRET efficiency. The method was tested with simulated data and then with DNA labeled with fluorescent dyes. The most probable joint distribution can be marginalized to obtain both the overall distribution of fluorescence photons and the apparent FRET efficiency distribution. This method proves to be ideal for determining the distance distribution of FRET-labeled biomolecules, and it successfully predicts the shape of the recovered distributions.

  1. Plasmonic biosensors.

    Science.gov (United States)

    Hill, Ryan T

    2015-01-01

    The unique optical properties of plasmon resonant nanostructures enable exploration of nanoscale environments using relatively simple optical characterization techniques. For this reason, the field of plasmonics continues to garner the attention of the biosensing community. Biosensors based on propagating surface plasmon resonances (SPRs) in films are the most well-recognized plasmonic biosensors, but there is great potential for the new, developing technologies to surpass the robustness and popularity of film-based SPR sensing. This review surveys the current plasmonic biosensor landscape with emphasis on the basic operating principles of each plasmonic sensing technique and the practical considerations when developing a sensing platform with the various techniques. The 'gold standard' film SPR technique is reviewed briefly, but special emphasis is devoted to the up-and-coming localized surface plasmon resonance and plasmonically coupled sensor technology. © 2014 Wiley Periodicals, Inc.

  2. Electrochemical biosensors

    CERN Document Server

    Cosnier, Serge

    2015-01-01

    "This is an excellent book on modern electrochemical biosensors, edited by Professor Cosnier and written by leading international experts. It covers state-of-the-art topics of this important field in a clear and timely manner."-Prof. Joseph Wang, UC San Diego, USA  "This book covers, in 13 well-illustrated chapters, the potential of electrochemical methods intimately combined with a biological component for the assay of various analytes of biological and environmental interest. Particular attention is devoted to the description of electrochemical microtools in close contact with a biological cell for exocytosis monitoring and to the use of nanomaterials in the electrochemical biosensor architecture for signal improvement. Interestingly, one chapter describes the concept and design of self-powered biosensors derived from biofuel cells. Each topic is reviewed by experts very active in the field. This timely book is well suited for providing a good overview of current research trends devoted to electrochemical...

  3. Highly sensitive "signal-on" electrochemiluminescent biosensor for the detection of DNA based on dual quenching and strand displacement reaction.

    Science.gov (United States)

    Lou, Jing; Wang, Zhaoyin; Wang, Xiao; Bao, Jianchun; Tu, Wenwen; Dai, Zhihui

    2015-10-07

    A "signal-on" electrochemiluminescent DNA biosensing platform was proposed based on the dual quenching and strand displacement reaction. This novel "signal-on" detection strategy revealed its sensitivity in achieving a detection limit of 2.4 aM and its selectivity in distinguishing single nucleotide polymorphism of target DNA.

  4. 48-spot single-molecule FRET setup with periodic acceptor excitation

    Science.gov (United States)

    Ingargiola, Antonino; Segal, Maya; Gulinatti, Angelo; Rech, Ivan; Labanca, Ivan; Maccagnani, Piera; Ghioni, Massimo; Weiss, Shimon; Michalet, Xavier

    2018-03-01

    Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

  5. Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor.

    Science.gov (United States)

    Xiang, Yang; Zhu, Xiaoyan; Huang, Qing; Zheng, Junsong; Fu, Weiling

    2015-04-15

    In this study, we developed a surface plasmon resonance (SPR) DNA biosensor array based on target-primed rolling circle amplification (RCA) for isothermal and rapid detection of two pathogenic mycobacteria, Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC).The species-specific padlock probe (PLP) was designed to target the sequence in 16S-23S rRNA gene internal transcribed spacer (ITS). After ligation, the circularized PLP could be primed by the target sequence to initial RCA. The RCA performed simultaneously with the cleavage reaction to produce small fragments of single strand DNA which immediately hybridized with the probe immobilized on the sensor chip without denaturation. This process caused SPR angle changes on the chip surface, which made the detection for analysis from the solution achievable, and dynamic real-time RCA monitoring of mycobacterium possible. Besides, Au nanoparticles (AuNPs) were directly assembled onto the surface of the sensor chip via hexanedithiol (HDT) for the enhancement of sensitivity as a label-free detection system. Experimental results show that the signal enhancement by the target-primed RCA together with AuNPs-embedded surface caused at least10-fold increased sensitivity as compared with conventional RCA on bare SPR chip method. Within 40min amplification duration as low as 20amol of synthetic targets and 10(4)CFUmL(-1) of genomic DNA from clinical samples can be detected. The proposed method not only provides a simple design idea for liquid-phase amplification monitoring, but also apply it in clinical pathogen detection, which holds great promise in ultrasensitive bioassay in the future. Copyright © 2014. Published by Elsevier B.V.

  6. FRET-based modified graphene quantum dots for direct trypsin quantification in urine

    Energy Technology Data Exchange (ETDEWEB)

    Poon, Chung-Yan; Li, Qinghua [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Zhang, Jiali; Li, Zhongping [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Dong, Chuan [Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Lee, Albert Wai-Ming; Chan, Wing-Hong [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Li, Hung-Wing, E-mail: hwli@hkbu.edu.hk [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong)

    2016-04-21

    A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening. - Highlights: • A FRET-based biosensor was developed for direct quantification of trypsin. • Fast and sensitive screening of pancreatic disease was facilitated. • The direct quantification of trypsin in urine samples was demonstrated.

  7. Standard guide for fretting fatigue testing

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 This guide defines terminology and covers general requirements for conducting fretting fatigue tests and reporting the results. It describes the general types of fretting fatigue tests and provides some suggestions on developing and conducting fretting fatigue test programs. 1.2 Fretting fatigue tests are designed to determine the effects of mechanical and environmental parameters on the fretting fatigue behavior of metallic materials. This guide is not intended to establish preference of one apparatus or specimen design over others, but will establish guidelines for adherence in the design, calibration, and use of fretting fatigue apparatus and recommend the means to collect, record, and reporting of the data. 1.3 The number of cycles to form a fretting fatigue crack is dependent on both the material of the fatigue specimen and fretting pad, the geometry of contact between the two, and the method by which the loading and displacement are imposed. Similar to wear behavior of materials, it is important t...

  8. Potential of cross-priming amplification and DNA-based lateral-flow strip biosensor for rapid on-site GMO screening.

    Science.gov (United States)

    Huang, Xin; Zhai, Congcong; You, Qimin; Chen, Hongjun

    2014-07-01

    The requirement to monitor the presence of genetically modified organisms (GMO) in a variety of marked products has generated an increasing demand for reliable, rapid, and time and cost-effective analytical methods. Here we report an on-site method for rapid detection of cauliflower mosaic virus promoter (CaMV 35S), a common element present in most GMO, using cross-priming amplification (CPA) technology. Detection was achieved using a DNA-based contamination-proof strip biosensor. The limit of detection was 30 copies for the pBI121 plasmid containing the CaMV 35S gene. The certified reference sample of GM maize line MON810 was detectable even at the low relative mass concentration of 0.05%. The developed CPA method had high specificity for the CaMV 35S gene, as compared with other GM lines not containing this gene and non-GM products. The method was further validated using nine real-world samples, and the results were confirmed by real-time PCR analysis. Because of its simplicity, rapidity, and high sensitivity, this method of detecting the CaMV 35S gene has great commercial prospects for rapid GMO screening of high-consumption food and agriculture products.

  9. Stuy on Fatigue Life of Aluminum Alloy Considering Fretting

    Science.gov (United States)

    Yang, Maosheng; Zhao, Hongqiang; Wang, Yunxiang; Chen, Xiaofei; Fan, Jiali

    2018-01-01

    To study the influence of fretting on Aluminum Alloy, a global finite element model considering fretting was performed using the commercial code ABAQUS. With which a new model for predicting fretting fatigue life has been presented based on friction work. The rationality and effectiveness of the model were validated according to the contrast of experiment life and predicting life. At last influence factor on fretting fatigue life of aerial aluminum alloy was investigated with the model. The results revealed that fretting fatigue life decreased monotonously with the increasing of normal load and then became constant at higher pressures. At low normal load, fretting fatigue life was found to increase with increase in the pad radius. At high normal load, however, the fretting fatigue life remained almost unchanged with changes in the fretting pad radius. The bulk stress amplitude had the dominant effect on fretting fatigue life. The fretting fatigue life diminished as the bulk stress amplitude increased.

  10. MnO2 nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

    Science.gov (United States)

    Ou, Min; Huang, Jin; Yang, Xiaohai; Quan, Ke; Yang, Yanjing; Xie, Nuli; Wang, Kemin

    2017-01-01

    The donor donor-acceptor (DD-A) FRET model has proven to have a higher FRET efficiency than donor-acceptor acceptor (D-AA), donor-acceptor (D-A), and donor donor-acceptor acceptor (DD-AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the "DD-A" FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary "D-A" model. Furthermore, MnO 2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn 2+ ions by intracellular GSH. The results indicated that the MnO 2 nanosheet mediated "DD-A" FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO 2 nanosheet mediated "DD-A" FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

  11. In-Channel-Grown Polypyrrole Nanowire for the Detection of DNA Hybridization in an Electrochemical Microfluidic Biosensor

    Directory of Open Access Journals (Sweden)

    Thi Luyen Tran

    2015-01-01

    Full Text Available A triple electrode setup with a Pt pseudo-reference electrode integrated in a polydimethylsiloxane- (PDMS- based microchamber was designed and fabricated. The integrated electrodes were deposited onto SiO2/Si substrate by sputtering. The PDMS microchamber was patterned using an SU-8 mold and sealed with electrodes in oxygen plasma. Polypyrrole nanowires (PPy NWs were electrochemically grown in situ at an accurate position of the working electrode in the sealed microchamber instead of in an open system. The DNA probe sequences were simply introduced into the channel to form bonds with the nanowires. A detection limit of 20 pM was achieved using a lock-in amplifier. The electrochemical characteristics produced by the hybridization of DNA strands in the microchamber showed a good signal/noise ratio and high sensitivity. Measurement of the DNA sensor in narrow space also required much less volume of the analytical sample compared with that in an open measuring cell. Results showed that this simple system can potentially fabricate nanostructures and detect bio/chemical molecules in a sealed system.

  12. Development and testing of a fluorescence biosensor for glucose sensing

    Science.gov (United States)

    Aloraefy, Mamdouh; Pfefer, Joshua; Ramella-Roman, Jessica; Sapsford, Kim

    2012-06-01

    Rapid, accurate, and minimally-invasive biosensors for glucose measurement have the potential to enhance management of diabetes mellitus and improve patient outcome in intensive care settings. Recent studies have indicated that implantable biosensors based on Förster Resonance Energy Transfer (FRET) can provide high sensitivity in quantifying glucose concentrations. However, standard approaches for determining the potential for interference from other biological constituents have not been established. The aim of this work was to design and optimize a FRET-based glucose sensor and assess its specificity to glucose. A sensor based on competitive binding between concanavalin A and dextran, labeled with long-wavelength acceptor and donor fluorophores, was developed. This process included optimization of dextran molecular weight and donor concentration, acceptor to donor ratio, and hydrogel concentration, as well as the number of polymer layers for encapsulation. The biosensor performance was characterized in terms of its response to clinically relevant glucose concentrations. The potential for interference and the development of test methods to evaluate this effect were studied using a potential clinical interferent, maltose. Results indicated that our biosensor had a prediction accuracy of better than 11% and that the robustness to maltose was highly dependent on glucose level.

  13. The Leakage determination on corrosion fretting machine

    International Nuclear Information System (INIS)

    Sriyono; Satmoko, Ari; Hafid, Abdul; Febrianto; Prasetio, Joko; Abtokhi; Sumarno, Edy; Handoyo, Ismu; Hidayati, Nur Rahmah; Histori

    1998-01-01

    Fretting machine is an experimental loop to learn fretting corrosion phenomena wich is caused by loading and vibration. On the steam generator, one of the corrosion process that's occurred, it can be caused by vibration between tubes and bending material. Because of high flow rate inside the tube, the high frequency vibration will appeared so it can make the corrosion on bending material more faster. This process can be simulate by fretting machine. This machine has already damage because of leakage. So it will be repaired by dismantling, radiography testing and redrawing. from the result of radiography, the leakage is caused by cracking on bellows seals of the dynamic main support

  14. Understanding and modeling Förster-type resonance energy transfer (FRET) introduction to FRET

    CERN Document Server

    Govorov, Alexander; Demir, Hilmi Volkan

    2016-01-01

    This Brief presents a historical overview of the Förster-type nonradiative energy transfer and a compilation of important progress in FRET research, starting from Förster until today, along with a summary of the current state-of-the-art. Here the objective is to provide the reader with a complete account of important milestones in FRET studies and FRET applications as well as a picture of the current status.

  15. Les vicissitudes du fret ferroviaire

    OpenAIRE

    DABLANC, L

    2010-01-01

    Dans beaucoup de pays européens, et plus encore en Amérique du Nord et en Asie, le transport de marchandises par le train a augmenté depuis dix ans. Cette activité réduit la part des marchandises acheminées par la route et contribue ainsi au développement durable : un camion émet 8 à 30 fois plus de dioxyde de carbone que le train, pour une distance et une quantité transportée équivalentes. Pourtant, la France a raté ce renouveau. Filiale du groupe public SNCF, la Société Fret SNCF, qui assur...

  16. A novel probe density controllable electrochemiluminescence biosensor for ultra-sensitive detection of Hg2+ based on DNA hybridization optimization with gold nanoparticles array patterned self-assembly platform.

    Science.gov (United States)

    Gao, Wenhua; Zhang, An; Chen, Yunsheng; Chen, Zixuan; Chen, Yaowen; Lu, Fushen; Chen, Zhanguang

    2013-11-15

    Biosensor based on DNA hybridization holds great potential to get higher sensitivity as the optimal DNA hybridization efficiency can be achieved by controlling the distribution and orientation of probe strands on the transducer surface. In this work, an innovative strategy is reported to tap the sensitivity potential of current electrochemiluminescence (ECL) biosensing system by dispersedly anchoring the DNA beacons on the gold nanoparticles (GNPs) array which was electrodeposited on the glassy carbon electrode surface, rather than simply sprawling the coil-like strands onto planar gold surface. The strategy was developed by designing a "signal-on" ECL biosensing switch fabricated on the GNPs nanopatterned electrode surface for enhanced ultra-sensitivity detection of Hg(2+). A 57-mer hairpin-DNA labeled with ferrocene as ECL quencher and a 13-mer DNA labeled with Ru(bpy)3(2+) as reporter were hybridized to construct the signal generator in off-state. A 31-mer thymine (T)-rich capture-DNA was introduced to form T-T mismatches with the loop sequence of the hairpin-DNA in the presence of Hg(2+) and induce the stem-loop open, meanwhile the ECL "signal-on" was triggered. The peak sensitivity with the lowest detection limit of 0.1 nM was achieved with the optimal GNPs number density while exorbitant GNPs deposition resulted in sensitivity deterioration for the biosensor. We expect the present strategy could lead the renovation of the existing probe-immobilized ECL genosensor design to get an even higher sensitivity in ultralow level of target detection such as the identification of genetic diseases and disorders in basic research and clinical application. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Fiber optic-based biosensor

    Science.gov (United States)

    Ligler, Frances S.

    1991-01-01

    The NRL fiber optic biosensor is a device which measures the formation of a fluorescent complex at the surface of an optical fiber. Antibodies and DNA binding proteins provide the mechanism for recognizing an analyze and immobilizing a fluorescent complex on the fiber surface. The fiber optic biosensor is fast, sensitive, and permits analysis of hazardous materials remote from the instrumentation. The fiber optic biosensor is described in terms of the device configuration, chemistry for protein immobilization, and assay development. A lab version is being used for assay development and performance characterization while a portable device is under development. Antibodies coated on the fiber are stable for up to two years of storage prior to use. The fiber optic biosensor was used to measure concentration of toxins in the parts per billion (ng/ml) range in under a minute. Immunoassays for small molecules and whole bacteria are under development. Assays using DNA probes as the detection element can also be used with the fiber optic sensor, which is currently being developed to detect biological warfare agents, explosives, pathogens, and toxic materials which pollute the environment.

  18. DNA biosensor combining single-wavelength colorimetry and a digital lock-in amplifier within a smartphone.

    Science.gov (United States)

    Wu, Tzu-Heng; Chang, Chia-Chen; Vaillant, Julien; Bruyant, Aurélien; Lin, Chii-Wann

    2016-11-15

    Smartphone camera based gold nanoparticle colorimetry (SCB-AuNP colorimetry) has shown good potential for point-of-care applications. However, due to the use of a camera as a photo-detector, there are major limitations to this technique such as a low bit resolution (∼8 bits mainstream) and a low data acquisition rate. These issues have limited the ultimate sensitivity of smartphone based colorimetry as well as the possibility to integrate efficiently a more sensitive approach such as detection based on a lock-in amplifier (LIA). In this paper, we improve the metrological performance of the smartphone to overcome existing issues by adding the LIA capability to AuNP sensing. In this work, instead of using the camera as a photo-detector, the audio jack is used as a photo-detector reader and function generator for driving a laser diode in order to achieve a smartphone based digital lock-in amplifier AuNP colorimetric (SBLIA-AuNP colorimetry) system. A full investigation on the SBLIA design, parameters and performance is comprehensively provided. It is found that the SBLIA can reduce most of the noise and provides a detection noise-to-signal ratio down to -63 dB, which is much better than the -49 dB of the state-of-the-art SCB based method. A DNA detection experiment is demonstrated to reveal the efficacy of the proposed metrological method. The results are compared to UV-visible spectrometry, which is the gold standard for colorimetric measurement. Based on our results, the SBLIA-AuNP colorimetric system has a detection limit of 0.77 nM on short strand DNA detection, which is 5.7 times better than the 4.36 nM limit of a commercial UV-visible spectrometer. Judging from the results, we believe that the sensitive SBLIA would be further extended to other optical diagnostic tools in the near future.

  19. Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

    Science.gov (United States)

    Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

    2000-01-01

    A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

  20. Gold nanoparticle-based electrochemical biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Pingarron, Jose M.; Yanez-Sedeno, Paloma; Gonzalez-Cortes, Araceli [Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid (Spain)

    2008-08-01

    The unique properties of gold nanoparticles to provide a suitable microenvironment for biomolecules immobilization retaining their biological activity, and to facilitate electron transfer between the immobilized proteins and electrode surfaces, have led to an intensive use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance with respect to other biosensor designs. Recent advances in this field are reviewed in this article. The advantageous operational characteristics of the biosensing devices designed making use of gold nanoparticles are highlighted with respect to non-nanostructured biosensors and some illustrative examples are commented. Electrochemical enzyme biosensors including those using hybrid materials with carbon nanotubes and polymers, sol-gel matrices, and layer-by-layer architectures are considered. Moreover, electrochemical immunosensors in which gold nanoparticles play a crucial role in the electrode transduction enhancement of the affinity reaction as well as in the efficiency of immunoreagents immobilization in a stable mode are reviewed. Similarly, recent advances in the development of DNA biosensors using gold nanoparticles to improve DNA immobilization on electrode surfaces and as suitable labels to improve detection of hybridization events are considered. Finally, other biosensors designed with gold nanoparticles oriented to electrically contact redox enzymes to electrodes by a reconstitution process and to the study of direct electron transfer between redox proteins and electrode surfaces have also been treated. (author)

  1. Synthetic biology for microbial heavy metal biosensors.

    Science.gov (United States)

    Kim, Hyun Ju; Jeong, Haeyoung; Lee, Sang Jun

    2018-02-01

    Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.

  2. Gold nanoparticle-based electrochemical biosensors

    International Nuclear Information System (INIS)

    Pingarron, Jose M.; Yanez-Sedeno, Paloma; Gonzalez-Cortes, Araceli

    2008-01-01

    The unique properties of gold nanoparticles to provide a suitable microenvironment for biomolecules immobilization retaining their biological activity, and to facilitate electron transfer between the immobilized proteins and electrode surfaces, have led to an intensive use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance with respect to other biosensor designs. Recent advances in this field are reviewed in this article. The advantageous operational characteristics of the biosensing devices designed making use of gold nanoparticles are highlighted with respect to non-nanostructured biosensors and some illustrative examples are commented. Electrochemical enzyme biosensors including those using hybrid materials with carbon nanotubes and polymers, sol-gel matrices, and layer-by-layer architectures are considered. Moreover, electrochemical immunosensors in which gold nanoparticles play a crucial role in the electrode transduction enhancement of the affinity reaction as well as in the efficiency of immunoreagents immobilization in a stable mode are reviewed. Similarly, recent advances in the development of DNA biosensors using gold nanoparticles to improve DNA immobilization on electrode surfaces and as suitable labels to improve detection of hybridization events are considered. Finally, other biosensors designed with gold nanoparticles oriented to electrically contact redox enzymes to electrodes by a reconstitution process and to the study of direct electron transfer between redox proteins and electrode surfaces have also been treated

  3. Polymer Based Biosensors for Medical Applications

    DEFF Research Database (Denmark)

    Cherré, Solène; Rozlosnik, Noemi

    2015-01-01

    , environmental monitoring and food safety. The detected element varies from a single molecule (such as glucose), a biopolymer (such as DNA or a protein) to a whole organism (such as bacteria). Due to their easy use and possible miniaturization, biosensors have a high potential to come out of the lab...... and be available for use by everybody. To fulfil these purposes, polymers represent very appropriate materials. Many nano- and microfabrication methods for polymers are available, allowing a fast and cheap production of devices. This chapter will present the general concept of a biosensor in a first part......The objective of this chapter is to give an overview about the newest developments in biosensors made of polymers for medical applications. Biosensors are devices that can recognize and detect a target with high selectivity. They are widely used in many fields such as medical diagnostic...

  4. Electrochemical Aptamer Scaffold Biosensors for Detection of Botulism and Ricin Proteins.

    Science.gov (United States)

    Daniel, Jessica; Fetter, Lisa; Jett, Susan; Rowland, Teisha J; Bonham, Andrew J

    2017-01-01

    Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of molecular targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins. Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin. This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

  5. Electronic Biosensors Based on III-Nitride Semiconductors.

    Science.gov (United States)

    Kirste, Ronny; Rohrbaugh, Nathaniel; Bryan, Isaac; Bryan, Zachary; Collazo, Ramon; Ivanisevic, Albena

    2015-01-01

    We review recent advances of AlGaN/GaN high-electron-mobility transistor (HEMT)-based electronic biosensors. We discuss properties and fabrication of III-nitride-based biosensors. Because of their superior biocompatibility and aqueous stability, GaN-based devices are ready to be implemented as next-generation biosensors. We review surface properties, cleaning, and passivation as well as different pathways toward functionalization, and critically analyze III-nitride-based biosensors demonstrated in the literature, including those detecting DNA, bacteria, cancer antibodies, and toxins. We also discuss the high potential of these biosensors for monitoring living cardiac, fibroblast, and nerve cells. Finally, we report on current developments of covalent chemical functionalization of III-nitride devices. Our review concludes with a short outlook on future challenges and projected implementation directions of GaN-based HEMT biosensors.

  6. Biosensors and preparation thereof

    NARCIS (Netherlands)

    2008-01-01

    A low-temp. prepn. method for a biosensor device with a layer of reagent on the sensor surface is disclosed. During manufg. biol. interaction between the biosensor substrate and the reagent layer material is reduced, e.g. by cooling the biosensor substrate and depositing the reagent layer on the

  7. Cholinesterase-based biosensors.

    Science.gov (United States)

    Štěpánková, Šárka; Vorčáková, Katarína

    2016-01-01

    Recently, cholinesterase-based biosensors are widely used for assaying anticholinergic compounds. Primarily biosensors based on enzyme inhibition are useful analytical tools for fast screening of inhibitors, such as organophosphates and carbamates. The present review is aimed at compilation of the most important facts about cholinesterase based biosensors, types of physico-chemical transduction, immobilization strategies and practical applications.

  8. Surface amplification of pencil graphite electrode with polypyrrole and reduced graphene oxide for fabrication of a guanine/adenine DNA based electrochemical biosensors for determination of didanosine anticancer drug

    Science.gov (United States)

    Karimi-Maleh, Hassan; Bananezhad, Asma; Ganjali, Mohammad R.; Norouzi, Parviz; Sadrnia, Abdolhossein

    2018-05-01

    Didanosine is nucleoside analog reverse transcriptase inhibitors with many side effects such as nausea and vomiting, stomach pain, tingling, burning and numbness and determination of this drug is very important in biological samples. This paper presents a DNA biosensor for determination of didanosine (DDI) in pharmaceutical samples. A pencil graphite electrode modified with conductive materials such as polypyrrole (PPy) and reduced graphene oxide (rGO) (PGE/PPy/rGO) was used for this goal. The double-stranded DNA was successfully immobilized on PGE/PPy/rGO. The PGE/PPy/rGO was characterized by microscopic and electrochemical methods. Then, the interaction of DDI with DNA was identified by decreases in the oxidation currents of guanine and adenine by differential pulse voltammetric (DPV) method. The dynamic range of DDI identified in the range of 0.02-50.0 μM and this electrode provided a low limit of detection (LOD = 8.0 nM) for DDI. The PGE/PPy/rGO loaded with ds-DNA was utilized for the measurement of DDI in real samples and obtained data were compared with HPLC method. The statistical tests such as F-test and t-test were used for confirming ability of PGE/PPy/rGO loaded with ds-DNA for analysis of DDI in real samples.

  9. Electroactive crown ester-Cu2+ complex with in-situ modification at molecular beacon probe serving as a facile electrochemical DNA biosensor for the detection of CaMV 35s.

    Science.gov (United States)

    Zhan, Fengping; Liao, Xiaolei; Gao, Feng; Qiu, Weiwei; Wang, Qingxiang

    2017-06-15

    A novel electrochemical DNA biosensor has been facilely constructed by in-situ assembly of electroactive 4'-aminobenzo-18-crown-6-copper(II) complex (AbC-Cu 2+ ) on the free terminal of the hairpin-structured molecule beacon. The 3'-SH modified molecule beacon probe was first immobilized on the gold electrode (AuE) surface through self-assembly chemistry of Au-S bond. Then the crow ester of AbC was covalently coupled with 5'-COOH on the molecule beacon, and served as a platform to attach the Cu 2+ by coordination with ether bond (-O-) of the crown cycle. Thus, an electroactive molecule beacon-based biosensing interface was constructed. In comparison with conventional methods for preparation of electroactive molecule beacon, the approach presented in this work is much simpler, reagent- and labor-saving. Selectivity study shows that the in-situ fabricated electroactive molecule beacon remains excellent recognition ability of pristine molecule beacon probe to well differentiate various DNA fragments. The target DNA can be quantatively determined over the range from 0.10pM to 0.50nM. The detection limit of 0.060pM was estimated based on signal-to-noise ratio of 3. When the biosensor was applied for the detection cauliflower mosaic virus 35s (CaMV 35s) in soybean extraction samples, satisfactory results are achieved. This work opens a new strategy for facilely fabricating electrochemical sensing interface, which also shows great potential in aptasensor and immurosensor fabrication. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Biosensors of bacterial cells.

    Science.gov (United States)

    Burlage, Robert S; Tillmann, Joshua

    2017-07-01

    Biosensors are devices which utilize both an electrical component (transducer) and a biological component to study an environment. They are typically used to examine biological structures, organisms and processes. The field of biosensors has now become so large and varied that the technology can often seem impenetrable. Yet the principles which underlie the technology are uncomplicated, even if the details of the mechanisms are elusive. In this review we confine our analysis to relatively current advancements in biosensors for the detection of whole bacterial cells. This includes biosensors which rely on an added labeled component and biosensors which do not have a labeled component and instead detect the binding event or bound structure on the transducer. Methods to concentrate the bacteria prior to biosensor analysis are also described. The variety of biosensor types and their actual and potential uses are described. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Biosensors for the Detection and Quantification of AI-2 Class Quorum-Sensing Compounds.

    Science.gov (United States)

    Rajamani, Sathish; Sayre, Richard

    2018-01-01

    Intercellular small-molecular-weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum-sensing molecules. These molecules mediate a variety of population-dependent responses including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules have important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused to the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. Ligand-insensitive LuxP mutant FRET protein sensors were also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2 compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of BAI-2.

  12. Prediction of fretting fatigue behavior under elastic-plastic conditions

    International Nuclear Information System (INIS)

    Shin, Ki Su

    2009-01-01

    Fretting fatigue generally leads to the degradation of the fatigue strength of a material due to cyclic micro-slip between two contacting materials. Fretting fatigue is regarded as an important issue in designing aerospace structures. While many studies have evaluated fretting fatigue behavior under elastic deformation conditions, few have focused on fretting fatigue behavior under elastic-plastic deformation conditions, especially the crack orientation and fatigue life prediction for Ti-6Al-4V. The primary goal of this study was to characterize the fretting fatigue crack initiation behavior in the presence of plasticity. Experimental tests were performed using pad configurations involving elastic-plastic deformations. To calculate stress distributions under elastic-plastic fretting fatigue conditions, FEA was also performed. Several parametric approaches were used to predict fretting fatigue life along with stress distribution resulting from FEA. However, those parameters using surface stresses were unable to establish an equivalence between elastic fretting fatigue data and elastic-plastic fretting fatigue data. Based on this observation, the critical distance methods, which are commonly used in notch analysis, were applied to the fretting fatigue problem. In conclusion, the effective strain range method when used in conjunction with the SMSSR parameter showed a good correlation of data points between the pad configurations involving elastic and elastic plastic deformations

  13. Fiber Optic Surface Plasmon Resonance-Based Biosensor Technique: Fabrication, Advancement, and Application.

    Science.gov (United States)

    Liang, Gaoling; Luo, Zewei; Liu, Kunping; Wang, Yimin; Dai, Jianxiong; Duan, Yixiang

    2016-05-03

    Fiber optic-based biosensors with surface plasmon resonance (SPR) technology are advanced label-free optical biosensing methods. They have brought tremendous progress in the sensing of various chemical and biological species. This review summarizes four sensing configurations (prism, grating, waveguide, and fiber optic) with two ways, attenuated total reflection (ATR) and diffraction, to excite the surface plasmons. Meanwhile, the designs of different probes (U-bent, tapered, and other probes) are also described. Finally, four major types of biosensors, immunosensor, DNA biosensor, enzyme biosensor, and living cell biosensor, are discussed in detail for their sensing principles and applications. Future prospects of fiber optic-based SPR sensor technology are discussed.

  14. Parallel multispot smFRET analysis using an 8-pixel SPAD array

    Science.gov (United States)

    Ingargiola, A.; Colyer, R. A.; Kim, D.; Panzeri, F.; Lin, R.; Gulinatti, A.; Rech, I.; Ghioni, M.; Weiss, S.; Michalet, X.

    2012-02-01

    Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

  15. A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET).

    Science.gov (United States)

    Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes; Cordes, Thorben; Weiss, Shimon

    2016-07-07

    Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

  16. Biosensors and bioelectronics

    CERN Document Server

    Karunakaran, Chandran; Benjamin, Robson

    2015-01-01

    Biosensors and Bioelectronics presents the rapidly evolving methodologies that are relevant to biosensors and bioelectronics fabrication and characterization. The book provides a comprehensive understanding of biosensor functionality, and is an interdisciplinary reference that includes a range of interwoven contributing subjects, including electrochemistry, nanoparticles, and conducting polymers. Authored by a team of bioinstrumentation experts, this book serves as a blueprint for performing advanced fabrication and characterization of sensor systems-arming readers with an application-based re

  17. Plasmonic Nanostructures for Biosensor Applications

    Science.gov (United States)

    Gadde, Akshitha

    Improving the sensitivity of existing biosensors is an active research topic that cuts across several disciplines, including engineering and biology. Optical biosensors are the one of the most diverse class of biosensors which can be broadly categorized into two types based on the detection scheme: label-based and label-free detection. In label-based detection, the target bio-molecules are labeled with dyes or tags that fluoresce upon excitation, indicating the presence of target molecules. Label-based detection is highly-sensitive, capable of single molecule detection depending on the detector type used. One method of improving the sensitivity of label-based fluorescence detection is by enhancement of the emission of the labels by coupling them with metal nanostructures. This approach is referred as plasmon-enhanced fluorescence (PEF). PEF is achieved by increasing the electric field around the nano metal structures through plasmonics. This increased electric field improves the enhancement from the fluorophores which in turn improves the photon emission from the fluorophores which, in turn, improves the limit of detection. Biosensors taking advantage of the plasmonic properties of metal films and nanostructures have emerged an alternative, low-cost, high sensitivity method for detecting labeled DNA. Localized surface plasmon resonance (LSPR) sensors employing noble metal nanostructures have recently attracted considerable attention as a new class of plasmonic nanosensors. In this work, the design, fabrication and characterization of plasmonic nanostructures is carried out. Finite difference time domain (FDTD) simulations were performed using software from Lumerical Inc. to design a novel LSPR structure that exhibit resonance overlapping with the absorption and emission wavelengths of quantum dots (QD). Simulations of a composite Au/SiO2 nanopillars on silicon substrate were performed using FDTD software to show peak plasmonic enhancement at QD emission wavelength

  18. Introduction to Biosensors From Electric Circuits to Immunosensors

    CERN Document Server

    Yoon, Jeong-Yeol

    2013-01-01

    Introduction to Biosensors: From Electric Circuits to Immunosensors discusses underlying circuitry of sensors for biomedical and biological engineers as well as biomedical sensing modalities for electrical engineers while providing an applications-based approach to the study of biosensors with over 13 extensive, hands-on labs. The material is presented using a building-block approach, beginning with the fundamentals of sensor design and temperature sensors and ending with more complicated biosensors. This book also: Provides electrical engineers with the specific knowledge they need to understand biological sensing modalities Provides biomedical engineers with a solid background in circuits and systems Includes complete coverage of temperature sensors, electrochemical sensors, DNA and immunosensors, piezoelectric sensors and immunosensing in a micofluidic device Introduction to Biosensors: From Electric Circuits to Immunosensors aims to provide an interdisciplinary approach to biosensors that will be apprecia...

  19. Introduction to biosensors from electric circuits to immunosensors

    CERN Document Server

    Yoon, Jeong-Yeol

    2016-01-01

    This book equips students with a thorough understanding of various types of sensors and biosensors that can be used for chemical, biological, and biomedical applications, including but not limited to temperature sensors, strain sensor, light sensors, spectrophotometric sensors, pulse oximeter, optical fiber probes, fluorescence sensors, pH sensor, ion-selective electrodes, piezoelectric sensors, glucose sensors, DNA and immunosensors, lab-on-a-chip biosensors, paper-based lab-on-a-chip biosensors, and microcontroller-based sensors. The author treats the study of biosensors with an applications-based approach, including over 15 extensive, hands-on labs given at the end of each chapter. The material is presented using a building-block approach, beginning with the fundamentals of sensor design and temperature sensors, and ending with more complicated biosensors. New to this second edition are sections on op-amp filters, pulse oximetry, meat quality monitoring, advanced fluorescent dyes, autofluorescence, various...

  20. Biosensors for Cell Analysis.

    Science.gov (United States)

    Zhou, Qing; Son, Kyungjin; Liu, Ying; Revzin, Alexander

    2015-01-01

    Biosensors first appeared several decades ago to address the need for monitoring physiological parameters such as oxygen or glucose in biological fluids such as blood. More recently, a new wave of biosensors has emerged in order to provide more nuanced and granular information about the composition and function of living cells. Such biosensors exist at the confluence of technology and medicine and often strive to connect cell phenotype or function to physiological or pathophysiological processes. Our review aims to describe some of the key technological aspects of biosensors being developed for cell analysis. The technological aspects covered in our review include biorecognition elements used for biosensor construction, methods for integrating cells with biosensors, approaches to single-cell analysis, and the use of nanostructured biosensors for cell analysis. Our hope is that the spectrum of possibilities for cell analysis described in this review may pique the interest of biomedical scientists and engineers and may spur new collaborations in the area of using biosensors for cell analysis.

  1. Impedimetric biosensors and immunosensors

    International Nuclear Information System (INIS)

    Prodromidis, M.I.

    2007-01-01

    The development of methods targeting the direct monitoring of antibody-antigen interactions is particularly attractive. The design of label-free affinity-based probing concepts is the objective of much current research, at both academic and industrial levels, towards establishing alternative methods to the already existing ELISA-based immunoassays. Among these, Electrochemical Impedance Spectroscopy (EIS) represents one of the most powerful methods, due to the ability of EIS-based sensors to be more easily integrated into multi-array or microprocessor, controlled diagnostic tools. During the last decade, EIS and the concept of biochemical capacitors have been widely used for probing various types of biomolecular interactions (immunosensors, DNA hybridization, protein-protein interactions). So far, impedimetric or capacitive immunosensors have been successfully applied at the academic level. However, no prototypes have been released into the market, since major fundamental issues still exist. Even though this fact has brought the reliability of impedimetric immunosensors into question, features associated with electrochemical approaches, namely the ability to be miniaturized, remote control of implanted sensors, low cost of electrode mass production and cost effective instrumentation (without need of high-energy sources) keep impedimetric sensors particularly attractive as compared to other approaches based on microbalances, surface plasmon resonance or ellipsometry. This lecture outlines the theoretical background of impedimetric immunosensors and presents different types of impedimetric biosensors as well as the instrumental approaches that have been so far proposed in the literature. (author)

  2. Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy

    Directory of Open Access Journals (Sweden)

    Daniel Sultanov

    2017-01-01

    Full Text Available DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose polymerase 1 increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA.

  3. A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.

    Directory of Open Access Journals (Sweden)

    Gerard N M van der Krogt

    Full Text Available We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.

  4. The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites

    Directory of Open Access Journals (Sweden)

    Abrenica Maria V

    2010-09-01

    Full Text Available Abstract Background Mycobacterium tuberculosis topoisomerase I (MtTOP1 and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex. Results We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate. Conclusions The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.

  5. CONSIDERATIONS REGARDING THE FRETTING PHENOMENON USING LEAF SPRINGS

    Directory of Open Access Journals (Sweden)

    Stefan GHIMIȘI

    2015-05-01

    Full Text Available The fretting phenomenon represents particulary and complex form of wear who is; generaly, and/or weary of fretting who is produced on the load contact in a relative oscialatory movement lay small amplitude.A simultaneoustly applied tangential force and normal into contact appears a adhesion force

  6. Wave propagation in coated cylinders with reference to fretting fatigue

    Indian Academy of Sciences (India)

    is to study stress wave propagation in cylinders with reference to high frequency fretting. ... The motivation for studying of fretting fatigue at higher frequency is to investigate the ... Hence focus in this work is given to thin rods and cylinders. The.

  7. Probing protein-lipid interactions by FRET between membrane fluorophores

    Science.gov (United States)

    Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

    2016-09-01

    Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

  8. BIOSENSORS FOR ENVIRONMENTAL APPLICATIONS

    Science.gov (United States)

    A review, with 19 references, is given on challenges and possible opportunities for the development of biosensors for environmental monitoring applications. The high cost and slow turnaround times typically associated with the measurement of regulated pollutants clearly indicates...

  9. Nanochannels Photoelectrochemical Biosensor.

    Science.gov (United States)

    Zhang, Nan; Ruan, Yi-Fan; Zhang, Li-Bin; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2018-02-06

    Nanochannels have brought new opportunities for biosensor development. Herein, we present the novel concept of a nanochannels photoelectrochemical (PEC) biosensor based on the integration of a unique Cu x O-nanopyramid-islands (NPIs) photocathode, an anodic aluminum oxide (AAO) membrane, and alkaline phosphatase (ALP) catalytic chemistry. The Cu x O-NPIs photocathode possesses good performance, and further assembly with AAO yields a designed architecture composed of vertically aligned, highly ordered nanoarrays on top of the Cu x O-NPIs film. After biocatalytic precipitation (BCP) was stimulated within the channels, the biosensor was used for the successful detection of ALP activity. This study has not only provided a novel paradigm for an unconventional nanochannels PEC biosensor, which can be used for general bioanalytical purposes, but also indicated that the new concept of nanochannel-semiconductor heterostructures is a step toward innovative biomedical applications.

  10. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

    Science.gov (United States)

    Bruno, John G.

    2013-01-01

    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  11. Triggered optical biosensor

    Science.gov (United States)

    Song, Xuedong; Swanson, Basil I.

    2001-10-02

    An optical biosensor is provided for the detection of a multivalent target biomolecule, the biosensor including a substrate having a bilayer membrane thereon, a recognition molecule situated at the surface, the recognition molecule capable of binding with the multivalent target biomolecule, the recognition molecule further characterized as including a fluorescence label thereon and as being movable at the surface and a device for measuring a fluorescence change in response to binding between the recognition molecule and the multivalent target biomolecule.

  12. Nuclear Fuel Fretting Mechanisms in a Room Temperature Unlubricated Condition

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Ho; Kim, Hyung Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2008-10-15

    Recently, efforts for evaluating the fretting wear mechanism have been carried out by many researchers in various conditions. In an unlubricated condition, especially, effects of a wear debris and/or its layer on the fretting wear behavior were proposed that the formation of a well-developed glaze layer has a beneficial effect for decreasing a friction coefficient. Otherwise, a wear rate was accelerated by a third-body abrasion. At this time, it is well known that wear debris behaviors are affected by test variables such as a temperature, environment, material characteristics, etc. In a nuclear fuel fretting, however, its contact condition is quite different when compared with general fretting wear studies and could be summarized as the following; first, a fuel rod is supported by spacer grid springs and dimples that were elastically deformable. This results in a unique friction loop and a different fretting mechanism when a fuel rod is vibrated due to a flow-induced vibration (FIV). Next, it is possible that some region of the wear scar area with a specific spring shape condition could be hidden due to different wear debris behavior. So, some of the wear debris layers could be found on the worn surfaces in previous studies even though fretting wear tests were performed in a water lubricated condition. Finally, initial contact condition could be changed both an actual operating condition in power plants (i.e. high temperature and pressurized water (HTHP) under severe irradiation conditions) and the fretting wear tests for evaluating the wear resistant spring in lab conditions (i.e. from room temperature to HTHP without irradiation conditions) due to material degradations and the formation of the wear scar, respectively. In summary, the spring shape effect and the variation of the contact condition with increasing fretting cycle should be evaluated in order to improve the wear resistance of the spacer grid spring. So, in this study, fretting wear tests have been

  13. Molecular Approaches to Optical Biosensors

    National Research Council Canada - National Science Library

    Fierke, Carol

    1998-01-01

    The goal of this proposal was to develop methodologies for the optimization of field-deployable optical biosensors, in general, and, in particular, to optimize a carbonic anhydrase-based fiber optic zinc biosensor...

  14. Construction and characterization of novel stress-responsive Deinococcal biosensors

    International Nuclear Information System (INIS)

    Joe, Min Ho; Lim, Sang Youg

    2012-01-01

    In this research, we constructed a recombinant whole-cell biosensor to detect mutagens (H2O2, mitomycin C, MNNG, bleomycin) using Deinococcus radiodurans and evaluated its possibility for actual application. We performed DNA microarray analysis and selected 10 candidate genes for biosensor recombinant plasmid construction. The expression of ddrA, ddrB, DR 0 161, DR 0 589, and pprA was highly increased after treatment of the target mutagens. Putative promoter region of the genes were used for LacZ-based biosensor plasmid construction by replacing groESL promoter of pRADZ3. Pormoter activity and specificity of the five recombinant LacZ-based biosensor strains harboring the recombinant plasmids was measured. The result indicated that the promoter region of ddrA is the most suitable promoter for the biosensor development. Red pigment-based biosensor plasmid was constructed by displacing lacZ with crtI. The sensor strain was constructed by transforming the sensor plasmid into crtI deleted mutant D. radiodurans strain. Finally, macroscopic detection of the target mutagens by the biosensor strain was evaluated. The strength of red pigment biosynthesis by this recombinant strain in response to the target mutagens was weaker than our expectation. Continuous damage to the sensor strain by the mutagens in the medium might be the main reason for this low red-pigment biosynthesis. Therefore, we propose that the LacZ-based biosensor is more effective than the biosensor using red pigment as indicator for the mutagen detection

  15. Construction and characterization of novel stress-responsive Deinococcal biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Joe, Min Ho; Lim, Sang Youg

    2012-01-15

    In this research, we constructed a recombinant whole-cell biosensor to detect mutagens (H2O2, mitomycin C, MNNG, bleomycin) using Deinococcus radiodurans and evaluated its possibility for actual application. We performed DNA microarray analysis and selected 10 candidate genes for biosensor recombinant plasmid construction. The expression of ddrA, ddrB, DR{sub 0}161, DR{sub 0}589, and pprA was highly increased after treatment of the target mutagens. Putative promoter region of the genes were used for LacZ-based biosensor plasmid construction by replacing groESL promoter of pRADZ3. Pormoter activity and specificity of the five recombinant LacZ-based biosensor strains harboring the recombinant plasmids was measured. The result indicated that the promoter region of ddrA is the most suitable promoter for the biosensor development. Red pigment-based biosensor plasmid was constructed by displacing lacZ with crtI. The sensor strain was constructed by transforming the sensor plasmid into crtI deleted mutant D. radiodurans strain. Finally, macroscopic detection of the target mutagens by the biosensor strain was evaluated. The strength of red pigment biosynthesis by this recombinant strain in response to the target mutagens was weaker than our expectation. Continuous damage to the sensor strain by the mutagens in the medium might be the main reason for this low red-pigment biosynthesis. Therefore, we propose that the LacZ-based biosensor is more effective than the biosensor using red pigment as indicator for the mutagen detection.

  16. Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes.

    Science.gov (United States)

    Li, Lanying; Wen, Yanli; Xu, Li; Xu, Qin; Song, Shiping; Zuo, Xiaolei; Yan, Juan; Zhang, Weijia; Liu, Gang

    2016-01-15

    Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Photoelectrochemical enzymatic biosensors.

    Science.gov (United States)

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-06-15

    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Mechanisms of fretting-fatigue of titanium alloys

    Energy Technology Data Exchange (ETDEWEB)

    Antoniou, R A; Radtke, T C [Defence Sci. and Technol. Organ., Melbourne, Vic. (Australia). Aeronautical and Maritime Res. Lab.

    1997-09-30

    The effect of continuous fretting in air at 20 C on fatigue performance has been studied for Ti-17 and Ti-6Al-4V, high strength titanium alloys used for gas-turbine fan and compressor disks and blades, respectively. The effect of fretting was to reduce the fatigue stress limit from 700 MPa for plain fatigue to 200 MPa for fretting-fatigue. A number of models, supported by metallographic and fractographic evidence, are proposed which explain (i) how the cyclic loading of individual asperities results in crack initiation; (ii) the formation of multiple cracks; (iii) the existence of non-propagating cracks; and (iv) how fretting influences crack propagation once fatigue cracks have formed. (orig.) 46 refs.

  19. Fluorescence resonance energy transfer (FRET) in chemistry and ...

    Indian Academy of Sciences (India)

    Förster distance dependence of the FRET rate. SANGEETA SAINI,1 HARJINDER SINGH2 and BIMAN BAGCHI1,*. 1Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560 012. 2Permanent address: Department of ...

  20. QD-Based FRET Probes at a Glance

    Directory of Open Access Journals (Sweden)

    Armen Shamirian

    2015-06-01

    Full Text Available The unique optoelectronic properties of quantum dots (QDs give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET are of special interest due to the multiple response mechanisms that may be utilized, which in turn imparts enhanced flexibility in their design. They may also function as ratiometric, or “color-changing” probes. In this review, we describe the fundamentals of FRET and provide examples of QD-FRET sensors as grouped by their response mechanisms such as link cleavage and structural rearrangement. An overview of early works, recent advances, and various models of QD-FRET sensors for the measurement of pH and oxygen, as well as the presence of metal ions and proteins such as enzymes, are also provided.

  1. Future of biosensors: a personal view.

    Science.gov (United States)

    Scheller, Frieder W; Yarman, Aysu; Bachmann, Till; Hirsch, Thomas; Kubick, Stefan; Renneberg, Reinhard; Schumacher, Soeren; Wollenberger, Ulla; Teller, Carsten; Bier, Frank F

    2014-01-01

    Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar" personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables" such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous" biosensors will emerge.

  2. Introduction to biosensors.

    Science.gov (United States)

    Bhalla, Nikhil; Jolly, Pawan; Formisano, Nello; Estrela, Pedro

    2016-06-30

    Biosensors are nowadays ubiquitous in biomedical diagnosis as well as a wide range of other areas such as point-of-care monitoring of treatment and disease progression, environmental monitoring, food control, drug discovery, forensics and biomedical research. A wide range of techniques can be used for the development of biosensors. Their coupling with high-affinity biomolecules allows the sensitive and selective detection of a range of analytes. We give a general introduction to biosensors and biosensing technologies, including a brief historical overview, introducing key developments in the field and illustrating the breadth of biomolecular sensing strategies and the expansion of nanotechnological approaches that are now available. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  3. Biosensors in forensic sciences

    Directory of Open Access Journals (Sweden)

    Frederickx, C.

    2011-01-01

    Full Text Available A biosensor is a device that uses biological materials to detect and monitor the presence of specific chemicals in an area. Traditional methods of volatile detection used by law enforcement agencies and rescue teams typically consist of reliance on canine olfaction. This concept of using dogs to detect specific substances is quite old. However, dogs have some limitations such as cost of training and time of conditioning. Thus, the possibility of using other organisms as biosensors including rats, dolphins, honeybees, and parasitic wasps for detecting explosives, narcotics and cadavers has been developed. Insects have several advantages unshared by mammals. Insects are sensitive, cheap to produce and can be conditioned with impressive speed for a specific chemical-detection task. Moreover, insects might be a preferred sensing method in scenarios that are deemed too dangerous to use mammals. The purpose of this review is to provide an overview of the biosensors used in forensic sciences.

  4. a Study on the Fretting Fatigue Life of Zircaloy Alloys

    Science.gov (United States)

    Kwon, Jae-Do; Park, Dae-Kyu; Woo, Seung-Wan; Chai, Young-Suck

    Studies on the strength and fatigue life of machines and structures have been conducted in accordance with the development of modern industries. In particular, fine and repetitive cyclic damage occurring in contact regions has been known to have an impact on fretting fatigue fractures. The main component of zircaloy alloy is Zr, and it possesses good mechanical characteristics at high temperatures. This alloy is used in the fuel rod material of nuclear power plants because of its excellent resistance. In this paper, the effect of the fretting damage on the fatigue behavior of the zircaloy alloy is studied. Further, various types of mechanical tests such as tension and plain fatigue tests are performed. Fretting fatigue tests are performed with a flat-flat contact configuration using a bridge-type contact pad and plate-type specimen. Through these experiments, it is found that the fretting fatigue strength decreases by about 80% as compared to the plain fatigue strength. Oblique cracks are observed in the initial stage of the fretting fatigue, in which damaged areas are found. These results can be used as the basic data for the structural integrity evaluation of corrosion-resisting alloys considering the fretting damages.

  5. Fuel bundle to pressure tube fretting in Bruce and Darlington

    Energy Technology Data Exchange (ETDEWEB)

    Norsworthy, A G; Ditschun, A [Atomic Energy of Canada Ltd., Mississauga, ON (Canada)

    1996-12-31

    As the fuel channel elongates due to creep, the fuel string moves relative to the inlet until the fuel pads at the inboard end eventually separate from the spacer sleeve, and the fuel resides on the burnish mark of the pressure tube. The bundle is then supported in a fashion which contributes to increased levels of vibration. Those pads which (due to geometric variation) have contact loads with the pressure tube within a certain range, vibrate, and cause significant fretting on the burnish mark, and further along at the midplane of the bundle. Inspection of the pressure tubes in Bruce A, Bruce B, and Darlington has revealed fret damage up to 0.55 mm at the burnish mark and slightly lower than this at the inlet bundle midplane. To date, all fret marks have been dealt with successfully without the need for tube replacement, but a program of work has been initiated to understand the mechanism and reduce the fretting. Such understanding is necessary to guide future design changes to the fuel bundle, to guide future inspection programs, to guide maintenance programs, and for longer term strategic planning. This paper discusses how the understanding of fretting has evolved and outlines a current hypothesis for the mechanism of fretting. The role of bundle geometry, excitation forces, and reactor conditions are reviewed, along with options under consideration to mitigate damage. (author). 4 refs., 2 tabs., 13 figs.

  6. Fuel bundle to pressure tube fretting in Bruce and Darlington

    International Nuclear Information System (INIS)

    Norsworthy, A.G.; Ditschun, A.

    1995-01-01

    As the fuel channel elongates due to creep, the fuel string moves relative to the inlet until the fuel pads at the inboard end eventually separate from the spacer sleeve, and the fuel resides on the burnish mark of the pressure tube. The bundle is then supported in a fashion which contributes to increased levels of vibration. Those pads which (due to geometric variation) have contact loads with the pressure tube within a certain range, vibrate, and cause significant fretting on the burnish mark, and further along at the midplane of the bundle. Inspection of the pressure tubes in Bruce A, Bruce B, and Darlington has revealed fret damage up to 0.55 mm at the burnish mark and slightly lower than this at the inlet bundle midplane. To date, all fret marks have been dealt with successfully without the need for tube replacement, but a program of work has been initiated to understand the mechanism and reduce the fretting. Such understanding is necessary to guide future design changes to the fuel bundle, to guide future inspection programs, to guide maintenance programs, and for longer term strategic planning. This paper discusses how the understanding of fretting has evolved and outlines a current hypothesis for the mechanism of fretting. The role of bundle geometry, excitation forces, and reactor conditions are reviewed, along with options under consideration to mitigate damage. (author). 4 refs., 2 tabs., 13 figs

  7. A new trend to determine biochemical parameters by quantitative FRET assays.

    Science.gov (United States)

    Liao, Jia-yu; Song, Yang; Liu, Yan

    2015-12-01

    Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

  8. Biosensors: Future Analytical Tools

    Directory of Open Access Journals (Sweden)

    Vikas

    2007-02-01

    Full Text Available Biosensors offer considerable promises for attaining the analytic information in a faster, simpler and cheaper manner compared to conventional assays. Biosensing approach is rapidly advancing and applications ranging from metabolite, biological/ chemical warfare agent, food pathogens and adulterant detection to genetic screening and programmed drug delivery have been demonstrated. Innovative efforts, coupling micromachining and nanofabrication may lead to even more powerful devices that would accelerate the realization of large-scale and routine screening. With gradual increase in commercialization a wide range of new biosensors are thus expected to reach the market in the coming years.

  9. All-Silica Hollow-Core Microstructured Bragg Fibers for Biosensor Application

    DEFF Research Database (Denmark)

    Passaro, Davide; Foroni, Matteo; Poli, Federica

    2008-01-01

    The possibility to exploit all-silica hollow-core-microstructured Bragg fibers to realize a biosensor useful to detect the DNA hybridization process has been investigated. A Bragg fiber recently fabricated has been considered for the analysis performed by means of a full-vector modal solver based...... layer on the inner surface of the fiber holes can modify the fundamental mode properties. The numerical analysis results have successfully demonstrated the DNA bio-sensor feasibility in hollow-core Bragg fibers....

  10. Surface stress-based biosensors.

    Science.gov (United States)

    Sang, Shengbo; Zhao, Yuan; Zhang, Wendong; Li, Pengwei; Hu, Jie; Li, Gang

    2014-01-15

    Surface stress-based biosensors, as one kind of label-free biosensors, have attracted lots of attention in the process of information gathering and measurement for the biological, chemical and medical application with the development of technology and society. This kind of biosensors offers many advantages such as short response time (less than milliseconds) and a typical sensitivity at nanogram, picoliter, femtojoule and attomolar level. Furthermore, it simplifies sample preparation and testing procedures. In this work, progress made towards the use of surface stress-based biosensors for achieving better performance is critically reviewed, including our recent achievement, the optimally circular membrane-based biosensors and biosensor array. The further scientific and technological challenges in this field are also summarized. Critical remark and future steps towards the ultimate surface stress-based biosensors are addressed. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Electrochemical biosensors for hormone analyses.

    Science.gov (United States)

    Bahadır, Elif Burcu; Sezgintürk, Mustafa Kemal

    2015-06-15

    Electrochemical biosensors have a unique place in determination of hormones due to simplicity, sensitivity, portability and ease of operation. Unlike chromatographic techniques, electrochemical techniques used do not require pre-treatment. Electrochemical biosensors are based on amperometric, potentiometric, impedimetric, and conductometric principle. Amperometric technique is a commonly used one. Although electrochemical biosensors offer a great selectivity and sensitivity for early clinical analysis, the poor reproducible results, difficult regeneration steps remain primary challenges to the commercialization of these biosensors. This review summarizes electrochemical (amperometric, potentiometric, impedimetric and conductometric) biosensors for hormone detection for the first time in the literature. After a brief description of the hormones, the immobilization steps and analytical performance of these biosensors are summarized. Linear ranges, LODs, reproducibilities, regenerations of developed biosensors are compared. Future outlooks in this area are also discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. FRET Response of a Modified Ribose Receptor Expressed in the Diatom Thalassiosira pseudonana

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Hanna

    2011-08-26

    The ability to insert complex proteins into silica has many applications including biosensing. Previous research has demonstrated how to direct proteins to the biosilica of diatoms [1]. Here, we show that a complex fusion protein that includes an enzyme, a bacterial ribose periplasmic binding protein, flanked by fluorescent proteins constituting a FRET pair can remain functional in the frustules of living diatoms. A Sil3 tag is attached to the N-terminal end to localize the fusion protein to frustules of the diatom Thalassiosira pseudonana. When ribose was applied, a larger decrease in FRET response was seen in transformed cells than in untransformed cells. Multiple forms of the expression vector were tested to find the optimal system; specifically, a one-vector system was compared to a two-vector system and the gDNA version of the Sil3 localization tag was compared to the cDNA version. The optimal system was found to be a one-vector system with the genomic version of the Sil3 tag to direct the protein to the frustules. Localization of the enzyme to the frustules was further confirmed through cell fluorescence imaging.

  13. Designed graphene-peptide nanocomposites for biosensor applications: A review

    International Nuclear Information System (INIS)

    Wang, Li; Zhang, Yujie; Wu, Aiguo; Wei, Gang

    2017-01-01

    The modification of graphene with biomacromolecules like DNA, protein, peptide, and others extends the potential applications of graphene materials in various fields. The bound biomacromolecules could improve the biocompatibility and bio-recognition ability of graphene-based nanocomposites, therefore could greatly enhance their biosensing performances on both selectivity and sensitivity. In this review, we presented a comprehensive introduction and discussion on recent advance in the synthesis and biosensor applications of graphene-peptide nanocomposites. The biofunctionalization of graphene with specifically designed peptides, and the synthesis strategies of graphene-peptide (monomer, nanofibrils, and nanotubes) nanocomposites were demonstrated. On the other hand, the fabrication of graphene-peptide nanocomposite based biosensor architectures for electrochemical, fluorescent, electronic, and spectroscopic biosensing were further presented. This review includes nearly all the studies on the fabrication and applications of graphene-peptide based biosensors recently, which will promote the future developments of graphene-based biosensors in biomedical detection and environmental analysis. - Highlights: • A comprehensive review on the fabrication and application of graphene-peptide nanocomposites was presented. • The design of peptide sequences for biofunctionalization of various graphene materials was presented. • Multi-strategies on the fabrication of biosensors with graphene-peptide nanocomposites were discussed. • Designed graphene-peptide nanocomposites showed wide biosensor applications.

  14. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    International Nuclear Information System (INIS)

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-01-01

    Highlights: ► An endothelial cell apoptosis assay using FRET-based biosensor was developed. ► The fluorescence of the cells changed from green to blue during apoptosis. ► This method was developed into a high-throughput assay in 96-well plates. ► This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  15. Studies of G-quadruplexes formed within self-assembled DNA mini-circles.

    Science.gov (United States)

    Klejevskaja, Beata; Pyne, Alice L B; Reynolds, Matthew; Shivalingam, Arun; Thorogate, Richard; Hoogenboom, Bart W; Ying, Liming; Vilar, Ramon

    2016-10-13

    We have developed self-assembled DNA mini-circles that contain a G-quadruplex-forming sequence from the c-Myc oncogene promoter and demonstrate by FRET that the G-quadruplex unfolding kinetics are 10-fold slower than for the simpler 24-mer G-quadruplex that is commonly used for FRET experiments.

  16. Implantable enzyme amperometric biosensors.

    Science.gov (United States)

    Kotanen, Christian N; Moussy, Francis Gabriel; Carrara, Sandro; Guiseppi-Elie, Anthony

    2012-05-15

    The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Intonation and compensation of fretted string instruments

    Science.gov (United States)

    Varieschi, Gabriele; Gower, Christina

    2011-04-01

    We discuss theoretical and physical models that are useful for analyzing the intonation of musical instruments such as guitars and mandolins and can be used to improve the tuning on these instruments. The placement of frets on the fingerboard is designed according to mathematical rules and the assumption of an ideal string. The analysis becomes more complicated when we include the effects of deformation of the string and inharmonicity due to other string characteristics. As a consequence, perfect intonation of all the notes on the instrument cannot be achieved, but complex compensation procedures can be introduced to minimize the problem. To test the validity of these procedures, we performed extensive measurements using standard monochord sonometers and other acoustical devices, confirming the correctness of our theoretical models. These experimental activities can be integrated into acoustics courses and laboratories and can become a more advanced version of basic experiments with monochords and sonometers. This work was supported by a grant from the Frank R. Seaver College of Science and Engineering, Loyola Marymount University.

  18. Assessment of fretting wear in Hanaro fuel

    International Nuclear Information System (INIS)

    Chae, Hee Taek; Lim, Kyeong Hwan; Kim, Hark Rho

    1999-06-01

    Since the first fuel loading on Feb. 1995, various zero-power tests were performed in HANARO and power ascending tests followed. After the initial fuel loading, Hanaro operation staffs inspected only two fuel bundles which were evaluated to have the highest power at the end of each cycle and they did not recognize anything peculiar in the inspected bundles. At the end of 1996, Hanaro staffs found severe wear damages in the fuel components. After that, the 4th cycle core was re-arranged with fresh fuels only to investigate wear phenomena on the fuel components. The fuel inspections have been performed 25 times periodically since the core re-configuration. In this report, fretting wear characteristics of the fuel assemblies were evaluated and summarized. Wear damages of the improved fuel assembly to resolve the wear problem were compared with those of the original fuel assembly. Based on the results of the fuel inspections, we suggest that fuel inspection need not be done for the first 60 pump operation days in order to reduce the potential of damage by a fuel handling error and an operator's burden of the fuel inspection. (author). 6 refs., 10 tabs., 5 figs

  19. Applicability of out-of-pile fretting wear tests to in-reactor fretting wear-induced failure time prediction

    Science.gov (United States)

    Kim, Kyu-Tae

    2013-02-01

    In order to investigate whether or not the grid-to-rod fretting wear-induced fuel failure will occur for newly developed spacer grid spring designs for the fuel lifetime, out-of-pile fretting wear tests with one or two fuel assemblies are to be performed. In this study, the out-of-pile fretting wear tests were performed in order to compare the potential for wear-induced fuel failure in two newly-developed, Korean PWR spacer grid designs. Lasting 20 days, the tests simulated maximum grid-to-rod gap conditions and the worst flow induced vibration effects that might take place over the fuel life time. The fuel rod perforation times calculated from the out-of-pile tests are greater than 1933 days for 2 μm oxidized fuel rods with a 100 μm grid-to-rod gap, whereas those estimated from in-reactor fretting wear failure database may be about in the range of between 60 and 100 days. This large discrepancy in fuel rod perforation may occur due to irradiation-induced cladding oxide microstructure changes on the one hand and a temperature gradient-induced hydrogen content profile across the cladding metal region on the other hand, which may accelerate brittleness in the grid-contacting cladding oxide and metal regions during the reactor operation. A three-phase grid-to-rod fretting wear model is proposed to simulate in-reactor fretting wear progress into the cladding, considering the microstructure changes of the cladding oxide and the hydrogen content profile across the cladding metal region combined with the temperature gradient. The out-of-pile tests cannot be directly applicable to the prediction of in-reactor fretting wear-induced cladding perforations but they can be used only for evaluating a relative wear resistance of one grid design against the other grid design.

  20. Influence of Fretting on Flexural Fatigue of 304 Stainless Steel and Mild Steel

    National Research Council Canada - National Science Library

    Bill, Robert

    1978-01-01

    Fretting fatigue experiments conducted on 304 stainless steel using a flexural-fatigue test arrangement with bolted-on fretting pads have demonstrated that fatigue life is reduced by at least a factor...

  1. Electrochemical biosensors in pharmaceutical analysis

    OpenAIRE

    Gil, Eric de Souza; Melo, Giselle Rodrigues de

    2010-01-01

    Given the increasing demand for practical and low-cost analytical techniques, biosensors have attracted attention for use in the quality analysis of drugs, medicines, and other analytes of interest in the pharmaceutical area. Biosensors allow quantification not only of the active component in pharmaceutical formulations, but also the analysis of degradation products and metabolites in biological fluids. Thus, this article presents a brief review of biosensor use in pharmaceutical analysis, fo...

  2. FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor

    Science.gov (United States)

    Du, Mengyan; Yang, Fangfang; Mai, Zihao; Qu, Wenfeng; Lin, Fangrui; Wei, Lichun; Chen, Tongsheng

    2018-04-01

    We here introduce a fluorescence resonance energy transfer (FRET) two-hybrid assay method to measure the maximal donor(D)- and acceptor(A)-centric FRET efficiency (ED,max and EA,max) of the D-A complex and its stoichiometry by linearly fitting the donor-centric FRET efficiency (ED) to the acceptor-to-donor concentration ratio (RC) and acceptor-centric FRET efficiency (EA) to 1/RC, respectively. We performed this method on a wide-field fluorescence microscope for living HepG2 cells co-expressing FRET tandem constructs and free donor/acceptor and obtained correct ED, EA, and stoichiometry values of those tandem constructs. Evaluation on the binding of Bad with Bcl-XL in Hela cells showed that Bad interacted strongly with Bcl-XL to form a Bad-Bcl-XL complex on mitochondria, and one Bad interacted mainly with one Bcl-XL molecule in healthy cells, while with multiple (maybe 2) Bcl-XL molecules in apoptotic cells.

  3. Carbon nanotube biosensors

    Science.gov (United States)

    Tîlmaciu, Carmen-Mihaela; Morris, May C.

    2015-01-01

    Nanomaterials possess unique features which make them particularly attractive for biosensing applications. In particular, carbon nanotubes (CNTs) can serve as scaffolds for immobilization of biomolecules at their surface, and combine several exceptional physical, chemical, electrical, and optical characteristics properties which make them one of the best suited materials for the transduction of signals associated with the recognition of analytes, metabolites, or disease biomarkers. Here we provide a comprehensive review on these carbon nanostructures, in which we describe their structural and physical properties, functionalization and cellular uptake, biocompatibility, and toxicity issues. We further review historical developments in the field of biosensors, and describe the different types of biosensors which have been developed over time, with specific focus on CNT-conjugates engineered for biosensing applications, and in particular detection of cancer biomarkers. PMID:26579509

  4. Carbon Nanotube Biosensors

    Directory of Open Access Journals (Sweden)

    Carmen-Mihaela eTilmaciu

    2015-10-01

    Full Text Available Nanomaterials possess unique features which make them particularly attractive for biosensing applications. In particular Carbon Nanotubes (CNTs can serve as scaffolds for immobilization of biomolecules at their surface, and combine several exceptional physical, chemical, electrical and optical characteristics properties which make them one of the best suited materials for the transduction of signals associated with the recognition of analytes, metabolites or disease biomarkers. Here we provide a comprehensive review on these carbon nanostructures, in which we will describe their structural and physical properties, discuss functionalization and cellular uptake, biocompatibility and toxicity issues. We further review historical developments in the field of biosensors, and describe the different types of biosensors which have been developed over time, with specific focus on CNT-conjugates engineered for biosensing applications, and in particular detection of cancer biomarkers.

  5. Recent advances in electrochemical biosensors based on graphene two-dimensional nanomaterials.

    Science.gov (United States)

    Song, Yang; Luo, Yanan; Zhu, Chengzhou; Li, He; Du, Dan; Lin, Yuehe

    2016-02-15

    Graphene as a star among two-dimensional nanomaterials has attracted tremendous research interest in the field of electrochemistry due to their intrinsic properties, including the electronic, optical, and mechanical properties associated with their planar structure. The marriage of graphene and electrochemical biosensors has created many ingenious biosensing strategies for applications in the areas of clinical diagnosis and food safety. This review provides a comprehensive overview of the recent advances in the development of graphene based electrochemical biosensors. Special attention is paid to graphene-based enzyme biosensors, immunosensors, and DNA biosensors. Future perspectives on high-performance graphene-based electrochemical biosensors are also discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Medical diagnosis and remote sensing at fiber-tip: picosecond resolved FRET sensor

    Science.gov (United States)

    Polley, Nabarun; Pal, Samir Kumar

    2016-03-01

    Förster Resonance Energy Transfer (FRET) strategy in popular in fiber-optic sensing. However, the steady state emission quenching of the donor is inadequate to conclude FRET. The resonance type energy transfer from one molecule (donor) to other (acceptor) should meet few key properties including donor to acceptor energy migration in non-radiative way. In the present study, we have coupled the evanescent field of an optical fiber to the covalently attached donor (dansyl) molecules at the fiber tip. By using picosecond resolved time correlated single photon counting (TCSPC) we have demonstrated that dansyl at the fiber tip transfers energy to a well known DNA-intercalating dye ethidium. Our ultrafast detection scheme selectively distinguishes the probe (dansyl) emission from the intrinsic emission of the fiber. We have also used the setup for the remote sensing of the dielectric constant (polarity) of an environment. We have finally implemented the detection mechanism to detect an industrial synthetic dye methylene blue (MB) in water.

  7. Biosensor. Seitai sensa

    Energy Technology Data Exchange (ETDEWEB)

    Karube, I [The Univ. of Tokyo, Tokyo (Japan). Research Center for Advanced Science and Technology

    1993-06-15

    Present state of the art of biosensors is described by taking taste sensors and odor sensors as examples. Bio-devices that response only to specific chemical substances are made using membranes that recognize particular molecules. Biosensors are constructed in combination of bio-devices with electronics devices that transduce the response of bio-devices to electric signals. Enzymes are used often as bio-devices to recognize molecules. They recognize strictly chemical substances and promote chemical reactions. Devices to measure electrochemically substances consumed or produced in the reactions serve as sensors. For taste sensors, inosinic acid or glutamic acid that is a component of taste, is recognized and measured. Combination of various bio-devices other than enzymes with various transducers makes it possible to produce biosensors based on a variety of principles. Odor sensors recognize odors by measuring frequency change of the electrode of quartz oscillator. The change occurs with weight change due to odorous substances absorbed on the oscillator electrode coated with lipids which exist in olfactory cells. 1 ref., 1 fig.

  8. Application of Influence Function Method to the Fretting Wear Problems

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Choon Yeol; Tian, Li Si; Bae, Joon Woo; Chai, Young Suck [Yeungnam University, Gyongsan (Korea, Republic of)

    2006-07-01

    Numerical analysis by influence function method (IFM) is demonstrated in this study in order to investigate the fretting wear problems on the secondary side of the steam generator, caused by flow induced vibration. Two-dimensional numerical contact model in terms of Cauchy integral equation is developed. The distributions of normal pressures, shear stresses and displacement fields are derived between two contact bodies which have similar elastic properties. The work rate model is adopted to find the wear amounts between two materials. The results are compared with the solutions by finite element analyses, which show the utilization of the present method to the fretting wear problems.

  9. Application of Influence Function Method to the Fretting Wear Problems

    International Nuclear Information System (INIS)

    Lee, Choon Yeol; Tian, Li Si; Bae, Joon Woo; Chai, Young Suck

    2006-01-01

    Numerical analysis by influence function method (IFM) is demonstrated in this study in order to investigate the fretting wear problems on the secondary side of the steam generator, caused by flow induced vibration. Two-dimensional numerical contact model in terms of Cauchy integral equation is developed. The distributions of normal pressures, shear stresses and displacement fields are derived between two contact bodies which have similar elastic properties. The work rate model is adopted to find the wear amounts between two materials. The results are compared with the solutions by finite element analyses, which show the utilization of the present method to the fretting wear problems

  10. Development of a pan-Babesia FRET-qPCR and a survey of livestock from five Caribbean islands.

    Science.gov (United States)

    Li, Jing; Kelly, Patrick; Zhang, Jilei; Xu, Chuanling; Wang, Chengming

    2015-09-30

    Babesia spp. are tick-borne protozoan hemoparasites and the second most common blood-borne parasites of mammals, in particular domestic animals. We used the Clustal Multiple Alignment program and 18S rRNA gene sequences of 22 Babesia species from GenBank to develop a PCR that could detect a wide variety of Babesia spp. in a single reaction. The pan-Babesia FRET-qPCR we developed reliably detected B. gibsoni, B. canis, B. vogeli, B. microti, B. bovis, and B. divergens under controlled conditions but did not react with closely related species, mainly Hepatozoon americanum, Theileria equi, and Toxoplasma gondii. When we tested the pan-Babesia FRET-qPCR on DNA of whole blood from 752 cattle, sheep, goats, donkeys and horses from five Caribbean islands, we detected Babesia spp. expected to be present in the animals, mainly B. bovis and B. bigemina in cattle and B. caballi in horses and donkeys. Further, we found that animals were not uncommonly infected with species of Babesia usually associated with other hosts, mainly B. vogeli and B. gibsoni in cattle, sheep and goats, B. rossi in goats, and B. caballi in goats and sheep. Finally, the pan-Babesia FRET-qPCR enabled us to identify unknown species of Babesia in cattle, goats, sheep and donkeys. Overall, 70 % (525/752) of the animals we tested were positive confirming earlier limited studies that infections with Babesia spp. are common in livestock in the Caribbean.

  11. A polyamidoamine dendrimer-streptavidin supramolecular architecture for biosensor development.

    Science.gov (United States)

    Soda, N; Arotiba, O A

    2017-12-01

    A novel polyamidoamine dendrimer-streptavidin supramolecular architecture suitable as a versatile platform for biosensor development is reported. The dendrimer was electrodeposited on a glassy carbon electrode via cyclic voltammetry. The dendrimer electrode was further modified with streptavidin by electrostatic attraction upon drop coating. The platform i.e. the dendrimer-streptavidin modified electrode was electrochemically interrogated in phosphate buffer, ferrocyanide and H 2 O 2 . The dendrimer-streptavidin platform was used in the preparation of a simple DNA biosensor as a proof of concept. The supramolecular architecture of dendrimer-streptavidin was stable, electroactive and thus lends itself as a versatile immobilisation layer for any biotinylated bioreceptors in biosensor development. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Dual door entry to exciplex emission in a chimeric DNA duplex containing non-nucleoside-nucleoside pair.

    Science.gov (United States)

    Bag, Subhendu Sekhar; Talukdar, Sangita; Kundu, Rajen; Saito, Isao; Jana, Subhashis

    2014-01-25

    Dual door entry to exciplex formation was established in a chimeric DNA duplex wherein a fluorescent non-nucleosidic base surrogate () is paired against a fluorescent nucleosidic base surrogate (). Packing of the nucleobases via intercalative stacking interactions led to an exciplex emission either via FRET from the donor or direct excitation of the FRET acceptor .

  13. Protein Detection with Aptamer Biosensors

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    2008-07-01

    Full Text Available Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers.

  14. Affinity biosensors: techniques and protocols

    National Research Council Canada - National Science Library

    Rogers, Kim R; Mulchandani, Ashok

    1998-01-01

    ..., and government to begin or expand their biosensors research. This volume, Methods in Biotechnology vol. 7: Affinity Biosensors: Techniques and Protocols, describes a variety of classical and emerging transduction technologies that have been interfaced to bioaffinity elements (e.g., antibodies and receptors). Some of the reas...

  15. Carbon Nanotube Biosensors for Space Molecule Detection and Clinical Molecular Diagnostics

    Science.gov (United States)

    Han, Jie

    2001-01-01

    Both space molecule detection and clinical molecule diagnostics need to develop ultra sensitive biosensors for detection of less than attomole molecules such as amino acids for DNA. However all the electrode sensor systems including those fabricated from the existing carbon nanotubes, have a background level of nA (nanoAmp). This has limited DNA or other molecule detection to nA level or molecules whose concentration is, much higher than attomole level. A program has been created by NASA and NCI (National Cancer Institute) to exploit the possibility of carbon nanotube based biosensors to solve this problem for both's interest. In this talk, I will present our effort on the evaluation and novel design of carbon nanotubes as electrode biosensors with strategies to minimize background currents while maximizing signal intensity.The fabrication of nanotube electrode arrays, immobilization of molecular probes on nanotube electrodes and in vitro biosensor testing will also be discussed.

  16. Application of the SSB biosensor to study in vitro transcription.

    Science.gov (United States)

    Cook, Alexander; Hari-Gupta, Yukti; Toseland, Christopher P

    2018-02-12

    Gene expression, catalysed by RNA polymerases (RNAP), is one of the most fundamental processes in living cells. The majority of methods to quantify mRNA are based upon purification of the nucleic acid which leads to experimental inaccuracies and loss of product, or use of high cost dyes and sensitive spectrophotometers. Here, we describe the use of a fluorescent biosensor based upon the single stranded binding (SSB) protein. In this study, the SSB biosensor showed similar binding properties to mRNA, to that of its native substrate, single-stranded DNA (ssDNA). We found the biosensor to be reproducible with no associated loss of product through purification, or the requirement for expensive dyes. Therefore, we propose that the SSB biosensor is a useful tool for comparative measurement of mRNA yield following in vitro transcription. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Development of Electrochemical Biosensors for Ultrasensitive Detection of Bacteria in the Environment

    DEFF Research Database (Denmark)

    Fapyane, Deby

    2018-01-01

    to those conventional methods, are intensively studied. Biosensor technology is one of the strategies for rapid monitoring of pathogens such as bacteria, virus, and parasites in the environment. Among them, the electrochemical biosensor offers simple, rapid, cost-effective and possibility...... for ultrasensitive detection of bacterial cells, DNA and rRNA. Several key operational parameters were assessed such as the optimization of probe design and labeling molecules. Here, more specifically we used two novel labels for the development of the electrochemical biosensor for bacteria detection; cellulase...

  18. Application of FRET probes in the analysis of neuronal plasticity

    Directory of Open Access Journals (Sweden)

    Yoshibumi eUeda

    2013-10-01

    Full Text Available Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since GFP was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET, which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo.

  19. Approximate stresses in 2-D flat elastic contact fretting problems

    Science.gov (United States)

    Urban, Michael Rene

    Fatigue results from the cyclic loading of a solid body. If the body subject to fatigue is in contact with another body and relative sliding motion occurs between these two bodies, then rubbing surface damage can accelerate fatigue failure. The acceleration of fatigue failure is especially important if the relative motion between the two bodies results in surface damage without excessive surface removal via wear. The situation just described is referred to as fretting fatigue. Understanding of fretting fatigue is greatly enhanced if the stress state associated with fretting can be characterized. For Hertzian contact, this can readily be done. Unfortunately, simple stress formulae are not available for flat body contact. The primary result of the present research is the development of a new, reasonably accurate, approximate closed form expression for 2-dimensional contact stresses which has been verified using finite element modeling. This expression is also combined with fracture mechanics to provide a simple method of determining when a crack is long enough to no longer be affected by the contact stress field. Lower bounds on fatigue life can then easily be calculated using fracture mechanics. This closed form expression can also be used to calculate crack propagation within the contact stress field. The problem of determining the cycles required to generate an initial crack and what to choose as an initial crack size is unresolved as it is in non-fretting fatigue.

  20. Rational design of FRET-based sensor proteins

    NARCIS (Netherlands)

    Merkx, M.

    2008-01-01

    Real-time imaging of molecular events inside living cells is important for understanding the basis of physiological processes and diseases. Genetically encoded sensors that use fluorescence resonance energy transfer (FRET) between two fluorescent proteins are attractive in this respect because they

  1. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee

    2016-03-01

    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  2. Innovations in biomedical nanoengineering: nanowell array biosensor

    Science.gov (United States)

    Seo, YoungTae; Jeong, Sunil; Lee, JuKyung; Choi, Hak Soo; Kim, Jonghan; Lee, HeaYeon

    2018-04-01

    Nanostructured biosensors have pioneered biomedical engineering by providing highly sensitive analyses of biomolecules. The nanowell array (NWA)-based biosensing platform is particularly innovative, where the small size of NWs within the array permits extremely profound sensing of a small quantity of biomolecules. Undoubtedly, the NWA geometry of a gently-sloped vertical wall is critical for selective docking of specific proteins without capillary resistances, and nanoprocessing has contributed to the fabrication of NWA electrodes on gold substrate such as molding process, e-beam lithography, and krypton-fluoride (KrF) stepper semiconductor method. The Lee group at the Mara Nanotech has established this NW-based biosensing technology during the past two decades by engineering highly sensitive electrochemical sensors and providing a broad range of detection methods from large molecules (e.g., cells or proteins) to small molecules (e.g., DNA and RNA). Nanosized gold dots in the NWA enhance the detection of electrochemical biosensing to the range of zeptomoles in precision against the complementary target DNA molecules. In this review, we discuss recent innovations in biomedical nanoengineering with a specific focus on novel NWA-based biosensors. We also describe our continuous efforts in achieving a label-free detection without non-specific binding while maintaining the activity and stability of immobilized biomolecules. This research can lay the foundation of a new platform for biomedical nanoengineering systems.

  3. Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules.

    Directory of Open Access Journals (Sweden)

    Antonino Ingargiola

    Full Text Available We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

  4. Inferring properties of disordered chains from FRET transfer efficiencies

    Science.gov (United States)

    Zheng, Wenwei; Zerze, Gül H.; Borgia, Alessandro; Mittal, Jeetain; Schuler, Benjamin; Best, Robert B.

    2018-03-01

    Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data.

  5. Instrumental biosensors: new perspectives for the analysis of biomolecular interactions.

    Science.gov (United States)

    Nice, E C; Catimel, B

    1999-04-01

    The use of instrumental biosensors in basic research to measure biomolecular interactions in real time is increasing exponentially. Applications include protein-protein, protein-peptide, DNA-protein, DNA-DNA, and lipid-protein interactions. Such techniques have been applied to, for example, antibody-antigen, receptor-ligand, signal transduction, and nuclear receptor studies. This review outlines the principles of two of the most commonly used instruments and highlights specific operating parameters that will assist in optimising experimental design, data generation, and analysis.

  6. Microbial biosensors for environmental monitoring

    Directory of Open Access Journals (Sweden)

    David VOGRINC

    2015-12-01

    Full Text Available Microbial biosensors are analytical devices capable of sensing substances in the environment due to the specific biological reaction of the microorganism or its parts. Construction of a microbial biosensor requires knowledge of microbial response to the specific analyte. Linking this response with the quantitative data, using a transducer, is the crucial step in the construction of a biosensor. Regarding the transducer type, biosensors are divided into electrochemical, optical biosensors and microbial fuel cells. The use of the proper configuration depends on the selection of the biosensing element. With the use of transgenic E. coli strains, bioluminescence or fluorescence based biosensors were developed. Microbial fuel cells enable the use of the heterogeneous microbial populations, isolated from wastewater. Different microorganisms are used for different pollutants – pesticides, heavy metals, phenolic compounds, organic waste, etc. Biosensing enables measurement of their concentration and their toxic or genotoxic effects on the microbes. Increasing environmental awareness has contributed to the increase of interest for biomonitoring. Although technologies, such as bioinformatics and genetic engineering, allow us to design complex and efficient microbial biosensors for environmental pollutants, the transfer of the laboratory work to the field still remains a problem to solve.

  7. rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

    Science.gov (United States)

    Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János

    2016-04-01

    Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

  8. Improved Biosensors for Soils

    Science.gov (United States)

    Silberg, J. J.; Masiello, C. A.; Cheng, H. Y.

    2014-12-01

    Microbes drive processes in the Earth system far exceeding their physical scale, affecting crop yields, water quality, the mobilization of toxic materials, and fundamental aspects of soil biogeochemistry. The tools of synthetic biology have the potential to significantly improve our understanding of microbial Earth system processes: for example, synthetic microbes can be be programmed to report on environmental conditions that stimulate greenhouse gas production, metal oxidation, biofilm formation, pollutant degradation, and microbe-plant symbioses. However, these tools are only rarely deployed in the lab. This research gap arises because synthetically programmed microbes typically report on their environment by producing molecules that are detected optically (e.g., fluorescent proteins). Fluorescent reporters are ideal for petri-dish applications and have fundamentally changed how we study human health, but their usefulness is quite limited in soils where detecting fluorescence is challenging. Here we describe the construction of gas-reporting biosensors, which release nonpolar gases that can be detected in the headspace of incubation experiments. These constructs can be used to probe microbial processes within soils in real-time noninvasive lab experiments. These biosensors can be combined with traditional omics-based approaches to reveal processes controlling soil microbial behavior and lead to improved environmental management decisions.

  9. Biosensors based on cantilevers.

    Science.gov (United States)

    Alvarez, Mar; Carrascosa, Laura G; Zinoviev, Kiril; Plaza, Jose A; Lechuga, Laura M

    2009-01-01

    Microcantilevers based-biosensors are a new label-free technique that allows the direct detection of biomolecular interactions in a label-less way and with great accuracy by translating the biointeraction into a nanomechanical motion. Low cost and reliable standard silicon technologies are widely used for the fabrication of cantilevers with well-controlled mechanical properties. Over the last years, the number of applications of these sensors has shown a fast growth in diverse fields, such as genomic or proteomic, because of the biosensor flexibility, the low sample consumption, and the non-pretreated samples required. In this chapter, we report a dedicated design and a fabrication process of highly sensitive microcantilever silicon sensors. We will describe as well an application of the device in the environmental field showing the immunodetection of an organic toxic pesticide as an example. The cantilever biofunctionalization process and the subsequent pesticide determination are detected in real time by monitoring the nanometer-scale bending of the microcantilever due to a differential surface stress generated between both surfaces of the device.

  10. Guided-Wave Optical Biosensors

    Science.gov (United States)

    Passaro, Vittorio M. N.; Dell'Olio, Francesco; Casamassima, Biagio; De Leonardis, Francesco

    2007-01-01

    Guided-wave optical biosensors are reviewed in this paper. Advantages related to optical technologies are presented and integrated architectures are investigated in detail. Main classes of bio receptors and the most attractive optical transduction mechanisms are discussed. The possibility to use Mach-Zehnder and Young interferometers, microdisk and microring resonators, surface plasmon resonance, hollow and antiresonant waveguides, and Bragg gratings to realize very sensitive and selective, ultra-compact and fast biosensors is discussed. Finally, CMOS-compatible technologies are proved to be the most attractive for fabrication of guided-wave photonic biosensors.

  11. Evaluation of surface characteristics under fretting of electrical contacts: Removal behaviour of hot dipped tin coating

    International Nuclear Information System (INIS)

    Park, Young Woo; Ramesh Bapu, G.N.K.; Lee, Kang Yong

    2009-01-01

    The fretting corrosion behaviour of hot dipped tin coating is investigated at low fretting cycles at ±25 μm displacement amplitude, 0.5N normal load, 3 Hz frequency, 45-50% relative humidity, and 25 ± 1 deg. C temperature. The typical characteristics of the change in contact resistance with fretting cycles are explained. The fretted surface is examined using laser scanning microscope, scanning electron microscope and energy dispersive X-ray analysis to assess the surface profile, extent of fretting damage, extent of oxidation and elemental distribution across the contact zone. The interdependence of extent of wear and oxidation increases the complexity of the fretting corrosion behaviour of tin coating. The variation of contact resistance clearly revealed the fretting of tin coating from 50 to 1200 cycles and the fretting of the substrate above 1200 cycles. The observed low and stable contact resistance region and the fluctuating resistance region at various fretting cycles are explained and substantiated with Scanning electron microscopy (SEM), laser scanning microscope (LSM) and energy dispersive analysis of X-rays (EDAX) analysis results of the fretted surface.

  12. A regenerative electrochemical biosensor for mercury(II) by using the insertion approach and dual-hairpin-based amplification

    International Nuclear Information System (INIS)

    Jia, Jing; Ling, Yu; Gao, Zhong Feng; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing

    2015-01-01

    Highlights: • The dual-hairpin structure as a signal amplifier is label-free and handy. • The strategy uses the insertion approach to improve the hybridization efficiency. • This biosensor has a low detection limit (28 pM) for detection of Hg 2+ . • This biosensor can be easily regenerated by using L-cysteine. - Abstract: A simple and effective biosensor for Hg 2+ determination was investigated. The novel biosensor was prepared by the insertion approach that the moiety-labeled DNA inserted into a loosely packed cyclic-dithiothreitol (DTT) monolayer, improving the hybridization efficiency. Electrochemical impedance spectroscopy studies of two biosensors (single-hairpin and dual-hairpin structure DNA modified electrodes) used for Hg 2+ detection indicated that the dual-hairpin modified electrode had a larger electron transfer resistance change (ΔR ct ). Consequently, the dual-hairpin structure was used as a signal amplifier for the preparation of a selective Hg 2+ biosensor. This biosensor exhibited an excellent selectivity toward Hg 2+ over Cd 2+ , Pd 2+ , Co 2+ etc. Also, a linear relation was observed between the ΔR ct and Hg 2+ concentrations in a range from 0.1 nM to 5 μM with a detection limit of 28 pM under optimum conditions. Moreover, the biosensor can be reused by using L-cysteine and successfully applied for detecting Hg 2+ in real samples

  13. Nanobioengineering and Characterization of a Novel Estrogen Receptor Biosensor

    Directory of Open Access Journals (Sweden)

    Wilfrid Boireau

    2008-07-01

    Full Text Available We constructed an original supramolecular assembly on a surface of sensor composed of an innovative combination of an engineered cytochrome b5 and a modified nucleic acid bound to a synthetic lipid hemimembrane. The protein/DNA block, called (PDNA 2, was synthesized and purified before its immobilization onto a hybrid bilayer reconstituted on a gold surface. Surface plasmon resonance (SPR and atomic force microscopy (AFM were engaged in parallel on the same substrates in order to better understand dynamic events that occur at the surface of the biosensor. Good correlations were obtained in terms of specificity and reversibility. These findings allow us to present a first application of such biosensor in the study of the interaction processes between nuclear receptor and DNA.

  14. Loading dynamics of a sliding DNA clamp.

    KAUST Repository

    Cho, Won-Ki

    2014-05-22

    Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single-molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single-molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction.

  15. A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH.

    Science.gov (United States)

    Rupprecht, Christian; Wingen, Marcus; Potzkei, Janko; Gensch, Thomas; Jaeger, Karl-Erich; Drepper, Thomas

    2017-09-20

    The intracellular pH is an important modulator of various bio(techno)logical processes such as enzymatic conversion of metabolites or transport across the cell membrane. Changes of intracellular pH due to altered proton distribution can thus cause dysfunction of cellular processes. Consequently, accurate monitoring of intracellular pH allows elucidating the pH-dependency of (patho)physiological and biotechnological processes. In this context, genetically encoded biosensors represent a powerful tool to determine intracellular pH values non-invasively and with high spatiotemporal resolution. We have constructed a toolbox of novel genetically encoded FRET-based pH biosensors (named Fluorescence Biosensors for pH or FluBpH) that utilizes the FMN-binding fluorescent protein EcFbFP as donor domain. In contrast to many fluorescent proteins of the GFP family, EcFbFP exhibits a remarkable tolerance towards acidic pH (pK a ∼3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pK a values of 5.7, 6.1 and 7.5 were used as pH-sensing FRET acceptor domains. The resulting biosensors FluBpH 5.7, FluBpH 6.1 and FluBpH 7.5 were calibrated in vitro and in vivo to accurately evaluate their pH indicator properties. To demonstrate the in vivo applicability of FluBpH, changes of intracellular pH were ratiometrically measured in E. coli cells during acid stress. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Detection Limits for Nanoscale Biosensors

    National Research Council Canada - National Science Library

    Sheehan, Paul E; Whitman, Lloyd J

    2005-01-01

    We examine through analytical calculations and finite element simulations how the detection efficiency of disk and wire-like biosensors in unmixed fluids varies with size from the micrometer to nanometer scales...

  17. Novel trends in affinity biosensors: current challenges and perspectives

    International Nuclear Information System (INIS)

    Arugula, Mary A; Simonian, Aleksandr

    2014-01-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives. (topical review)

  18. DNA Diagnostics: Optical or by Electronics?

    KAUST Repository

    Khan, Hadayat Ullah; Knoll, Wolfgang

    2016-01-01

    In this paper, we very briefly review DNA biosensors based on optical and electrical detection principles, referring mainly to our past work applying both techniques but here using nearly identical sensor chip surface architectures, i.e., capture

  19. Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

    Directory of Open Access Journals (Sweden)

    Ruud G J Detert Oude Weme

    Full Text Available Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET. Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM. For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

  20. Micro- and nanogap based biosensors

    OpenAIRE

    Hammond, Jules L.

    2017-01-01

    Biosensors are used for the detection of a range of analytes for applications in healthcare, food production, environmental monitoring and biodefence. However, many biosensing platforms are large, expensive, require skilled operators or necessitate the analyte to be labelled. Direct electrochemical detection methods present a particularly attractive platform due to the simplified instrumentation when compared to other techniques such as fluorescence-based biosensors. With modern integrated ci...

  1. Burst pressure and leak rate from fretted SG tubes

    International Nuclear Information System (INIS)

    Hwang, Seong Sik; Jung, Man Kyo; Kim, Hong Pyo; Kim, Joung Soo

    2005-01-01

    Steam generator(SG) tubes of a pressurized water reactor(PWR) have suffered from various types of corrosion, such as pitting, wastage and stress corrosion cracking (SCC) on both the primary and secondary side. Recently, fretting/wear degradation at the tube support region has been reported in some Korean nuclear power plants. In order to prevent the primary coolant from leaking to the secondary side, the tubes are repaired by a sleeving or plugging. It is important to establish the repair criteria to assure a reactor integrity and yet maintain the plugging ratio within the limits needed for an efficient operation. The objective of the burst test is to obtain a relationship between the burst/leak rate and the shape of the fretted flaws machined with an electro discharge machining (EDM)

  2. Recent advances in transition-metal dichalcogenides based electrochemical biosensors: A review.

    Science.gov (United States)

    Wang, Yi-Han; Huang, Ke-Jing; Wu, Xu

    2017-11-15

    Layered transition metal dichalcogenides (TMDCs) comprise a category of two-dimensional (2D) materials that offer exciting properties, including large surface area, metallic and semi-conducting electrical capabilities, and intercalatable morphologies. Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. TMDCs nanomaterials have been widely applied in various electrochemical biosensors with high sensitivity and selectivity. The marriage of TMDCs and electrochemical biosensors has created many productive sensing strategies for applications in the areas of clinical diagnosis, environmental monitoring and food safety. In recent years, an increasing number of TMDCs-based electrochemical biosensors are reported, suggesting TMDCs offers new possibilities of improving the performance of electrochemical biosensors. This review summarizes recent advances in electrochemical biosensors based on TMDCs for detection of various inorganic and organic analytes in the last five years, including glucose, proteins, DNA, heavy metal, etc. In addition, we also point out the challenges and future perspectives related to the material design and development of TMDCs-based electrochemical biosensors. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Enhancement in sensitivity of graphene-based zinc oxide assisted bimetallic surface plasmon resonance (SPR) biosensor

    Science.gov (United States)

    Kumar, Rajeev; Kushwaha, Angad S.; Srivastava, Monika; Mishra, H.; Srivastava, S. K.

    2018-03-01

    In the present communication, a highly sensitive surface plasmon resonance (SPR) biosensor with Kretschmann configuration having alternate layers, prism/zinc oxide/silver/gold/graphene/biomolecules (ss-DNA) is presented. The optimization of the proposed configuration has been accomplished by keeping the constant thickness of zinc oxide (32 nm), silver (32 nm), graphene (0.34 nm) layer and biomolecules (100 nm) for different values of gold layer thickness (1, 3 and 5 nm). The sensitivity of the proposed SPR biosensor has been demonstrated for a number of design parameters such as gold layer thickness, number of graphene layer, refractive index of biomolecules and the thickness of biomolecules layer. SPR biosensor with optimized geometry has greater sensitivity (66 deg/RIU) than the conventional (52 deg/RIU) as well as other graphene-based (53.2 deg/RIU) SPR biosensor. The effect of zinc oxide layer thickness on the sensitivity of SPR biosensor has also been analysed. From the analysis, it is found that the sensitivity increases significantly by increasing the thickness of zinc oxide layer. It means zinc oxide intermediate layer plays an important role to improve the sensitivity of the biosensor. The sensitivity of SPR biosensor also increases by increasing the number of graphene layer (upto nine layer).

  4. A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

    Directory of Open Access Journals (Sweden)

    Ramesh K. Jha

    2018-06-01

    Full Text Available Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators. Keywords: Whole cell biosensor, Aromatic catabolism, Transcription factor, PcaU, Shikimate

  5. Recycling microcavity optical biosensors.

    Science.gov (United States)

    Hunt, Heather K; Armani, Andrea M

    2011-04-01

    Optical biosensors have tremendous potential for commercial applications in medical diagnostics, environmental monitoring, and food safety evaluation. In these applications, sensor reuse is desirable to reduce costs. To achieve this, harsh, wet chemistry treatments are required to remove surface chemistry from the sensor, typically resulting in reduced sensor performance and increased noise due to recognition moiety and optical transducer degradation. In the present work, we suggest an alternative, dry-chemistry method, based on O2 plasma treatment. This approach is compatible with typical fabrication of substrate-based optical transducers. This treatment completely removes the recognition moiety, allowing the transducer surface to be refreshed with new recognition elements and thus enabling the sensor to be recycled.

  6. Electrochemical and AFM Characterization of G-Quadruplex Electrochemical Biosensors and Applications

    Science.gov (United States)

    2018-01-01

    Guanine-rich DNA sequences are able to form G-quadruplexes, being involved in important biological processes and representing smart self-assembling nanomaterials that are increasingly used in DNA nanotechnology and biosensor technology. G-quadruplex electrochemical biosensors have received particular attention, since the electrochemical response is particularly sensitive to the DNA structural changes from single-stranded, double-stranded, or hairpin into a G-quadruplex configuration. Furthermore, the development of an increased number of G-quadruplex aptamers that combine the G-quadruplex stiffness and self-assembling versatility with the aptamer high specificity of binding to a variety of molecular targets allowed the construction of biosensors with increased selectivity and sensitivity. This review discusses the recent advances on the electrochemical characterization, design, and applications of G-quadruplex electrochemical biosensors in the evaluation of metal ions, G-quadruplex ligands, and other small organic molecules, proteins, and cells. The electrochemical and atomic force microscopy characterization of G-quadruplexes is presented. The incubation time and cations concentration dependence in controlling the G-quadruplex folding, stability, and nanostructures formation at carbon electrodes are discussed. Different G-quadruplex electrochemical biosensors design strategies, based on the DNA folding into a G-quadruplex, the use of G-quadruplex aptamers, or the use of hemin/G-quadruplex DNAzymes, are revisited. PMID:29666699

  7. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy

    Directory of Open Access Journals (Sweden)

    Maria E. Valieva

    2017-01-01

    Full Text Available A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD and the high mobility group (HMG domain of the structure-specific recognition protein 1 (SSRP1 subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16 and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3. Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

  8. Surveillance of siRNA integrity by FRET imaging

    Science.gov (United States)

    Järve, Anne; Müller, Julius; Kim, Il-Han; Rohr, Karl; MacLean, Caroline; Fricker, Gert; Massing, Ulrich; Eberle, Florian; Dalpke, Alexander; Fischer, Roger; Trendelenburg, Michael F.; Helm, Mark

    2007-01-01

    Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes. PMID:17890733

  9. Experimental fretting-wear studies of steam generator materials

    International Nuclear Information System (INIS)

    Fisher, N.J.; Chow, A.B.; Weckwerth, M.K.

    1994-01-01

    Flow-induced vibration of steam generator tubes results in fretting-wear damage due to impacting and rubbing of the tubes against their supports. This damage can be predicted by computing tube response to flow-induced excitation forces using analytical techniques, and then relating this response to resultant wear damage using experimentally-derived wear coefficients. Fretting-wear of steam generator materials has been studied experimentally at Chalk River Laboratories for two decades. Tests are conducted in machines that simulate steam generator environmental conditions and tube-to-support dynamic interactions. Different tube and support materials, tube-to-support clearances and tube support geometries have been studied. As well, the effect of environmental conditions, such as temperature, oxygen content, pH and chemistry control additive, have been investigated. Early studies showed that damage was related to contact force as long as other parameters, such as geometry and motion were held constant. Later studies have shown that damage is related to a parameter called work-rate, which combines both contact force and sliding distance. Results of short- and long-term fretting-wear tests for CANDU steam generator materials at realistic environmental conditions are presented. These results demonstrate that work-rate is appropriate correlating parameter for impact-sliding interaction

  10. Aspects of fretting wear of sprayed cermet coatings

    International Nuclear Information System (INIS)

    Chivers, T.C.

    1985-01-01

    Two experimental fretting programmes which investigated aspects of fretting wear of sprayed cermet coatings are reviewed. These programmes were conducted in support of components used in the advanced gas-cooled reactor. It is speculated that the results from these programmes are compatible with a simple two-stage wear model. This model assumes that an initial wear process occurs which is dominated by an interlocking and removal of asperities. Such a phase will be dependent on the superficial contact areas and possibly the interfacial load, but the latter aspect is not considered. This initial wear is of very short duration and is followed by a mild, oxidative, wear mode. Coatings data are also compared with those for structural steels. In short-term low temperature tests it appears that structural steels have comparable performance with the cermet coatings but it is argued that this is an artefact of the wear process. However, at high temperatures (600 0 C) wear of stainless steel could not be determined, the specimens showing a net weight gain. It is concluded that for in-reactor fretting applications cermet coatings will have advantages over structural steels at low temperatures. Even in high temperature regions some operation at low temperatures is experienced and consequently cermet coatings may be useful here also. (orig.)

  11. Protecting AREVA ATRIUM™ BWR fuel from debris fretting failure

    International Nuclear Information System (INIS)

    Cole, Steven E.; Garner, Norman L.; Lippert, Hans-Joachim; Graebert, Rüdiger; Mollard, Pierre; Hahn, Gregory C.

    2014-01-01

    Historically, debris fretting has been the leading cause of fuel rod failure in BWR fuel assemblies, costing the industry millions of dollars in lost generation and negatively impacting the working area of plant site personnel. In this paper the focus will be on recent BWR fuel product innovation designed to eliminate debris related failures. Experience feedback from more than three decades of operation history with non-line-of-sight FUELGUARD™ lower tie plate debris filters will be presented. The development and relative effectiveness of successive generations of filtration technology will be discussed. It will be shown that modern, state of the art debris filters are an effective defense against debris fretting failure. Protective measures extend beyond inlet nozzle debris filters. The comprehensive debris resistance features built into AREVA’s newest fuel design, the ATRIUM™ 11, reduce the overall risk of debris entrapment as well as providing a degree of protection from debris that may fall down on the fuel assembly from above, e.g., during refueling operations. The positive recent experience in a debris sensitive plant will be discussed showing that the combination of advanced fuel technology and a robust foreign material exclusion program at the reactor site can eliminate the debris fretting failure mechanism. (author)

  12. Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

    Directory of Open Access Journals (Sweden)

    Uhna Sung

    Full Text Available FRET (Förster Resonance Energy Transfer-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different "Nabi1" constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms and signal decay (~3 ms. We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP and mRuby2 (acceptor FP to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz.

  13. Fretting and wear of stainless and ferritic steels in LMFBR steam generators

    International Nuclear Information System (INIS)

    Lewis, M.W.J.; Campbell, C.S.

    1981-01-01

    Steam generators for LMFBR's may be subject to both fretting wear as a result of flow-induced vibrations and to wear from larger amplitude sliding movements from thermal changes. Results of tests simulating the latter are given for stainless and ferritic steels. For the assessment of fretting wear damage, vibration assessments must be combined with data on specific wear rates. Test mechanisms used to study fretting in sodium covering impact, impact-slide and pure rubbing are described and results presented. (author)

  14. Prediction of pressure tube fretting-wear damage due to fuel vibration

    International Nuclear Information System (INIS)

    Yetisir, M.; Fisher, N.J.

    1997-01-01

    Fretting marks between fuel bundle bearing pads and pressure tubes have been observed at the inlet end of some Darlington Nuclear Generating Station (NGS) and Bruce NGS fuel channels. The excitation mechanisms that lead to fretting are not fully understood. In this paper, the possibility of bearing pad-to-pressure tube fretting due to turbulence-induced motion of the fuel element is investigated. Numerical simulations indicate that this mechanism by itself is not likely to cause the level of fretting experienced in Darlington and Bruce NGSs. (orig.)

  15. Characteristics of CANDU fuel bundles that caused pressure tube fretting at the bundle midplane

    Energy Technology Data Exchange (ETDEWEB)

    Dennier, D; Manzer, A M [Atomic Energy of Canada Ltd., Mississauga, ON (Canada); Koehn, E [Ontario Hydro, Toronto, ON (Canada)

    1996-12-31

    Detailed measurements on new bundles, and those that caused fretting during in- and out-reactor tests, have given insight into the factors responsible for fretting at the midplane of the inlet bundle. Bottom fuel elements that were attached near radial endplate spokes and had inboard bearing pads in the rolled joint cavity produced a significant portion of the observed fret marks. These elements are influenced by several driving forces that deflect the centre bearing pads towards the pressure tube surface. The evidence suggests that slight changes in bundle design may be possible to reduce pressure tube fretting. (author). 4 refs., 3 tabs., 8 figs.

  16. Roughness Influence on Initiation of Fretting Fatigue Scar of Ti-6Al-4V Alloy

    Science.gov (United States)

    Capitanu, L.; Badita, L. L.; Florescu, V.; Tiganesteanu, C.

    2018-01-01

    This paper reports on the experimental studies undertaken to detect the early stage when appears the fretting wear of the Ti-6Al-4V alloy used for the hip prostheses. Wear is a critical aspect for estimating the fretting fatigue. Studies were performed on samples of special shape, in order to be able to study the influence of in contact surfaces roughness on the durability to fretting. Fretting buffers, with roughnesses Ra of the contact surface of 0.015 and 0.045 μm, and Ti-6Al-4V samples with roughnesses Ra = 0.045 μm, Ra = 0.075 μm and Ra = 0.19 μm, were used. Testing periods of 3 seconds, 1 minute and 5 minutes were selected to capture the moment of the fretting scar appearance, long before these initiate the eventual fretting cracking. Simultaneously with fretting wear of the surface, the friction coefficient was also measured. From the in time evolution determinations of the fretting wear, it resulted that, under the experimental conditions used, the minimum wear occurs at a certain value of the roughness and not at the minimum roughness. Surprisingly, the minimum friction coefficient does not coincide with the minimum fretting wear.

  17. Prediction of pressure tube fretting-wear damage due to fuel vibration

    Energy Technology Data Exchange (ETDEWEB)

    Yetisir, M; Fisher, N J [Atomic Energy of Canada Ltd., Chalk River, ON (Canada)

    1996-12-31

    Fretting marks between fuel bundle bearing pads and pressure tubes have been observed at the inlet end of some Darlington NGS (nuclear generating station) and Bruce NGS fuel channels. The excitation mechanisms that lead to fretting are not fully understood. In this paper, the possibility of bearing pad-to-pressure tube fretting due to turbulence-induced motion of the fuel element is investigated. Numerical simulations indicate that this mechanism by itself is not likely to cause the level of fretting experienced in Darlington and Bruce NGS`s (nuclear generating stations). (author). 12 refs., 2 tabs., 11 figs.

  18. A communication theoretical analysis of FRET-based mobile ad hoc molecular nanonetworks.

    Science.gov (United States)

    Kuscu, Murat; Akan, Ozgur B

    2014-09-01

    Nanonetworks refer to a group of nanosized machines with very basic operational capabilities communicating to each other in order to accomplish more complex tasks such as in-body drug delivery, or chemical defense. Realizing reliable and high-rate communication between these nanomachines is a fundamental problem for the practicality of these nanonetworks. Recently, we have proposed a molecular communication method based on Förster Resonance Energy Transfer (FRET) which is a nonradiative excited state energy transfer phenomenon observed among fluorescent molecules, i.e., fluorophores. We have modeled the FRET-based communication channel considering the fluorophores as single-molecular immobile nanomachines, and shown its reliability at high rates, and practicality at the current stage of nanotechnology. In this study, for the first time in the literature, we investigate the network of mobile nanomachines communicating through FRET. We introduce two novel mobile molecular nanonetworks: FRET-based mobile molecular sensor/actor nanonetwork (FRET-MSAN) which is a distributed system of mobile fluorophores acting as sensor or actor node; and FRET-based mobile ad hoc molecular nanonetwork (FRET-MAMNET) which consists of fluorophore-based nanotransmitter, nanoreceivers and nanorelays. We model the single message propagation based on birth-death processes with continuous time Markov chains. We evaluate the performance of FRET-MSAN and FRET-MAMNET in terms of successful transmission probability and mean extinction time of the messages, system throughput, channel capacity and achievable communication rates.

  19. Understanding and modeling Förster-type resonance energy transfer (FRET)

    CERN Document Server

    Hernández Martínez, Pedro Ludwig; Demir, Hilmi Volkan

    2017-01-01

    This Brief presents a complete study of the generalized theory of Förster-type energy transfer in nanostructures with mixed dimensionality. Here the aim is to obtain a generalized theory of FRET including a comprehensive set of analytical equations for all combinations and configurations of nanostructures and deriving generic expressions for the dimensionality involved. In this brief, the modification of FRET mechanism with respect to the nanostructure serving as the donor vs. the acceptor will be included, focusing on the rate’s distance dependency and the role of the effective dielectric function in FRET, which will be a unique, useful source for those who study and model FRET.

  20. Loading dynamics of a sliding DNA clamp.

    KAUST Repository

    Cho, Won-Ki; Jergic, Slobodan; Kim, Daehyung; Dixon, Nicholas E; Lee, Jong-Bong

    2014-01-01

    8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction.

  1. Multiplex and high-throughput DNA detection using surface plasmon mediated fluorescence

    Science.gov (United States)

    Mei, Zhong

    The overall objective of this research project was to develop a user-friendly and sensitive biosensor for nucleic acid aptamers with multiplexing and high-throughput capability. The sensing was based on the fluorescence signals emitted by the fluorophores coupling with plamonic nanoparticle (gold nanorod) deposited on a patterned substrate. Gold nanorods (GNRs) were synthesized using a binary mixture of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL) in seed mediated growth method. Polytetrafluoroethylene (PTFE) printed glass slides were selectively coated with a gold thin-film to define hydrophilic areas for GNR deposition. Due to the wettablity contrast, GNR solution dropped on the slide was induced to assemble exclusively in the hydrophilic spots. By controlling temperature and humidity of the evaporation process, vertically-standing GNR arrays were achieved on the pattered slide. Fluorescence was conjugated to GNR surface via DNA double strand with tunable length. Theoretical simulation predicted a flat layer ( 30 nm thick) of uniform "hot spots" presented on the GNR tips, which could modify the nearby fluorescence. Experimentally, the vertical GNR arrays yielded metallic enhanced fluorescence (MEF) effect, which was dependent on the spectrum overlap and GNR-fluorophore distance. Specifically, the maximum enhancement of Quasar 670 and Alexa 750 was observed when it was coupled with GNR664 (plasmonic wavelength 664 nm) and GNR778 respectively at a distance of 16 nm, while the carboxyfluorescein (FAM) was at maximal intensity when attached to gold nanosphere520. This offers an opportunity for multiplexed DNA sensing. Based on this, we developed a novel GNR mediated fluorescence biosensor for DNA detection. Fluorescence labeled haipin-DNA probes were introduced to designated spots of GNR array with the matching LSPR wavelengths on the substrate. The fluorescence was quenched originally because of Forster resonance energy transfer (FRET) effect

  2. NANOSCALE BIOSENSORS IN ECOSYSTEM EXPOSURE RESEARCH

    Science.gov (United States)

    This powerpoint presentation presented information on nanoscale biosensors in ecosystem exposure research. The outline of the presentation is as follows: nanomaterials environmental exposure research; US agencies involved in nanosensor research; nanoscale LEDs in biosensors; nano...

  3. BIOSENSORS FOR ENVIRONMENTAL MONITORING: A REGULATORY PERSPECTIVE

    Science.gov (United States)

    Biosensors show the potential to complement laboratory-based analytical methods for environmental applications. Although biosensors for potential environmental-monitoring applications have been reported for a wide range of environmental pollutants, from a regulatory perspective, ...

  4. Porous silicon localization for implementation in matrix biosensors

    International Nuclear Information System (INIS)

    Benilov, A.; Cabrera, M.; Skryshevsky, V.; Martin, J.-R.

    2007-01-01

    The search of appropriate substrates and methods of surface DNA functionalisation is one of the important tasks of semiconductor biosensors. In this work we develop a method of light-assisted porous silicon etching in order to localize porous silicon spots on silicon substrate for matrix fluorophore-labeled DNA sensors implementation. The principal difference of porous spots localization proposed is considered for n- and p-type Si substrates under the condition of supplementary illumination. The tuning of the porous profile via applying of lateral electric field is proposed and experimentally proved

  5. Biosensor for metal analysis and speciation

    Science.gov (United States)

    Aiken, Abigail M.; Peyton, Brent M.; Apel, William A.; Petersen, James N.

    2007-01-30

    A biosensor for metal analysis and speciation is disclosed. The biosensor comprises an electron carrier immobilized to a surface of an electrode and a layer of an immobilized enzyme adjacent to the electrode. The immobilized enzyme comprises an enzyme having biological activity inhibited by a metal to be detected by the biosensor.

  6. Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.

    Science.gov (United States)

    Jenison, Robert; Jaeckel, Heidi; Klonoski, Joshua; Latorra, David; Wiens, Jacinta

    2014-08-07

    Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60 000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105).

  7. Effects of fretting fatigue on the residual stress of shot peened Ti-6Al-4V samples

    International Nuclear Information System (INIS)

    Martinez, S.A.; Sathish, S.; Blodgett, M.P.; Mall, S.; Namjoshi, S.

    2005-01-01

    X-ray diffraction residual stress measurement has been utilized as nondestructive tool for the characterization of fretting fatigue damage in shot peened samples of Ti-6Al-4V. Prior to fretting fatigue damage, compressive residual stresses were found to be uniform over the entire face of the sample and independent of the measurement direction. After fretting fatigue, inside and in the vicinity of the fretting damage zone large relaxation of compressive residual stress was observed. An anisotropic residual stress distribution has been observed in the fretting fatigue damaged region. Residual stress measurements in interrupted fretting fatigue experiments showed that the relaxation of residual stress increases as the number of fretting fatigue cycles increase. The results are discussed in the light of their importance in establishing X-ray diffraction residual stress measurement technique as a nondestructive tool to characterize fretting fatigue damage

  8. Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers

    KAUST Repository

    Sun, Lu; Frykholm, Karolin; Fornander, Louise H.; Svedhem, Sofia; Westerlund, Fredrik; Å kerman, Bjö rn

    2014-01-01

    © 2014 American Chemical Society. Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions

  9. Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

    Science.gov (United States)

    Laine, Romain; Stuckey, Daniel W.; Manning, Hugh; Warren, Sean C.; Kennedy, Gordon; Carling, David

    2012-01-01

    We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that m

  10. Multi step FRET among three laser dyes Pyrene, Acriflavine and Rhodamine B

    International Nuclear Information System (INIS)

    Saha, Jaba; Dey, Dibyendu; Roy, Arpan Datta; Bhattacharjee, D.; Hussain, Syed Arshad

    2016-01-01

    Fluorescence Resonance Energy Transfer (FRET) system using three dyes has been demonstrated. It has been observed that multi step energy transfer occurred from Pyrene to Rhodamine B via Acriflavine. Here Acriflavine acts as an antenna to receive energy from Pyrene and transfer the same to Rhodamine B. This multi step FRET system is advantageous compared to the conventional FRET as this can be used to study molecular level interaction beyond conventional FRET distance (1–10 nm) as well as studying multi-branched macromolecules. The introduction of clay enhances the FRET efficiencies among the dye pair, which is an advantage to make the multi step system more useful. Similar approach can be used for increasing FRET efficiencies by using other dyes. - Highlights: • Multi-step FRET occurred from Pyrene (Py) to Rhodamine B (RhB) via Acriflavine (Acf). • Acf acts as an antenna to receive energy from Py and to transfer energy to RhB. • Multi-step FRET can be used to study molecular level interaction beyond 1–10 nm. • Incorporation of nanoclay laponite enhances the energy transfer efficiency.

  11. Nucleic Acid Base Analog FRET-Pair Facilitating Detailed Structural Measurements in Nucleic Acid Containing Systems

    DEFF Research Database (Denmark)

    Börjesson, Karl; Preus, Søren; El-Sagheer, Afaf

    2009-01-01

    We present the first nucleobase analog fluorescence resonance energy transfer (FRET)-pair. The pair consists of tCO, 1,3-diaza-2-oxophenoxazine, as an energy donor and the newly developed tC(nitro), 7-nitro-1,3-diaza-2-oxophenothiazine, as an energy acceptor. The FRET-pair successfully monitors d...

  12. A Lateral Flow Biosensor for the Detection of Single Nucleotide Polymorphisms.

    Science.gov (United States)

    Zeng, Lingwen; Xiao, Zhuo

    2017-01-01

    A lateral flow biosensor (LFB) is introduced for the detection of single nucleotide polymorphisms (SNPs). The assay is composed of two steps: circular strand displacement reaction and lateral flow biosensor detection. In step 1, the nucleotide at SNP site is recognized by T4 DNA ligase and the signal is amplified by strand displacement DNA polymerase, which can be accomplished at a constant temperature. In step 2, the reaction product of step 1 is detected by a lateral flow biosensor, which is a rapid and cost effective tool for nuclei acid detection. Comparing with conventional methods, it requires no complicated machines. It is suitable for the use of point of care diagnostics. Therefore, this simple, cost effective, robust, and promising LFB detection method of SNP has great potential for the detection of genetic diseases, personalized medicine, cancer related mutations, and drug-resistant mutations of infectious agents.

  13. Antibody Immobilization on Conductive Polymer Coated Nonwoven Fibers for Biosensors

    Directory of Open Access Journals (Sweden)

    Shannon K. MCGRAW

    2011-12-01

    Full Text Available This work is being performed to develop rapid and novel electrochemical biosensors for foodborne pathogen detection. This research focuses on electrotextile platforms to perform both capture and sensing functions in a single component. The biosensor uses nonwoven fiber membranes coated with conductive polymer and functionalized with antibodies for biological capture. This study examines three methods for antibody immobilization: passive adsorption, glutaraldehyde cross-linking, and EDC/Sulfo-NHS cross-linking. Antibodies are immobilized onto the conductive fiber surfaces for the specific capture of a target pathogen. The immobilization and capture capabilities of each method are analyzed through the use of two different fluorescent reporters: FITC and PicoGreen DNA stain. Fluorescence is measured using a fluorescent plate reader and then imaged using a fluorescent microscope. The effect of a blocking agent on specificity is also evaluated. It is found that glutaraldehyde with blocking is the best immobilization method with PicoGreen being the best fluorescent reporter.

  14. Micro-and nanoelectromechanical biosensors

    CERN Document Server

    Nicu, Liviu

    2014-01-01

    Most books dedicated to the issues of bio-sensing are organized by the well-known scheme of a biosensor. In this book, the authors have deliberately decided to break away from the conventional way of treating biosensing research by uniquely addressing biomolecule immobilization methods on a solid surface, fluidics issues and biosensing-related transduction techniques, rather than focusing simply on the biosensor. The aim is to provide a contemporary snapshot of the biosensing landscape without neglecting the seminal references or products where needed, following the downscaling (from the micr

  15. Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

    Science.gov (United States)

    Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

    2010-02-01

    An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

  16. Electrophoresis- and FRET-Based Measures of Serpin Polymerization.

    Science.gov (United States)

    Faull, Sarah V; Brown, Anwen E; Haq, Imran; Irving, James A

    2017-01-01

    Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

  17. A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors

    DEFF Research Database (Denmark)

    Vedel, Line; Bräuner-Osborne, Hans; Mathiesen, Jesper Mosolff

    2015-01-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or ...... also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS....

  18. Biosensors in Clinical Practice: Focus on Oncohematology

    Directory of Open Access Journals (Sweden)

    Agostino Cortelezzi

    2013-05-01

    Full Text Available Biosensors are devices that are capable of detecting specific biological analytes and converting their presence or concentration into some electrical, thermal, optical or other signal that can be easily analysed. The first biosensor was designed by Clark and Lyons in 1962 as a means of measuring glucose. Since then, much progress has been made and the applications of biosensors are today potentially boundless. This review is limited to their clinical applications, particularly in the field of oncohematology. Biosensors have recently been developed in order to improve the diagnosis and treatment of patients affected by hematological malignancies, such as the biosensor for assessing the in vitro pre-treatment efficacy of cytarabine in acute myeloid leukemia, and the fluorescence resonance energy transfer-based biosensor for assessing the efficacy of imatinib in chronic myeloid leukemia. The review also considers the challenges and future perspectives of biosensors in clinical practice.

  19. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    International Nuclear Information System (INIS)

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  20. A luminescent nisin biosensor

    Science.gov (United States)

    Immonen, Nina; Karp, Matti

    2006-02-01

    Nisin is a lantibiotic, an antibacterial peptide produced by certain Lactococcus lactis strains that kills or inhibits the growth of other bacteria. Nisin is widely used as a food preservative, and its long-time use suggests that it can be generally regarded as safe. We have developed a method for determining the amount of nisin in food samples that is based on luminescent biosensor bacteria. Bacterial luciferase operon luxABCDE was inserted into plasmid pNZ8048, and the construct was transformed by electroporation into Lc. lactis strain NZ9800, whose ability to produce nisin has been erased by deletion of the gene nisA. The operon luxABCDE has been modified to be functional in gram-positive bacteria to confer a bioluminescent phenotype without the requirement of adding an exogenous substrate. In the plasmid pNZ8048, the operon was placed under control of the nisin-inducible nisA promoter. The chromosomal nisRK genes of Lc. lactis NZ9800 allow it to sense nisin in the environment and relay this signal via signal transduction proteins NisK and NisR to initiate transcription from nisA promoter. In the case of our sensor bacteria, this leads to production of luciferase and, thus, luminescence that can be directly measured from living bacteria. Luminescence can be detected as early as within minutes of induction. The nisin assay described here provides a detection limit in the sub-picogram level per ml, and a linear area between 1 - 1000 pg/ml. The sensitivity of this assay exceeds the performance of all previously published methods.

  1. Label-free aptamer biosensor for selective detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Na, Weidan; Liu, Xiaotong; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2015-10-29

    We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd{sup 2+} on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results. - Highlights: • A novel strategy for the detection of thrombin was established based on BSA-CdS QDs. • DNA could serve as the co-ligands to stabilize CdS QDs and enhance the fluorescence intensity. • Thrombin could change the structure of DNA1 and quench the fluorescence of CdS QDs. • Thrombin in real sample was detected with satisfactory results.

  2. Inter-Dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV Algorithm

    Directory of Open Access Journals (Sweden)

    Henning Höfig

    2014-11-01

    Full Text Available Förster resonance energy transfer (FRET is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers.

  3. Simulation of FRET dyes allows quantitative comparison against experimental data

    Science.gov (United States)

    Reinartz, Ines; Sinner, Claude; Nettels, Daniel; Stucki-Buchli, Brigitte; Stockmar, Florian; Panek, Pawel T.; Jacob, Christoph R.; Nienhaus, Gerd Ulrich; Schuler, Benjamin; Schug, Alexander

    2018-03-01

    Fully understanding biomolecular function requires detailed insight into the systems' structural dynamics. Powerful experimental techniques such as single molecule Förster Resonance Energy Transfer (FRET) provide access to such dynamic information yet have to be carefully interpreted. Molecular simulations can complement these experiments but typically face limits in accessing slow time scales and large or unstructured systems. Here, we introduce a coarse-grained simulation technique that tackles these challenges. While requiring only few parameters, we maintain full protein flexibility and include all heavy atoms of proteins, linkers, and dyes. We are able to sufficiently reduce computational demands to simulate large or heterogeneous structural dynamics and ensembles on slow time scales found in, e.g., protein folding. The simulations allow for calculating FRET efficiencies which quantitatively agree with experimentally determined values. By providing atomically resolved trajectories, this work supports the planning and microscopic interpretation of experiments. Overall, these results highlight how simulations and experiments can complement each other leading to new insights into biomolecular dynamics and function.

  4. Nanopatterned Bulk Metallic Glass Biosensors.

    Science.gov (United States)

    Kinser, Emily R; Padmanabhan, Jagannath; Yu, Roy; Corona, Sydney L; Li, Jinyang; Vaddiraju, Sagar; Legassey, Allen; Loye, Ayomiposi; Balestrini, Jenna; Solly, Dawson A; Schroers, Jan; Taylor, André D; Papadimitrakopoulos, Fotios; Herzog, Raimund I; Kyriakides, Themis R

    2017-12-22

    Nanopatterning as a surface area enhancement method has the potential to increase signal and sensitivity of biosensors. Platinum-based bulk metallic glass (Pt-BMG) is a biocompatible material with electrical properties conducive for biosensor electrode applications, which can be processed in air at comparably low temperatures to produce nonrandom topography at the nanoscale. Work presented here employs nanopatterned Pt-BMG electrodes functionalized with glucose oxidase enzyme to explore the impact of nonrandom and highly reproducible nanoscale surface area enhancement on glucose biosensor performance. Electrochemical measurements including cyclic voltammetry (CV) and amperometric voltammetry (AV) were completed to compare the performance of 200 nm Pt-BMG electrodes vs Flat Pt-BMG control electrodes. Glucose dosing response was studied in a range of 2 mM to 10 mM. Effective current density dynamic range for the 200 nm Pt-BMG was 10-12 times greater than that of the Flat BMG control. Nanopatterned electrode sensitivity was measured to be 3.28 μA/cm 2 /mM, which was also an order of magnitude greater than the flat electrode. These results suggest that nonrandom nanotopography is a scalable and customizable engineering tool which can be integrated with Pt-BMGs to produce biocompatible biosensors with enhanced signal and sensitivity.

  5. Improved Ion-Channel Biosensors

    Science.gov (United States)

    Nadeau, Jay; White, Victor; Dougherty, Dennis; Maurer, Joshua

    2004-01-01

    An effort is underway to develop improved biosensors of a type based on ion channels in biomimetic membranes. These sensors are microfabricated from silicon and other materials compatible with silicon. As described, these sensors offer a number of advantages over prior sensors of this type.

  6. An electromagnetic system for biosensors

    NARCIS (Netherlands)

    2008-01-01

    The invention relates to an electromagnetic system for biosensors, in which the system can switch quickly between high magnetic gradients, without the need of movement of mech. elements. This is realized by two independent emu which are sepd. in the region of the pole shoes over a gap, in which a

  7. Development of Biosensors From Graphene

    Institute of Scientific and Technical Information of China (English)

    高瑞红; 孙红; 李霄寒; 于冲

    2017-01-01

    Graphene's success has stimulated great interest and research in the synthesis and characterization of graphene -like 2D materials, single and few -atom -thick layers of van der Waals materials, which show fascinating and technologically useful properties.This review presents an overview of recent electrochemical sensors and biosensors based on graphene and on graphene-like 2D materials.

  8. Biosensors and multiple mycotoxin analysis

    NARCIS (Netherlands)

    Gaag, B. van der; Spath, S.; Dietrich, H.; Stigter, E.; Boonzaaijer, G.; Osenbruggen, T. van; Koopal, K.

    2003-01-01

    An immunochemical biosensor assay for the detection of multiple mycotoxins in a sample is described.The inhibition assay is designed to measure four different mycotoxins in a single measurement, following extraction, sample clean-up and incubation with an appropriate cocktail of anti-mycotoxin

  9. A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction.

    Science.gov (United States)

    Xiao, Zhuo; Lie, Puchang; Fang, Zhiyuan; Yu, Luxin; Chen, Junhua; Liu, Jie; Ge, Chenchen; Zhou, Xuemeng; Zeng, Lingwen

    2012-09-04

    A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.

  10. Denaturation strategies for detection of double stranded PCR products on GMR magnetic biosensor array

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Lee, Jung-Rok; Guldberg, Per

    2017-01-01

    Microarrays and other surface-based nucleic acid detection schemes rely on the hybridization of the target to surface-bound detection probes. We present the first comparison of two strategies to detect DNA using a giant magnetoresistive (GMR) biosensor platform starting from an initially double...

  11. Printable Electrochemical Biosensors: A Focus on Screen-Printed Electrodes and Their Application

    Directory of Open Access Journals (Sweden)

    Keiichiro Yamanaka

    2016-10-01

    Full Text Available In this review we present electrochemical biosensor developments, focusing on screen-printed electrodes (SPEs and their applications. In particular, we discuss how SPEs enable simple integration, and the portability needed for on-field applications. First, we briefly discuss the general concept of biosensors and quickly move on to electrochemical biosensors. Drawing from research undertaken in this area, we cover the development of electrochemical DNA biosensors in great detail. Through specific examples, we describe the fabrication and surface modification of printed electrodes for sensitive and selective detection of targeted DNA sequences, as well as integration with reverse transcription-polymerase chain reaction (RT-PCR. For a more rounded approach, we also touch on electrochemical immunosensors and enzyme-based biosensors. Last, we present some electrochemical devices specifically developed for use with SPEs, including USB-powered compact mini potentiostat. The coupling demonstrates the practical use of printable electrode technologies for application at point-of-use. Although tremendous advances have indeed been made in this area, a few challenges remain. One of the main challenges is application of these technologies for on-field analysis, which involves complicated sample matrices.

  12. A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity.

    Science.gov (United States)

    Slattery, Scott D; Hahn, Klaus M

    2014-12-01

    Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single-chain and dual-chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for effects of upstream molecules. Copyright © 2014 John Wiley & Sons, Inc.

  13. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

    Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  14. Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Fluorescence resonance energy transfer (FRET between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

  15. A Study on Fretting Behavior in Room Temperature for Inconel Alloy 690

    Science.gov (United States)

    Kwon, Jae Do; Chai, Young Suck; Bae, Yong Tak; Choi, Sung Jong

    The initial crack under fretting condition occurs at lower stress amplitude and lower cycles of cyclic loading than that under plain fatigue condition. The fretting damage, for example, can be observed in fossil and nuclear power plant, aircraft, automobile and petroleum chemical plants etc. INCONEL alloy 690 is a high-chromium nickel alloy having excellent resistance to many corrosive aqueous media and high-temperature atmospheres. This alloy is used extensively in the industries of nuclear power, chemicals, heat-treatment and electronics. In this paper, the effect of fretting damage on fatigue behavior for INCONEL alloy 690 was studied. Also, various kinds of tests on mechanical properties such as hardness, tension and plain fatigue tests are performed. Fretting fatigue tests were carried out with flat-flat contact configuration using a bridge type contact pad and plate type specimen. Through these experiments, it is found that the fretting fatigue strength decreased about 43% compared to the plain fatigue strength. In fretting fatigue, the wear debris is observed on the contact surface, and the oblique micro-cracks are initiated at an earlier stage. These results can be used as the basic data in a structural integrity evaluation of heat and corrosion resistant alloy considering fretting damages.

  16. Impact Fretting Wear Behavior of Alloy 690 Tubes in Dry and Deionized Water Conditions

    Institute of Scientific and Technical Information of China (English)

    Zhen-Bing Cai; Jin-Fang Peng; Hao Qian; Li-Chen Tang; Min-Hao Zhu

    2017-01-01

    The impact fretting wear has largely occurred at nuclear power device induced by the flow-induced vibration,and it will take potential hazards to the service of the equipment.However,the present study focuses on the tangential fretting wear of alloy 690 tubes.Research on impact fretting wear of alloy 690 tubes is limited and the related research is imminent.Therefore,impact fretting wear behavior of alloy 690 tubes against 304 stainless steels is investigated.Deionized water is used to simulate the flow environment of the equipment,and the dry environment is used for comparison.Varied analytical techniques are employed to characterize the wear and tribochemical behavior during impact fretting wear.Characterization results indicate that cracks occur at high impact load in both water and dry equipment;however,the water as a medium can significantly delay the cracking time.The crack propagation behavior shows a jagged shape in the water,but crack extended disorderly in dry equipment because the water changed the stress distribution and retarded the friction heat during the wear process.The SEM and XPS analysis shows that the main failure mechanisms of the tube under impact fretting are fatigue wear and friction oxidation.The effect of medium(water) on fretting wear is revealed,which plays a potential and promising role in the service of nuclear power device and other flow equipments.

  17. MIL-L-87177 and CLT:X-10 Lubricants Improve Electrical Connector Fretting Corrosion Behavior

    International Nuclear Information System (INIS)

    AUKLAND, NEIL R.; HANLON, JAMES T.

    1999-01-01

    We have conducted a fretting research project using MIL-L-87177 and CLT: X-10 lubricants on Nano-miniature connectors. When they were fretted without lubricant, individual connectors first exceeded our 0.5 ohm failure criteria from 2,341 to 45,238 fretting cycles. With additional fretting, their contact resistance increased to more than 100,000 ohms. Unmodified MIL-L-87177 lubricant delayed the onset of first failure to between 430,000 and over 20,000,000 fretting cycles. MIL-L-87177 modified by addition of Teflon powder delayed first failure to beyond 5 million fretting cycles. Best results were obtained when Teflon was used and also when both the straight and modified lubricants were poured into and then out of the connector. CLT: X-10 lubricant delayed the onset of first failure to beyond 55 million cycles in one test where a failure was actually observed and to beyond 20 million cycles in another that was terminated without failure. CLT: X-10 recovered an unlubricated connector driven deeply into failure, with six failed pins recovering immediately and four more recovering during an additional 420 thousand fretting cycles. MIL-L-87177 was not able to recover a connector under similar conditions

  18. Influence of plasma molybdenizing and shot-peening on fretting damage behavior of titanium alloy

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Chang-bin, E-mail: tcbtop@126.com [School of Metallurgy and Engineering, Xi’an University of Architecture and Technology, Xi’an, Shaanxi 710055 (China); Institute of Corrosion and Protection, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China); Liu, Dao-xin, E-mail: liudaox@nwpu.edu.cn [Institute of Corrosion and Protection, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China); Tang, Bin [Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan Shanxi 030024 (China); Zhang, Xiao-hua [Institute of Corrosion and Protection, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China); Qin, Lin [Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan Shanxi 030024 (China); Liu, Cheng-song [Institute of Corrosion and Protection, Northwestern Polytechnical University, Xi’an, Shaanxi 710072 (China)

    2016-12-30

    Highlights: • Plasma molybdenizing increases FW resistance of Ti6Al4V, but reduces its FF life. • Shot-peened plasmamolybdenizing surface enhances FW and FF resistance of Ti6Al4V. • Combined treatment yields low surface-roughness & high hardness gradient distribution. • Combined treatment yields beneficial residual compressive stress & good toughness. • Anti-wear & -fatigue performance improvements for surface engineering applications. - Abstract: Effect of plasma molybdenizing and shot-peening on fretting wear and fretting fatigue behaviors of Ti6Al4V alloy was investigated. The plasma molybdenized layer composed of a dense molybdenum deposition layer and a Mo–Ti solid–solution layer can increase surface hardness by 2.8 times and cause its volume loss by fretting wear to decrease to 1/14 compared with that of the substrate. Plasma molybdenized treatment results in a significant decrease in resistance of the substrate to fretting fatigue. It is ascribed that the molybdenized layer with high hardness yields a low toughness, and its high surface roughness leads to a micro-notched effect. However, proper combination plasma molybdenizing and subsequent shot-peening may enhance the simultaneous fretting fatigue and fretting wear resistance of Ti6Al4V significantly, which can decrease the fretting wear volume loss to 1/27, and may increase the fretting fatigue life by more than 69 times. A synergistic improvement in fretting fatigue of the titanium alloy by combining surface alloying with shot-peening can be achieved. The results indicate that a beneficial residual compressive stress distribution, high surface hardness with suitable hardness gradient distribution, good apparent toughness, relatively low surface roughness, and excellent surface integrity are achieved.

  19. Capacitive Biosensors and Molecularly Imprinted Electrodes.

    Science.gov (United States)

    Ertürk, Gizem; Mattiasson, Bo

    2017-02-17

    Capacitive biosensors belong to the group of affinity biosensors that operate by registering direct binding between the sensor surface and the target molecule. This type of biosensors measures the changes in dielectric properties and/or thickness of the dielectric layer at the electrolyte/electrode interface. Capacitive biosensors have so far been successfully used for detection of proteins, nucleotides, heavy metals, saccharides, small organic molecules and microbial cells. In recent years, the microcontact imprinting method has been used to create very sensitive and selective biorecognition cavities on surfaces of capacitive electrodes. This chapter summarizes the principle and different applications of capacitive biosensors with an emphasis on microcontact imprinting method with its recent capacitive biosensor applications.

  20. An overview of the Canadian program to investigate vibration and fretting in nuclear fuel assemblies

    International Nuclear Information System (INIS)

    Oldaker, I.E.; Lane, A.D.; Forrest, C.F.

    The development of a model that would allow the fuel designer to predict the occurrence of fretting could materially reduce the amount of development testing of a new fuel design. To achieve this, we are working in several areas: to identify and measure the phenomena that excite fuel to vibrate, and to study their relation to reactor design features; to predict the vibratory response of a fuel assembly as a function of its design and environment, and; to study the relationship between vibration and fretting to determine when vibration results in fretting. (author)

  1. Fretting wear damage of HexTOOL{sup TM} composite depending on the different fibre orientations

    Energy Technology Data Exchange (ETDEWEB)

    Terekhina, S; Salvia, M; Fouvry, S [Laboratoire de Tribologie et Dynamique des Systemes, UMR CNRS ECL ENISE ENSMSE 5513, Ecole Centrale de Lyon, 69134 Ecully cedex (France); Malysheva, G; Tarasova, T, E-mail: svetlana.terekhina@ec-lyon.fr, E-mail: svetlanaterekhina@yandex.ru [Bauman Moscow State Technical University, 105005 Moscow, 5, 2nd Baumanskaya str (Russian Federation)

    2009-09-15

    The composites have drawn considerable interest in the mould processes. The vibrations and fatigue stresses induced in the moulds made evident to characterize the composite HexTOOL{sup TM} under fretting conditions. Fretting is a small-amplitude oscillatory motion between contacting surfaces. The running conditions fretting maps (RCFM) of composite at ambient conditions were established. The influence of different fiber orientations of HexTOOL{sup TM} composite on the wear kinetics was shown. An energy wear approach was developed. According to results of dynamic mechanical analysis (DMA), the viscoelastic properties of composite material were obtained.

  2. Turbulence induced Fretting-wear characteristics of steam generator helical tubes

    International Nuclear Information System (INIS)

    Jhung, Myung Jo; Jo, Jong Chull; Kim, Hho Jung; Yune, Young Gill; Yu, Seon Oh

    2005-01-01

    This study addresses safety assessment of the potential for fretting-wear damages on steam generator helical tubes due to turbulence-induced vibration in operating nuclear power plants. To get the natural frequency, corresponding mode shape and participation factor, modal analyses are performed for helical type tubes with various conditions. Special emphases are put on the effects of coil diameter and the number of turns on the modal and fretting wear characteristics of tubes. Also, investigated are the effects of external pressure on the tube modal characteristics as well as the effects of turbulence induced vibration on the fretting-wear characteristics of tubes

  3. Electrochemical paper-based peptide nucleic acid biosensor for detecting human papillomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Teengam, Prinjaporn [Program in Petrochemistry, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, 10330 (Thailand); Siangproh, Weena [Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110 (Thailand); Tuantranont, Adisorn [Nanoelectronics and MEMS Laboratory, National Electronics and Computer Technology Center, Pathumthani, 12120 (Thailand); Henry, Charles S. [Department of Chemistry, Colorado State University, Fort Collins, CO, 80523 (United States); Vilaivan, Tirayut [Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, 10330 (Thailand); Chailapakul, Orawon, E-mail: corawon@chula.ac.th [Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry, Chulalongkorn University, Pathumwan, Bangkok, 10330 (Thailand); Nanotec-CU Center of Excellence on Food and Agriculture, Bangkok, 10330 (Thailand)

    2017-02-01

    A novel paper-based electrochemical biosensor was developed using an anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe (AQ-PNA) and graphene-polyaniline (G-PANI) modified electrode to detect human papillomavirus (HPV). An inkjet printing technique was employed to prepare the paper-based G-PANI-modified working electrode. The AQ-PNA probe baring a negatively charged amino acid at the N-terminus was immobilized onto the electrode surface through electrostatic attraction. Electrochemical impedance spectroscopy (EIS) was used to verify the AQ-PNA immobilization. The paper-based electrochemical DNA biosensor was used to detect a synthetic 14-base oligonucleotide target with a sequence corresponding to human papillomavirus (HPV) type 16 DNA by measuring the electrochemical signal response of the AQ label using square-wave voltammetry before and after hybridization. It was determined that the current signal significantly decreased after the addition of target DNA. This phenomenon is explained by the rigidity of PNA-DNA duplexes, which obstructs the accessibility of electron transfer from the AQ label to the electrode surface. Under optimal conditions, the detection limit of HPV type 16 DNA was found to be 2.3 nM with a linear range of 10–200 nM. The performance of this biosensor on real DNA samples was tested with the detection of PCR-amplified DNA samples from the SiHa cell line. The new method employs an inexpensive and disposable device, which easily incinerated after use and is promising for the screening and monitoring of the amount of HPV-DNA type 16 to identify the primary stages of cervical cancer. - Highlights: • A paper-based DNA biosensor using AQ-PNA probe and G-PANI modified electrode was first developed. • This developed DNA biosensor was highly specific over the non-complementary DNA. • This sensor was successfully applied to detect the HPV-DNA type 16 obtained from cancer cell lines. • This sensor is inexpensive and

  4. Recent Development in Optical Fiber Biosensors

    Directory of Open Access Journals (Sweden)

    Catalina Bosch Ojeda

    2007-06-01

    Full Text Available Remarkable developments can be seen in the field of optical fibre biosensors in the last decade. More sensors for specific analytes have been reported, novel sensing chemistries or transduction principles have been introduced, and applications in various analytical fields have been realised. This review consists of papers mainly reported in the last decade and presents about applications of optical fiber biosensors. Discussions on the trends in optical fiber biosensor applications in real samples are enumerated.

  5. Biosensors based on nanomaterials and nanodevices

    CERN Document Server

    Li, Jun

    2013-01-01

    Biosensors Based on Nanomaterials and Nanodevices links interdisciplinary research from leading experts to provide graduate students, academics, researchers, and industry professionals alike with a comprehensive source for key advancements and future trends in nanostructured biosensor development. It describes the concepts, principles, materials, device fabrications, functions, system integrations, and applications of various types of biosensors based on signal transduction mechanisms, including fluorescence, photonic crystal, surface-enhanced Raman scattering, electrochemistry, electro-lumine

  6. BioSentinel: Developing a Space Radiation Biosensor

    Science.gov (United States)

    Santa Maria, Sergio R.

    2015-01-01

    BioSentinel is an autonomous fully self-contained science mission that will conduct the first study of the biological response to space radiation outside low Earth orbit (LEO) in over 40 years. The 4-unit (4U) BioSentinel biosensor system, is housed within a 6-Unit (6U) spacecraft, and uses yeast cells in multiple independent microfluidic cards to detect and measure DNA damage that occurs in response to ambient space radiation. Cell growth and metabolic activity will be measured using a 3-color LED detection system and a metabolic indicator dye with a dedicated thermal control system per fluidic card.

  7. Failure of fretted steam generator tubes under accident conditions

    International Nuclear Information System (INIS)

    Forrest, C.F.

    1996-10-01

    Tests were carried out with a bank of tubes in a water tunnel to determine the tolerance of flawed nuclear reactor steam generator tubes to accident conditions which would result in high cross-flow velocities. Fourteen specimen tubes were tested, each having one or two types of defect machined into the surface simulating fretting-wear type scars found in some operating steam generators. The tubes were tested at flow velocities sufficient to induce high fluid elastic-type vibrations. Seven of the tubes failed near the thinnest section of the defects during the one-hour tests, due to impacting and/or rubbing between the tube and the support. Strain gauges, displacement transducers, force gauges and an accelerometer were used on the target tube and/or the tube immediately downstream of it to measure their vibrational characteristics

  8. Portable evanescent wave fiber biosensor for highly sensitive detection of Shigella

    Science.gov (United States)

    Xiao, Rui; Rong, Zhen; Long, Feng; Liu, Qiqi

    2014-11-01

    A portable evanescent wave fiber biosensor was developed to achieve the rapid and highly sensitive detection of Shigella. In this study, a DNA probe was covalently immobilized onto fiber-optic biosensors that can hybridize with a fluorescently labeled complementary DNA. The sensitivity of detection for synthesized oligonucleotides can reach 10-10 M. The surface of the sensor can be regenerated with 0.5% sodium dodecyl sulfate solution (pH 1.9) for over 30 times without significant deterioration of performance. The total analysis time for a single sample, including the time for measurement and surface regeneration, was less than 6 min. We employed real-time polymerase chain reaction (PCR) and compared the results of both methods to investigate the actual Shigella DNA detection capability of the fiber-optic biosensor. The fiber-optic biosensor could detect as low as 102 colony-forming unit/mL Shigella. This finding was comparable with that by real-time PCR, which suggests that this method is a potential alternative to existing detection methods.

  9. A novel gold nanoparticle-DNA aptamer-based plasmonic chip for rapid and sensitive detection of bacterial pathogens

    DEFF Research Database (Denmark)

    Sun, Yi; Phuoc Long, Truong; Wolff, Anders

    2016-01-01

    Gold nanoparticles (AuNPs)-based biosensors are emerging technologies for rapid detection of pathogens. However, it is very challenging to develop chip-based AuNP-biosensors for whole cells. This paper describes a novel AuNPs-DNA aptamer-based plasmonic assay which allows DNA aptamers...

  10. Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

    Science.gov (United States)

    Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

    2016-11-25

    Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

  11. A regenerative electrochemical biosensor for mercury(II) by using the insertion approach and dual-hairpin-based amplification

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Jing; Ling, Yu; Gao, Zhong Feng [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: luohq@swu.edu.cn [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: linb@swu.edu.cn [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2015-09-15

    Highlights: • The dual-hairpin structure as a signal amplifier is label-free and handy. • The strategy uses the insertion approach to improve the hybridization efficiency. • This biosensor has a low detection limit (28 pM) for detection of Hg{sup 2+}. • This biosensor can be easily regenerated by using L-cysteine. - Abstract: A simple and effective biosensor for Hg{sup 2+} determination was investigated. The novel biosensor was prepared by the insertion approach that the moiety-labeled DNA inserted into a loosely packed cyclic-dithiothreitol (DTT) monolayer, improving the hybridization efficiency. Electrochemical impedance spectroscopy studies of two biosensors (single-hairpin and dual-hairpin structure DNA modified electrodes) used for Hg{sup 2+} detection indicated that the dual-hairpin modified electrode had a larger electron transfer resistance change (ΔR{sub ct}). Consequently, the dual-hairpin structure was used as a signal amplifier for the preparation of a selective Hg{sup 2+} biosensor. This biosensor exhibited an excellent selectivity toward Hg{sup 2+} over Cd{sup 2+}, Pd{sup 2+}, Co{sup 2+} etc. Also, a linear relation was observed between the ΔR{sub ct} and Hg{sup 2+} concentrations in a range from 0.1 nM to 5 μM with a detection limit of 28 pM under optimum conditions. Moreover, the biosensor can be reused by using L-cysteine and successfully applied for detecting Hg{sup 2+} in real samples.

  12. Fretting friction and wear characteristics of magnetorheological fluid under different magnetic field strengths

    International Nuclear Information System (INIS)

    Zhang, P.; Lee, K.H.; Lee, C.H.

    2017-01-01

    A magnetorheological fluid (MRF) performs differently under different magnetic field strength. This study examined the fretting friction and wear characteristics of MRFs under a range of magnetic field strengths and oscillation frequencies. The fretting friction and wear behaviors of MRF are investigated using a fretting friction and wear tester. The surfaces of specimen are examined by optical microscopy and 3D surface profilometer before and after the tests and wear surface profiles, the wear volume loss and wear coefficient for each magnetic field strength are evaluated. The results show that the friction and wear properties of MRF change according to the magnetic field strength and oscillation frequency. - Highlights: • Fretting friction and wear characteristics of MRF is examined. • The friction coefficients increased with increasing magnetic field strength. • The coefficient of friction decreased with increasing oscillation frequency. • Wear volume and coefficient become worse with increasing magnetic field strength.

  13. FLIM-FRET image analysis of tryptophan in prostate cancer cells

    Science.gov (United States)

    Periasamy, Ammasi; Alam, Shagufta R.; Svindrych, Zdenek; Wallrabe, Horst

    2017-07-01

    A region of interest (ROI) based quantitative FLIM-FRET image analysis is developed to quantitate the autofluorescence signals of the essential amino acid tryptophan as a biomarker to investigate the metabolism in prostate cancer cells.

  14. Dichotomy Boundary at Aeolis Mensae, Mars: Fretted Terrain Developed in a Sedimentary Deposit

    Science.gov (United States)

    Irwin, R. P., III; Watters, T. R.; Howard, A. D.; Maxwell, T. A.; Craddock, R. A.

    2003-03-01

    Fretted terrain in Aeolis Mensae, Mars, developed in a sedimentary deposit. A thick, massive unit with a capping layer or duricrust overlies a more durable layered sequence. Wind, collapse, and minor fluvial activity contributed to degradation.

  15. Methodological considerations for global analysis of cellular FLIM/FRET measurements

    Science.gov (United States)

    Adbul Rahim, Nur Aida; Pelet, Serge; Kamm, Roger D.; So, Peter T. C.

    2012-02-01

    Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

  16. Combined effects of fretting and pollutant particles on the contact resistance of the electrical connectors

    Directory of Open Access Journals (Sweden)

    Zhigang Kong

    2017-06-01

    Full Text Available Usually, when electrical connectors operate in vibration environments, fretting will be produced at the contact interfaces. In addition, serious environmental pollution particles will affect contact resistance of the connectors. The fretting will worsen the reliability of connectors with the pollutant particles. The combined effects of fretting and quartz particles on the contact resistance of the gold plating connectors are studied with a fretting test system. The results show that the frequencies have obvious effect on the contact resistance. The higher the frequency, the higher the contact resistance is. The quartz particles cause serious wear of gold plating, which make the nickel and copper layer exposed quickly to increase the contact resistance. Especially in high humidity environments, water supply certain adhesion function and make quartz particles easy to insert or cover the contact surfaces, and even cause opening resistance.

  17. Single-molecule three-color FRET with both negligible spectral overlap and long observation time.

    Directory of Open Access Journals (Sweden)

    Sanghwa Lee

    Full Text Available Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF microscopy.

  18. Fretting corrosion tests on orthopedic plates and screws made of ASTM F138 stainless steel

    OpenAIRE

    Santos,Claudio Teodoro dos; Barbosa,Cássio; Monteiro,Maurício de Jesus; Abud,Ibrahim de Cerqueira; Caminha,Ieda Maria Vieira; Roesler,Carlos Rodrigo de Mello

    2015-01-01

    Introduction Although there has been significant progress in the design of implants for osteosynthesis, the occurrence of failures in these medical devices are still frequent. These implants are prone to suffer from fretting corrosion due to micromotion that takes place between the screw heads and plate holes. Consequently, fretting corrosion has been the subject of research in order to understand its influence on the structural integrity of osteosynthesis implants. The aim of this paper is t...

  19. An experimental study on the key fretting variables for flexible marine risers

    OpenAIRE

    O’Halloran, S.M.; Harte, A.M.; Shipway, P.H.; Leen, S.B.

    2018-01-01

    This paper presents an experimental investigation into the effects of contact conformity, contact pressure and displacement amplitude on the gross-slip fretting behaviour grease-lubricated cylinder-on-flat contacts in the context of flexible marine riser pressure armour wire, and compares behaviour with that observed in unlubricated conditions. Characterisation of friction and wear is critical to fretting fatigue life prediction in flexible risers since friction directly controls trailing-edg...

  20. EXPERIMENTAL INVESTIGTION OF THE FRETTING PHENOMENON-DEPENDENCE OF NUMBERS CYCLES

    Directory of Open Access Journals (Sweden)

    Ştefan GHIMISI

    2014-12-01

    Full Text Available The present paper argues that adhesion forces and elastic deformation in the contact zone may contribute significantly to the relative displacement during fretting of metals. A simultaneously applied tangential force and normal into contact appears a adhesion force. A tangential force whose magnitude is less equal on greater than the force of limiting friction will not give rise on give rise to a sliding motion.It is determined the energy loss dissipated per fretting cycle.

  1. Two-dimensional Forster resonance energy transfer (2-D FRET) and the membrane raft hypothesis

    OpenAIRE

    Acasandrei, Maria; Dale, Robert; VAN DE VEN, Martin; AMELOOT, Marcel

    2006-01-01

    A model for analyzing Forster resonance energy transfer (FRET) data in relation to the cell plasma membrane raft hypothesis is developed to take into account: (a) the distribution of FRET donors and acceptors at the surface of probing antibody fragments specific for a putative raft component; (b) partitioning of the raft component between raft and non-raft areas of the membrane; and (c) the dependence of the raft partition on the expression level of the considered component. Analysis of relev...

  2. Fretting wear characteristic tests of X2-GEN midgrid for SMART under a FIV rod trace

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Ho; Lee, Kang Hee; Kim, Jae Yong; Kim, Hyung Kyu [KAERI, Daejeon (Korea, Republic of)

    2011-12-15

    The KEPCO Nuclear Fuel Co. requested the fretting wear characteristic tests of a X2-GEN midgrid under a FIV rod trace at room temperature air. The following results were obtained for the fretting wear test. {center_dot} Fretting wear tests under a FIV rod trace Based on the result of the fretting wear tests of the X2-GEN and 17ACE7 1x1 mid-grid under a FIV rod trace, X2-GEN mid-grid showed a slightly severe wear volume rather than 17ACE7 spring. But, maximum wear depth shows an opposite behavior. This is due to spring shape effect. The fretting wear mechanisms at each mid-grid were influenced by each spring shape, that are depended on the different impacting behavior under a FIV rod motion. Up to 5x105 cycles, wear characteristics of each mid-grid shows a relatively similar wear rate. Consequently, it is necessary to further study for examining exact fretting wear behavior under a FIV rod tra

  3. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    Science.gov (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. © 2016 Society for Laboratory Automation and Screening.

  4. Standard test method for damage to contacting solid surfaces under fretting conditions

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 This test method covers the studying or ranking the susceptibility of candidate materials to fretting corrosion or fretting wear for the purposes of material selection for applications where fretting corrosion or fretting wear can limit serviceability. 1.2 This test method uses a tribological bench test apparatus with a mechanism or device that will produce the necessary relative motion between a contacting hemispherical rider and a flat counterface. The rider is pressed against the flat counterface with a loading mass. The test method is intended for use in room temperature air, but future editions could include fretting in the presence of lubricants or other environments. 1.3 The purpose of this test method is to rub two solid surfaces together under controlled fretting conditions and to quantify the damage to both surfaces in units of volume loss for the test method. 1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.5...

  5. Antibody orientation on biosensor surfaces: a minireview

    NARCIS (Netherlands)

    Trilling, A.K.; Beekwilder, M.J.; Zuilhof, H.

    2013-01-01

    Detection elements play a key role in analyte recognition in biosensors. Therefore, detection elements with high analyte specificity and binding strength are required. While antibodies (Abs) have been increasingly used as detection elements in biosensors, a key challenge remains – the immobilization

  6. A New Laccase Based Biosensor for Tartrazine

    Directory of Open Access Journals (Sweden)

    Siti Zulaikha Mazlan

    2017-12-01

    Full Text Available Laccase enzyme, a commonly used enzyme for the construction of biosensors for phenolic compounds was used for the first time to develop a new biosensor for the determination of the azo-dye tartrazine. The electrochemical biosensor was based on the immobilization of laccase on functionalized methacrylate-acrylate microspheres. The biosensor membrane is a composite of the laccase conjugated microspheres and gold nanoparticles (AuNPs coated on a carbon-paste screen-printed electrode. The reaction involving tartrazine can be catalyzed by laccase enzyme, where the current change was measured by differential pulse voltammetry (DPV at 1.1 V. The anodic peak current was linear within the tartrazine concentration range of 0.2 to 14 μM (R2 = 0.979 and the detection limit was 0.04 μM. Common food ingredients or additives such as glucose, sucrose, ascorbic acid, phenol and sunset yellow did not interfere with the biosensor response. Furthermore, the biosensor response was stable up to 30 days of storage period at 4 °C. Foods and beverage were used as real samples for the biosensor validation. The biosensor response to tartrazine showed no significant difference with a standard HPLC method for tartrazine analysis.

  7. Background reduction in a young interferometer biosensor

    NARCIS (Netherlands)

    Mulder, H. K P; Subramaniam, V.; Kanger, J. S.

    2014-01-01

    Integrated optical Young interferometer (IOYI) biosensors are among the most sensitive label-free biosensors. Detection limits are in the range of 20 fg/mm2. The applicability of these sensors is however strongly hampered by the large background that originates from both bulk refractive index

  8. A New Laccase Based Biosensor for Tartrazine.

    Science.gov (United States)

    Mazlan, Siti Zulaikha; Lee, Yook Heng; Hanifah, Sharina Abu

    2017-12-09

    Laccase enzyme, a commonly used enzyme for the construction of biosensors for phenolic compounds was used for the first time to develop a new biosensor for the determination of the azo-dye tartrazine. The electrochemical biosensor was based on the immobilization of laccase on functionalized methacrylate-acrylate microspheres. The biosensor membrane is a composite of the laccase conjugated microspheres and gold nanoparticles (AuNPs) coated on a carbon-paste screen-printed electrode. The reaction involving tartrazine can be catalyzed by laccase enzyme, where the current change was measured by differential pulse voltammetry (DPV) at 1.1 V. The anodic peak current was linear within the tartrazine concentration range of 0.2 to 14 μM ( R ² = 0.979) and the detection limit was 0.04 μM. Common food ingredients or additives such as glucose, sucrose, ascorbic acid, phenol and sunset yellow did not interfere with the biosensor response. Furthermore, the biosensor response was stable up to 30 days of storage period at 4 °C. Foods and beverage were used as real samples for the biosensor validation. The biosensor response to tartrazine showed no significant difference with a standard HPLC method for tartrazine analysis.

  9. Nanomaterials based biosensors for cancer biomarker detection

    International Nuclear Information System (INIS)

    Malhotra, Bansi D; Kumar, Saurabh; Pandey, Chandra Mouli

    2016-01-01

    Biosensors have enormous potential to contribute to the evolution of new molecular diagnostic techniques for patients suffering with cancerous diseases. A major obstacle preventing faster development of biosensors pertains to the fact that cancer is a highly complex set of diseases. The oncologists currently rely on a few biomarkers and histological characterization of tumors. Some of the signatures include epigenetic and genetic markers, protein profiles, changes in gene expression, and post-translational modifications of proteins. These molecular signatures offer new opportunities for development of biosensors for cancer detection. In this context, conducting paper has recently been found to play an important role towards the fabrication of a biosensor for cancer biomarker detection. In this paper we will focus on results of some of the recent studies obtained in our laboratories relating to fabrication and application of nanomaterial modified paper based biosensors for cancer biomarker detection. (paper)

  10. Functionalized Palladium Nanoparticles for Hydrogen Peroxide Biosensor

    Directory of Open Access Journals (Sweden)

    H. Baccar

    2011-01-01

    Full Text Available We present a comparison between two biosensors for hydrogen peroxide (H2O2 detection. The first biosensor was developed by the immobilization of Horseradish Peroxidase (HRP enzyme on thiol-modified gold electrode. The second biosensor was developed by the immobilization of cysteamine functionalizing palladium nanoparticles on modified gold surface. The amino groups can be activated with glutaraldehyde for horseradish peroxidase immobilization. The detection of hydrogen peroxide was successfully observed in PBS for both biosensors using the cyclic voltammetry and the chronoamperometry techniques. The results show that the limit detection depends on the large surface-to-volume ratio attained with palladium nanoparticles. The second biosensor presents a better detection limit of 7.5 μM in comparison with the first one which is equal to 75 μM.

  11. A new assay format for NF-kappaB based on a DNA triple helix and a fluorescence resonance energy transfer.

    Science.gov (United States)

    Altevogt, Dominik; Hrenn, Andrea; Kern, Claudia; Clima, Lilia; Bannwarth, Willi; Merfort, Irmgard

    2009-10-07

    Herein we report a feasibility study for a new concept to detect DNA binding protein NF-kappaB based on a DNA triple helix formation in combination with a fluorescence resonance energy transfer (FRET). The new principle avoids expensive antibodies and radioactivity and might have implications for assays of other DNA binding proteins.

  12. Quantum dot-based microfluidic biosensor for cancer detection

    Energy Technology Data Exchange (ETDEWEB)

    Ghrera, Aditya Sharma [Biomedical Instrumentation Section, CSIR-National Physical Laboratory, New Delhi-110012 (India); School of Engineering and Technology, ITM University, Gurgaon-122017 (India); Pandey, Chandra Mouli; Ali, Md. Azahar [Biomedical Instrumentation Section, CSIR-National Physical Laboratory, New Delhi-110012 (India); Malhotra, Bansi Dhar, E-mail: bansi.malhotra@gmail.com [Department of Biotechnology, Delhi Technological University, Delhi-110042 (India)

    2015-05-11

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

  13. Quantum dot-based microfluidic biosensor for cancer detection

    Science.gov (United States)

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

    2015-05-01

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

  14. Quantum dot-based microfluidic biosensor for cancer detection

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  15. A combined wear-fatigue design methodology for fretting in the pressure armour layer of flexible marine risers

    OpenAIRE

    O'Halloran, S.M.; Shipway, P.H.; Connaire, A.D.; Leen, Sean B.; Harte, A.M.

    2017-01-01

    This paper presents a combined experimental and computational methodology for fretting wear-fatigue prediction of pressure armour wire in flexible marine risers. Fretting wear, friction and fatigue parameters of pressure armour material have been characterised experimentally. A combined fretting wear-fatigue finite element model has been developed using an adaptive meshing technique and the effect of bending-induced tangential slip has been characterised. It has been shown that a surface dama...

  16. Electrochemical biosensors in pharmaceutical analysis

    Directory of Open Access Journals (Sweden)

    Eric de Souza Gil

    2010-09-01

    Full Text Available Given the increasing demand for practical and low-cost analytical techniques, biosensors have attracted attention for use in the quality analysis of drugs, medicines, and other analytes of interest in the pharmaceutical area. Biosensors allow quantification not only of the active component in pharmaceutical formulations, but also the analysis of degradation products and metabolites in biological fluids. Thus, this article presents a brief review of biosensor use in pharmaceutical analysis, focusing on enzymatic electrochemical sensors.Em virtude do aumento da demanda por técnicas analíticas simples e de baixo custo, os biossensores têm atraído a atenção para a análise de fármacos, medicamentos e outros analitos de interesse em controle de qualidade de medicamentos. Os biossensores permitem a quantificação não somente de princípio ativo em formulações farmacêuticas, mas também de produtos de degradação e metabólitos em fluídos biológicos, bem como análise de amostras de interesse clínico e industrial, além de possibilitar a determinação de enantiômeros. Desta forma, este artigo objetiva fazer uma breve revisão a respeito do emprego de biossensores em análise farmacêutica, com ênfase em sensores eletroquímicos enzimáticos.

  17. An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

    Science.gov (United States)

    Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

    2012-12-01

    Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

  18. Residual stress relaxation due to fretting fatigue in shot peened surfaces of Ti-6Al-4V

    International Nuclear Information System (INIS)

    Martinez, S.A.; Blodgett, M.P.; Mall, S.; Sathish, S.; Namjoshi, S.

    2003-01-01

    Fretting fatigue occurs at locations where the materials are sliding against each other under load. In order to enhance the fatigue life under fretting conditions the surface of the component is shot peened. In general, the shot peening process produces a compressive stress on the surface of the material, thereby increasing the resistance of the material to crack initiation. This paper presents the relaxation of residual stress caused during fretting fatigue. X-ray diffraction has been utilized as the method to measure residual stress in fretting fatigued samples of Ti-6Al-4V

  19. Steam generator fretting-wear damage: A summary of recent findings

    International Nuclear Information System (INIS)

    Guerout, F.M.; Fisher, N.J.

    1999-01-01

    Flow-induced vibration of steam generator (SG) tubes may sometimes result in fretting-wear damage at the tube-to-support locations. Fretting-wear damage predictions are largely based on experimental data obtained at representative test conditions. Fretting-wear of SG materials has been studied at the Chalk River Laboratories for two decades. Tests are conducted in fretting-wear test machines that simulate SG environmental conditions and tube-to-support dynamic interactions. A new high-temperature force and displacement measuring system was developed to monitor tube-to-support interaction (i.e., work-rate) at operating conditions. This improvement in experimental fretting-wear technology was used to perform a comprehensive study of the effect of various environment and design parameters on SG tube wear damage. This paper summarizes the results of tests performed over the past 4 yr to study the effect of temperature, water chemistry, support geometry, and tube material on fretting-wear. The results show a significant effect of temperature on tube wear damage. Therefore, fretting-wear tests must be performed at operating temperatures in order to be relevant. No significant effect of the type of water treatment on tube wear damage was observed. For predominantly impacting motion, the wear of SG tubes in contact with 410 stainless steel is similar regardless of whether Alloy 690 or Alloy 800 is used as tubing material or whether lattice bars or broached hole supports are used. Based on results presented in this paper, an average wear coefficient value is recommended that is used for the prediction of SG tube wear depth versus time

  20. Determination of a Wear Initiation Cycle by using a Contact Resistance Measurement in Nuclear Fuel Fretting

    International Nuclear Information System (INIS)

    Lee, Young Ho; Kim, Hyung Kyu

    2008-01-01

    In nuclear fuel fretting, the improving of the contact condition with a modified spring shape is a useful method for increasing the wear resistance of the nuclear fuel rod. This is because the fretting wear resistance between the fuel rod and grid spring is mainly affected by the grid spring shape rather than the environment, the contact modes, etc. In addition, the wear resistance is affected by the wear debris behavior between contact surfaces. So, it is expected that the wear initiation of each spring shape should be determined in order to evaluate a wear resistance. However, it is almost impossible to measure the wear behavior in contact surfaces on a real time basis because the contact surfaces are always hidden. Besides, the results of the worn surface observation after the fretting wear tests are restricted to archive the information on the wear debris behavior and the formation mechanism of the wear scar. In order to evaluate the wear behavior during the fretting wear tests, it is proposed that the contact resistance measurement is a useful method for examining the wear initiation cycle and modes. Generally, fretting wear damages are rapidly progressed by a localized plastic deformation between the contact surfaces, crack initiation and fracture of the deformed surface with a strain hardening difference between a surface and a subsurface and finally a detachment of wear debris. After this, wear debris is easily oxidized by frictional heat, test environment, etc. At this time, a small amount of electric current applied between the contact surfaces will be influenced by the wear debris, which could be an obstacle to an electric current flow. So, it is possible to archive the information on the wear behavior by measuring the contact resistance. In order to determine the wear initiation cycle during the fretting wear tests, in this study, fretting wear tests have been performed by applying a constant electric current in room temperature air

  1. Effect of mixed alloy combinations on fretting corrosion performance of spinal screw and rod implants.

    Science.gov (United States)

    Mali, Sachin A; Singh, Vaneet; Gilbert, Jeremy L

    2017-07-01

    Spinal implants are made from a variety of materials to meet the unique mechanical demands of each application. However, the medical device community has raised concern about mixing dissimilar metals in an implant because of fear of inducing corrosion. There is a lack of systematic studies on the effects of mixing metals on performance of spinal implants, especially in fretting corrosion conditions. Hence, the goal was to determine whether mixing stainless steel (SS316L), titanium alloy (Ti6Al4V) and cobalt chromium (CoCrMo) alloy components in a spinal implant leads to any increased risk of corrosion degradation. Spinal constructs consisting of single assembly screw-connector-rod components were tested using a novel short-term cyclic fretting corrosion test method. A total of 17 alloy component combinations (comprised of SS316L, Ti6Al4V-anodized and CoCrMo alloy for rod, screws and connectors) were tested under three anatomic orientations. Spinal constructs having all SS316L were most susceptible to fretting-initiated crevice corrosion attack and showed higher average fretting currents (∼25 - 30 µA), whereas constructs containing all Ti6Al4V components were less susceptible to fretting corrosion with average fretting currents in the range of 1 - 6 µA. Mixed groups showed evidence of fretting corrosion but they were not as severe as all SS316L group. SEM results showed evidence of severe corrosion attack in constructs having SS316L components. There also did not appear to be any galvanic effects of combining alloys together. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1169-1177, 2017. © 2016 Wiley Periodicals, Inc.

  2. Fretting and Corrosion Damage in Taper Adapter Sleeves for Ceramic Heads: A Retrieval Study.

    Science.gov (United States)

    MacDonald, Daniel W; Chen, Antonia F; Lee, Gwo-Chin; Klein, Gregg R; Mont, Michael A; Kurtz, Steven M; Cates, Harold E; Kraay, Matthew J; Rimnac, Clare M

    2017-09-01

    During revision surgery with a well-fixed stem, a titanium sleeve can be used in conjunction with a ceramic head to achieve better stress distribution across the taper surface. In vitro testing suggests that corrosion is not a concern in sleeved ceramic heads; however, little is known about the in vivo fretting corrosion of the sleeves. The purpose of this study was to investigate fretting corrosion in sleeved ceramic heads in retrieved total hip arthroplasties. Thirty-seven sleeved ceramic heads were collected during revision. The femoral heads and sleeves were implanted 0.0-3.3 years. The implants were revised predominantly for instability, infection, and loosening. Fifty percent of the retrievals were implanted during a primary surgery. Fretting corrosion was assessed using the Goldberg-Higgs semiquantitative scoring system. Mild-to-moderate fretting corrosion scores (score = 2-3) were observed in 92% of internal tapers, 19% of external tapers, and 78% of the stems. Severe fretting corrosion was observed in 1 stem trunnion that was previously retained during revision surgery and none of the retrieved sleeves. There was no difference in corrosion damage of sleeves used in primary or revision surgery. The fretting corrosion scores in this study were predominantly mild and lower than reported fretting scores of cobalt-chrome heads in metal-on-polyethylene bearings. Although intended for use in revisions, we found that the short-term in vivo corrosion behavior of the sleeves was similar in both primary and revision surgery applications. From an in vivo corrosion perspective, sleeves are a reasonable solution for restoring the stem taper during revision surgery. Copyright © 2017. Published by Elsevier Inc.

  3. Quantitative Analysis of Tau-Microtubule Interaction Using FRET

    Directory of Open Access Journals (Sweden)

    Isabelle L. Di Maïo

    2014-08-01

    Full Text Available The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αβ-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and β-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau.

  4. APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

    Directory of Open Access Journals (Sweden)

    Heidi J Chial

    2010-08-01

    Full Text Available Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes. Furthermore, the results of our experiments

  5. Comparative advantages of mechanical biosensors.

    Science.gov (United States)

    Arlett, J L; Myers, E B; Roukes, M L

    2011-04-01

    Mechanical interactions are fundamental to biology. Mechanical forces of chemical origin determine motility and adhesion on the cellular scale, and govern transport and affinity on the molecular scale. Biological sensing in the mechanical domain provides unique opportunities to measure forces, displacements and mass changes from cellular and subcellular processes. Nanomechanical systems are particularly well matched in size with molecular interactions, and provide a basis for biological probes with single-molecule sensitivity. Here we review micro- and nanoscale biosensors, with a particular focus on fast mechanical biosensing in fluid by mass- and force-based methods, and the challenges presented by non-specific interactions. We explain the general issues that will be critical to the success of any type of next-generation mechanical biosensor, such as the need to improve intrinsic device performance, fabrication reproducibility and system integration. We also discuss the need for a greater understanding of analyte-sensor interactions on the nanoscale and of stochastic processes in the sensing environment.

  6. Biosensor approach to psychopathology classification.

    Directory of Open Access Journals (Sweden)

    Misha Koshelev

    2010-10-01

    Full Text Available We used a multi-round, two-party exchange game in which a healthy subject played a subject diagnosed with a DSM-IV (Diagnostic and Statistics Manual-IV disorder, and applied a Bayesian clustering approach to the behavior exhibited by the healthy subject. The goal was to characterize quantitatively the style of play elicited in the healthy subject (the proposer by their DSM-diagnosed partner (the responder. The approach exploits the dynamics of the behavior elicited in the healthy proposer as a biosensor for cognitive features that characterize the psychopathology group at the other side of the interaction. Using a large cohort of subjects (n = 574, we found statistically significant clustering of proposers' behavior overlapping with a range of DSM-IV disorders including autism spectrum disorder, borderline personality disorder, attention deficit hyperactivity disorder, and major depressive disorder. To further validate these results, we developed a computer agent to replace the human subject in the proposer role (the biosensor and show that it can also detect these same four DSM-defined disorders. These results suggest that the highly developed social sensitivities that humans bring to a two-party social exchange can be exploited and automated to detect important psychopathologies, using an interpersonal behavioral probe not directly related to the defining diagnostic criteria.

  7. Simulation of Biosensor using FEM

    International Nuclear Information System (INIS)

    Sheeparamatti, B G; Hebbal, M S; Sheeparamatti, R B; Math, V B; Kadadevaramath, J S

    2006-01-01

    Bio-Micro Electro Mechanical Systems/Nano Electro Mechanical Systems include a wide variety of sensors, actuators, and complex micro/nano devices for biomedical applications. Recent advances in biosensors have shown that sensors based on bending of microfabricated cantilevers have potential advantages over earlier used detection methods. Thus, a simple cantilever beam can be used as a sensor for biomedical, chemical and environmental applications. Here, microfabricated multilayered cantilever beam is exposed to sensing environment. Lower layer being pure structural silicon or polymer and upper layer is of polymer with antigen/antibody immobilized in it. Obviously, it has an affinity towards its counterpart i.e. antibody/antigen. In the sensing environment, if counter elements exists, they get captured by this sensing beam head, and the cantilever beam deflects. This deflection can be sensed and the presence of counter elements in the environment can be predicted. In this work, a finite element model of a biosensor for sensing antibody/antigen reaction is developed and simulated using ANSYS/Multiphysics. The optimal dimensions of the microcantilever beam are selected based on permissible deflection range with the aid of MATLAB. In the model analysis, both weight and surface stress effects on the cantilever are considered. Approximate weights are taken into account because of counter elements, considering their molecular weight and possible number of elements required for sensing. The results obtained in terms of lateral deflection are presented

  8. Electronic transport in methylated fragments of DNA

    International Nuclear Information System (INIS)

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

    2015-01-01

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics

  9. Electronic transport in methylated fragments of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L., E-mail: umbertofulco@gmail.com; Albuquerque, E. L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Freire, V. N. [Departamento de Física, Universidade Federal do Ceará, 60455-760 Fortaleza, CE (Brazil); Caetano, E. W. S. [Instituto Federal de Educação, Ciência e Tecnologia do Ceará, 60040-531 Fortaleza, CE (Brazil); Moura, F. A. B. F. de; Lyra, M. L. [Instituto de Física, Universidade Federal de Alagoas, 57072-900 Maceió-AL (Brazil)

    2015-11-16

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  10. Prospects of conducting polymers in biosensors

    International Nuclear Information System (INIS)

    Malhotra, Bansi D.; Chaubey, Asha; Singh, S.P.

    2006-01-01

    Applications of conducting polymers to biosensors have recently aroused much interest. This is because these molecular electronic materials offer control of different parameters such as polymer layer thickness, electrical properties and bio-reagent loading, etc. Moreover, conducting polymer based biosensors are likely to cater to the pressing requirements such as biocompatibility, possibility of in vivo sensing, continuous monitoring of drugs or metabolites, multi-parametric assays, miniaturization and high information density. This paper deals with the emerging trends in conducting polymer based biosensors during the last about 5 years

  11. Design Strategies for Aptamer-Based Biosensors

    Science.gov (United States)

    Han, Kun; Liang, Zhiqiang; Zhou, Nandi

    2010-01-01

    Aptamers have been widely used as recognition elements for biosensor construction, especially in the detection of proteins or small molecule targets, and regarded as promising alternatives for antibodies in bioassay areas. In this review, we present an overview of reported design strategies for the fabrication of biosensors and classify them into four basic modes: target-induced structure switching mode, sandwich or sandwich-like mode, target-induced dissociation/displacement mode and competitive replacement mode. In view of the unprecedented advantages brought about by aptamers and smart design strategies, aptamer-based biosensors are expected to be one of the most promising devices in bioassay related applications. PMID:22399891

  12. Improved biosensor-based detection system

    DEFF Research Database (Denmark)

    2015-01-01

    Described is a new biosensor-based detection system for effector compounds, useful for in vivo applications in e.g. screening and selecting of cells which produce a small molecule effector compound or which take up a small molecule effector compound from its environment. The detection system...... comprises a protein or RNA-based biosensor for the effector compound which indirectly regulates the expression of a reporter gene via two hybrid proteins, providing for fewer false signals or less 'noise', tuning of sensitivity or other advantages over conventional systems where the biosensor directly...

  13. Molecularly imprinted fluorescent probe based on FRET for selective and sensitive detection of doxorubicin

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zhifeng, E-mail: 897061147@qq.com [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China); Deng, Peihong; Li, Junhua [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China); Xu, Li [Department of Applied Chemistry, College of Materials and Energy, South China Agricultural University, Guangzhou 510642 (China); Tang, Siping [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China)

    2017-04-15

    Highlights: • FRET-based molecularly imprinted probe for detection of doxorubicin was prepared. • The detection limit of the probe was 13.8 nM for doxorubicin. • The FRET-based probe had a higher selectivity for the template than ordinary MIMs. - Abstract: In this work, a new type of fluorescent probe for detection of doxorubicin has been constructed by the combined use of fluorescence resonance energy transfer (FRET) technology and molecular imprinting technique (MIT). Using doxorubicin as the template, the molecularly imprinted polymer thin layer was fabricated on the surfaces of carbon dot (CD) modified silica by sol-gel polymerization. The excitation energy of the fluorescent donor (CDs) could be transferred to the fluorescent acceptor (doxorubicin). The FRET based fluorescent probe demonstrated high sensitivity and selectivity for doxorubicin. The detection limit was 13.8 nM. The fluorescent probe was successfully applied for detecting doxorubicin in doxorubicin-spiked plasmas with a recovery of 96.8–103.8%, a relative standard deviation (RSD) of 1.3–2.8%. The strategy for construction of FRET-based molecularly imprinted materials developed in this work is very promising for analytical applications.

  14. A Study on Surface Modification of Al7075-T6 Alloy against Fretting Fatigue Phenomenon

    Directory of Open Access Journals (Sweden)

    E. Mohseni

    2014-01-01

    Full Text Available Aircraft engines, fuselage, automobile parts, and energy saving strategies in general have promoted the interest and research in the field of lightweight materials, typically on alloys based on aluminum. Aluminum alloy itself does not have suitable wear resistance; therefore, it is necessary to enhance surface properties for practical applications, particularly when aluminum is in contact with other parts. Fretting fatigue phenomenon occurs when two surfaces are in contact with each other and one or both parts are subjected to cyclic load. Fretting drastically decreases the fatigue life of materials. Therefore, investigating the fretting fatigue life of materials is an important subject. Applying surface modification methods is anticipated to be a supreme solution to gradually decreasing fretting damage. In this paper, the authors would like to review methods employed so far to diminish the effect of fretting on the fatigue life of Al7075-T6 alloy. The methods include deep rolling, shot peening, laser shock peening, and thin film hard coatings. The surface coatings techniques are comprising physical vapor deposition (PVD, hard anodizing, ion-beam-enhanced deposition (IBED, and nitriding.

  15. Experimental facility design for study of fretting in steam generator tubes

    International Nuclear Information System (INIS)

    Balbiani, J.P.; Bergant, M.; Yawny, A.

    2012-01-01

    The design of an experimental facility for fretting wear testing of steam generator tubes under pressurized water up to 340 o C, is presented. The main component of the device consists in an autoclave which permits to recreate steam generator operating conditions. CAD CATIA V5R18, CAE ABAQUS and ASME Sec. VII Div. 1 (Rules for Construction of Pressure Vessels) were used along the design process. The design of the autoclave included the pressure vessel itself and the necessary flanges and nozzles. In addition, an axial dynamic sealing system was designed to allow for actuation from outside the pressure boundary. Complementary, typical tube - support contact conditions were analyzed and the principal variables affecting their mutual interaction determined. In addition, a simple device which allows performing fretting wear testing on steam generator tubes in air at room temperature was fabricated and the feasibility of a quantitative assessment of different aspects related with the fretting induced damage was explored. Characterization techniques available at Centro Atomico Bariloche, like light microscopy, scanning electron microscopy (SEM), energy dispersive analysis of X-ray (EDAX) and surface damage analysis by optic profilometry were shown to be appropriate for this aim. The designed facility will allow evaluating fretting damage of tubes - support combinations that might be used on the steam generator of the prototype reactor CAREM-25. It is also expected it could be applied to characterize fretting severity in other applications (nuclear fuel elements) (author)

  16. In Situ Probing Intracellular Drug Release from Redox-Responsive Micelles by United FRET and AIE.

    Science.gov (United States)

    Wang, Xuelin; Li, Juanjuan; Yan, Qi; Chen, Yanrui; Fan, Aiping; Wang, Zheng; Zhao, Yanjun

    2018-03-01

    Redox-responsive micelles are versatile nanoplatforms for on-demand drug delivery, but the in situ evaluation of drug release is challenging. Fluorescence resonance energy transfer (FRET) technique shows potential for addressing this, while the aggregation-caused quenching effect limits the assay sensitivity. The aim of the current work is to combine aggregation-induced emission (AIE) probe with FRET to realize drug release assessment from micelles. Tetraphenylethene (TPE) is selected as AIE dye and curcumin (Cur) is chosen as the model drug as well as FRET receptor. The drug is covalently linked to a block copolymer via the disulfide bond linker and TPE is also chemically linked to the polymer via an amide bond; the obtained amphiphilic polymer conjugate self-assembles into micelles with a hydrodynamic size of ≈125 nm. Upon the supplement of glutathione or tris(2-carboxyethyl)phosphine) trigger (10 × 10 -3 m), the drug release induces the fluorescence increase of both TPE and Cur. Accompanied with the FRET decay, absorption enhancement and particle size increase are observed. The same phenomenon is observed in MCF-7 cells. The FRET-AIE approach can be a useful addition to the spectrum of available methods for monitoring drug release from stimuli-responsive nanomedicine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. An Epidermal Biosensor for Carcinoembryonic Antigen

    National Research Council Canada - National Science Library

    Schwartz, Pauline

    2001-01-01

    ...). An epidermal biosensor is a new approach for the early continuous, in vivo detection of the onset of disease by the using genetically modified skin cells to respond to molecules secreted by tumor cells...

  18. An Epidermal Biosensor for Carcinoembryonic Antigen

    National Research Council Canada - National Science Library

    Schwartz, Pauline

    2003-01-01

    ...) An epidermal biosensor was conceived as a new approach for the early continuous, in vivo detection of the onset of disease by the using genetically modified skin cells to respond to molecules secreted by tumor cells...

  19. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    Science.gov (United States)

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  20. Biosensors in immunology: the story so far

    NARCIS (Netherlands)

    Pathak, S.S.; Savelkoul, H.F.J.

    1997-01-01

    Optical biosensors are finding a range of applications in immunology. They enable biomolecular interactions to be characterized in real time without the need to label reactants, and, because individual binding steps can be visualized, are particularly suited to complex assays

  1. Biosensors a promising future in measurements

    International Nuclear Information System (INIS)

    Saleem, Muhammad

    2013-01-01

    A biosensor is an analytical device which can be used to convert the existence of a molecule or compound into a measurable and useful signal. Biosensors use stimulus to translate changes to recognisable signals and have great importance to society. Applications include diagnosis tools for diseases, security appliances, and other biomedical equipments. Biosensors can also be used in the detection of pathogens and other microbes in foodstuffs, drugs and processing industries. Enormous progress and advancement has been witnessed in this area. Research and development in micro level systems serves to interface biology with novel materials such as nanomaterial. Development of high speed and accurate electronic devices tfor use in medicine and energy storage (such as biofuel cells) is one of the target areas. This paper discusses the importance, use and current and future trend in the application of biosensors

  2. Fretting fatigue behaviour of Ni-free high-nitrogen stainless steel in a simulated body fluid

    Directory of Open Access Journals (Sweden)

    Norio Maruyama, Sachiko Hiromoto, Eiji Akiyama and Morihiko Nakamura

    2013-01-01

    Full Text Available Fretting fatigue behaviour of Ni-free high-nitrogen steel (HNS with a yield strength of about 800 MPa, which was prepared by nitrogen gas pressurized electroslag remelting, was studied in air and in phosphate-buffered saline (PBS(-. For comparison, fretting fatigue behaviour of cold-rolled SUS316L steel (SUS316L(CR with similar yield strength was examined. The plain fatigue limit of HNS was slightly lower than that of SUS316L(CR although the former had a higher tensile strength than the latter. The fretting fatigue limit of HNS was higher than that of SUS316L(CR both in air and in PBS(-. A decrease in fatigue limit of HNS by fretting was significantly smaller than that of SUS316L(CR in both environments, indicating that HNS has better fretting fatigue resistance than SUS316L(CR. The decrease in fatigue limit by fretting is discussed taking into account the effect of friction stress due to fretting and the additional influences of wear, tribocorrosion and plastic deformation in the fretted area.

  3. Fluorophores, environments, and quantification techniques in the analysis of transmembrane helix interaction using FRET.

    Science.gov (United States)

    Khadria, Ambalika S; Senes, Alessandro

    2015-07-01

    Förster resonance energy transfer (FRET) has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane (TM) helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interactions in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments, and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring TM helix-helix associations. © 2015 Wiley Periodicals, Inc.

  4. Calculated and experimental research of WWER-1000 assembly vibration and fretting damage

    International Nuclear Information System (INIS)

    Drozdov, Y.; Afanasyev, A.; Makarov, V.; Tutnov, A.; Tutnov, A.; Alekseev, E.

    2008-01-01

    The report covers the methods and results of the latest analytical and experimental studies of fretting corrosion and natural vibrations of a WWER-1000 reactor fuel assemblies (FA). The process of fretting-corrosion was investigated using a multi-specimen facility that simulated fragments of fuel rod-to-spacer grid and lower support grid mating units. A computational model was developed for vibrations in the mechanical system of a fuel rod fragment and a spacer grid fragment. A calculational and experimental modal analysis of a FA was performed. Natural frequencies, modes and decrements of FA vibrations were determined and a satisfactory coincidence of analytical and experimental results was obtained. The assessment of fretting-corrosion process dynamics was made and its dependences on operational factors were obtained. (authors)

  5. Fretting Wear Behaviors of Aluminum Cable Steel Reinforced (ACSR Conductors in High-Voltage Transmission Line

    Directory of Open Access Journals (Sweden)

    Xingchi Ma

    2017-09-01

    Full Text Available This work reports the fretting wear behavior of aluminum cable steel reinforced (ACSR conductors for use in high-voltage transmission line. Fretting wear tests of Al wires were conducted on a servo-controlled fatigue testing machine with self-made assistant apparatus, and their fretting process characteristics, friction force, wear damage, and wear surface morphology were detailed analyzed. The results show that the running regime of Al wires changes from a gross slip regime to a mixed regime more quickly as increasing contact load. With increasing amplitudes, gross slip regimes are more dominant under contact loads of lower than 30 N. The maximum friction force is relatively smaller in the NaCl solution than in a dry friction environment. The primary wear mechanisms in dry friction environments are abrasive wear and adhesive wear whereas abrasive wear and fatigue damage are dominant in NaCl solution.

  6. Biosensors for cardiac biomarkers detection: a review

    OpenAIRE

    Qureshi, Anjum; Gürbüz, Yaşar; Gurbuz, Yasar; Kolkar Mohammed, Javed Hussain Niazi

    2012-01-01

    The cardiovascular disease (CVD) is considered as a major threat to global health. Therefore, there is a growing demand for a range of portable, rapid and low cost biosensing devices for the detection of CVD. Biosensors can play an important role in the early diagnosis of CVD without having to rely on hospital visits where expensive and time-consuming laboratory tests are recommended. Over the last decade, many biosensors have been developed to detect a wide range of cardiac marker to reduce ...

  7. Fuel-element vibration and bearing pad to pressure tube fretting

    International Nuclear Information System (INIS)

    Fisher, N.J.; Taylor, C.E.; Pettigrew, M.J.

    1990-08-01

    Fuel channel operation under boiling condition results in increased flow velocities, which may lead to unacceptable fuel-element vibration and bearing pad to pressure tube fretting. The existing endurance test database does not fully cover the range of future channel operating conditions. In particular, after refuelling, some channels for future designs may operate with two-phase flow conditions outside the range of endurance test conditions. Full-scale endurance testing at realistic steam-water conditions involves substantial energy costs. Therefore, fundamental laboratory investigations were conducted to define and endurance test matrix which adequately envelops the future range of operating conditions while minimizing both the number of tests and the energy requirement of individual tests. The main focus of the laboratory investigations was to establish the relationships between: fuel channel flow conditions and fuel-element vibration; and fuel-element vibration and bearing pad to pressure tube fretting. The vibration response of a single fuel element was measured over a wide range of operating conditions covering realistic fuel channel conditions and simulated endurance testing conditions. For higher void fractions, the vibration amplitudes measured in air/water were much higher than in steam/water, while for low void fractions, the amplitudes were similar. The measured amplitudes in steam/water varied very little over the range of temperature and pressure investigated. The effects of temperature, pressure tube oxide thickness, vibration amplitude and bearing pad manufacturer on pressure tube fretting were investigated. The fretting rate is extremely temperature dependent. For vibration amplitudes about three or four times greater than expected in-reactor conditions, peak fretting rates were observed in the 225 to 286 degrees C temperature range. Fretting rates were seven times less at the higher temperatures of 300 and 315 degrees C, and the lower temperatures

  8. Fluorescence resonance energy transfer (FRET-based subcellular visualization of pathogen-induced host receptor signaling

    Directory of Open Access Journals (Sweden)

    Zimmermann Timo

    2009-11-01

    Full Text Available Abstract Background Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET to visualize pathogen-initiated signaling events in human cells. Results Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3. In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2 domain of Hck (Hck-SH2 was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet-Hck-SH2 and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. Conclusion These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

  9. Yeast-based biosensors: design and applications.

    Science.gov (United States)

    Adeniran, Adebola; Sherer, Michael; Tyo, Keith E J

    2015-02-01

    Yeast-based biosensing (YBB) is an exciting research area, as many studies have demonstrated the use of yeasts to accurately detect specific molecules. Biosensors incorporating various yeasts have been reported to detect an incredibly large range of molecules including but not limited to odorants, metals, intracellular metabolites, carcinogens, lactate, alcohols, and sugars. We review the detection strategies available for different types of analytes, as well as the wide range of output methods that have been incorporated with yeast biosensors. We group biosensors into two categories: those that are dependent upon transcription of a gene to report the detection of a desired molecule and those that are independent of this reporting mechanism. Transcription-dependent biosensors frequently depend on heterologous expression of sensing elements from non-yeast organisms, a strategy that has greatly expanded the range of molecules available for detection by YBBs. Transcription-independent biosensors circumvent the problem of sensing difficult-to-detect analytes by instead relying on yeast metabolism to generate easily detected molecules when the analyte is present. The use of yeast as the sensing element in biosensors has proven to be successful and continues to hold great promise for a variety of applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  10. Biosensors-on-chip: a topical review

    International Nuclear Information System (INIS)

    Chen, Sensen; Shamsi, Mohtashim H

    2017-01-01

    This review will examine the integration of two fields that are currently at the forefront of science, i.e. biosensors and microfluidics. As a lab-on-a-chip (LOC) technology, microfluidics has been enriched by the integration of various detection tools for analyte detection and quantitation. The application of such microfluidic platforms is greatly increased in the area of biosensors geared towards point-of-care diagnostics. Together, the merger of microfluidics and biosensors has generated miniaturized devices for sample processing and sensitive detection with quantitation. We believe that microfluidic biosensors (biosensors-on-chip) are essential for developing robust and cost effective point-of-care diagnostics. This review is relevant to a variety of disciplines, such as medical science, clinical diagnostics, LOC technologies including MEMs/NEMs, and analytical science. Specifically, this review will appeal to scientists working in the two overlapping fields of biosensors and microfluidics, and will also help new scientists to find their directions in developing point-of-care devices. (topical review)

  11. Nanostructured Tip-Shaped Biosensors: Application of Six Sigma Approach for Enhanced Manufacturing.

    Science.gov (United States)

    Kahng, Seong-Joong; Kim, Jong-Hoon; Chung, Jae-Hyun

    2016-12-23

    Nanostructured tip-shaped biosensors have drawn attention for biomolecule detection as they are promising for highly sensitive and specific detection of a target analyte. Using a nanostructured tip, the sensitivity is increased to identify individual molecules because of the high aspect ratio structure. Various detection methods, such as electrochemistry, fluorescence microcopy, and Raman spectroscopy, have been attempted to enhance the sensitivity and the specificity. Due to the confined path of electrons, electrochemical measurement using a nanotip enables the detection of single molecules. When an electric field is combined with capillary action and fluid flow, target molecules can be effectively concentrated onto a nanotip surface for detection. To enhance the concentration efficacy, a dendritic nanotip rather than a single tip could be used to detect target analytes, such as nanoparticles, cells, and DNA. However, reproducible fabrication with relation to specific detection remains a challenge due to the instability of a manufacturing method, resulting in inconsistent shape. In this paper, nanostructured biosensors are reviewed with our experimental results using dendritic nanotips for sequence specific detection of DNA. By the aid of the Six Sigma approach, the fabrication yield of dendritic nanotips increases from 20.0% to 86.6%. Using the nanotips, DNA is concentrated and detected in a sequence specific way with the detection limit equivalent to 1000 CFU/mL. The pros and cons of a nanotip biosensor are evaluated in conjunction with future prospects.

  12. A signal-on electrogenerated chemiluminescent biosensor for lead ion based on DNAzyme

    International Nuclear Information System (INIS)

    Ma Fen; Sun Bo; Qi Honglan; Zhang Hongge; Gao Qiang; Zhang Chengxiao

    2011-01-01

    A highly reproducible and sensitive signal-on electrogenerated chemiluminescence (ECL) biosensor based on the DNAzyme for the determination of lead ion was developed. The ECL biosensor was fabricated by covalently coupling 5'-amino-DNAzyme-tagged with ruthenium bis (2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid)-ethylenediamine (Ru1-17E') onto the surface of graphite electrode modified with 4-aminobenzoic acid, and then a DNA substrate with a ribonucleotide adenosine hybridized with Ru1-17E' on the electrode. Upon binding of Pb 2+ to the Ru1-17E' to form a complex which catalyzed the cleavage of the DNA substrate, the double-stranded DNA was dissociated and thus led to a high ECL signal. The signal linearly increases with the concentration of Pb 2+ in the range from 5.0 to 80 pM with a detection limit of 1.4 pM and a relative standard derivation of 2.3%. This work demonstrates that using DNAzyme tagged with ruthenium complex as an ECL probe and covalently coupling method for the fabrication of the ECL biosensor with high sensitivity, good stability and significant regeneration ability is promising approach.

  13. Precision and accuracy in smFRET based structural studies—A benchmark study of the Fast-Nano-Positioning System

    Science.gov (United States)

    Nagy, Julia; Eilert, Tobias; Michaelis, Jens

    2018-03-01

    Modern hybrid structural analysis methods have opened new possibilities to analyze and resolve flexible protein complexes where conventional crystallographic methods have reached their limits. Here, the Fast-Nano-Positioning System (Fast-NPS), a Bayesian parameter estimation-based analysis method and software, is an interesting method since it allows for the localization of unknown fluorescent dye molecules attached to macromolecular complexes based on single-molecule Förster resonance energy transfer (smFRET) measurements. However, the precision, accuracy, and reliability of structural models derived from results based on such complex calculation schemes are oftentimes difficult to evaluate. Therefore, we present two proof-of-principle benchmark studies where we use smFRET data to localize supposedly unknown positions on a DNA as well as on a protein-nucleic acid complex. Since we use complexes where structural information is available, we can compare Fast-NPS localization to the existing structural data. In particular, we compare different dye models and discuss how both accuracy and precision can be optimized.

  14. Angle-resolved diffraction grating biosensor based on porous silicon

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Changwu; Li, Peng [School of Physical Science and Technology, Xinjiang University, Urumqi 830046 (China); Jia, Zhenhong, E-mail: jzhh@xju.edu.cn; Liu, Yajun; Mo, Jiaqing; Lv, Xiaoyi [College of Information Science and Engineering, Xinjiang University, Urumqi 830046 (China)

    2016-03-07

    In this study, an optical biosensor based on a porous silicon composite structure was fabricated using a simple method. This structure consists of a thin, porous silicon surface diffraction grating and a one-dimensional porous silicon photonic crystal. An angle-resolved diffraction efficiency spectrum was obtained by measuring the diffraction efficiency at a range of incident angles. The angle-resolved diffraction efficiency of the 2nd and 3rd orders was studied experimentally and theoretically. The device was sensitive to the change of refractive index in the presence of a biomolecule indicated by the shift of the diffraction efficiency spectrum. The sensitivity of this sensor was investigated through use of an 8 base pair antifreeze protein DNA hybridization. The shifts of the angle-resolved diffraction efficiency spectrum showed a relationship with the change of the refractive index, and the detection limit of the biosensor reached 41.7 nM. This optical device is highly sensitive, inexpensive, and simple to fabricate. Using shifts in diffraction efficiency spectrum to detect biological molecules has not yet been explored, so this study establishes a foundation for future work.

  15. Biosensor

    DEFF Research Database (Denmark)

    2002-01-01

    The invention relates to a biochemical assay for wide class of hydrophobic Coenzyme A esters wherein the analyte is caused to react with a specifically binding, modified protein, and thereby causing a detectable signal. A one step assay for hydrophobic carboxylic acid esters in whole blood, serum...

  16. Biosensors

    Indian Academy of Sciences (India)

    and an electronic component to transduce and detect the signal. A variety of .... aliphatic aldehyde as fol- lows: FMNH2 + .... microorganisms by the use of high temperature. ... ISFET. The oxidation of hypoxanthine to uric acid by xanthine.

  17. Uzawa algorithm to solve elastic and elastic-plastic fretting wear problems within the bipotential framework

    Science.gov (United States)

    Ning, Po; Feng, Zhi-Qiang; Quintero, Juan Antonio Rojas; Zhou, Yang-Jing; Peng, Lei

    2018-03-01

    This paper deals with elastic and elastic-plastic fretting problems. The wear gap is taken into account along with the initial contact distance to obtain the Signorini conditions. Both the Signorini conditions and the Coulomb friction laws are written in a compact form. Within the bipotential framework, an augmented Lagrangian method is applied to calculate the contact forces. The Archard wear law is then used to calculate the wear gap at the contact surface. The local fretting problems are solved via the Uzawa algorithm. Numerical examples are performed to show the efficiency and accuracy of the proposed approach. The influence of plasticity has been discussed.

  18. A dansyl-rhodamine ratiometric fluorescent probe for Hg2+ based on FRET mechanism.

    Science.gov (United States)

    Xie, Puhui; Guo, Fengqi; Wang, Lingyu; Yang, Sen; Yao, Denghui; Yang, Guoyu

    2015-03-01

    Based on resonance energy transfer (FRET) from dansyl to rhodamine 101, a new fluorescent probe (compound 1) containing rhodamine 101 and a dansyl unit was synthesized for detecting Hg(2+) through ratiometric sensing in DMSO aqueous solutions. This probe shows a fast, reversible and selective response toward Hg(2+) in a wide pH range. Hg(2+) induced ring-opening reactions of the spirolactam rhodamine moiety of 1, leading to the formation of fluorescent derivatives that can serve as the FRET acceptors. Very large stokes shift (220 nm) was observed in this case. About 97-fold increase in fluorescence intensity ratio was observed upon its binding with Hg(2+).

  19. Fretting fatigue behavior of high-strength steel monostrands under bending load

    DEFF Research Database (Denmark)

    Winkler, Jan; Georgakis, Christos T.; Fischer, Gregor

    2015-01-01

    In this paper, the fretting fatigue behavior of pretensioned high-strength steel monostrands is investigated. To measure the local deformations on the strands, a novel method based on the digital image correlation (DIC) technique was used to quantify the relative movement between individual wires...... along the length of the monostrand. Information about the monostrand bending stiffness and the extent of relative displacement between core and outer wires of a monostrand undergoing flexural deformations is provided. From the series of dynamic fatigue tests, a fretting fatigue spectrum is derived...

  20. Fretting wear damage of steam generator tubes and its prediction modeling

    International Nuclear Information System (INIS)

    Che Honglong; Lei Mingkai

    2013-01-01

    The steam generator is the key equipment used for the energy transition in nuclear power plant. Since the high-temperature and high-pressure fluid flows with high speed, the steam generator tubes will be excited and vibrate, leading to the tremendous fretting wear problem on the tubes, sometimes even leading to tube cracking. This paper introduces typical fretting wear cases, the result of corresponding simulation wear experiment and damage mechanism which combining mechanical wear and erosion-corrosion. Work rate model could give a reasonable life prediction about the steam generator tube, and this predictive model has been used in nuclear power plant safety assessment. (authors)

  1. Theoretical-experimental analysis of the fretting/impact wear in fuel rods

    International Nuclear Information System (INIS)

    Pecos, Luis F.

    2001-01-01

    Nuclear power plant fuel elements are subjected to flow induced vibrations. A consequence of these vibrations is impact/fretting wear in fuel rods or sliding shoes. Because of the difficulties to assert the mechanism of impact/fretting wear phenomenon it is necessary to use semiempirical formulations in order to predict the wear rate of the components. The results of a series of experiments with Zr-4 specimens are presented in this work. A parameter called 'work-rate' was used to normalize the wear rates and interpret the results in terms of wear coefficient. (author) [es

  2. The Necessity of a New Type Test Rig for the Development of an Evaluation Method in Grid Fretting Problems

    International Nuclear Information System (INIS)

    Lee, Young-Ho; Kim, Hyung-Kyu

    2007-01-01

    A grid fretting problem is recognized as one of the most important degradation mechanisms even though the examination results of fretting experiments could be applied to the development and design of spacer grid structures. This is because it is difficult to develop a fretting wear model for a grid fretting problem due to the various wear mechanisms involved according to the mechanical and environmental variables, the contact condition with a spring/dimple and the material properties. A number of spring shapes has been developed in KAERI and their performance tests such as fretting wear, flow-induced vibration (FIV) tests, etc. have been carried out from a part unit to a full assembly scale. From the unit part fretting test results, one of the noticeable results is that the contacting force (normal load) was gradually decreased with increasing number of fretting cycles due to a depth increase and this behavior was closely related to the contacting spring shape. When considering the actual contact condition between a fuel rod and a spring/dimple, if a fretting wear progresses due to a FIV under a specific normal load exerted on the fuel rod by an elastic deformation of the spring, the contacting force between the fuel rod and dimple that are located in the opposite side should be decreased. Consequently, an evaluation of developed spacer grids against fretting wear damage should be performed with the results of 1x1 cell unit experiments because a contacting force is one of the most important variables that influences a fretting wear mechanism. The discussion was focused on the development procedure of a new test rig and its performance by using a 1x1 cell unit test rig. (authors)

  3. Recovery Based Nanowire Field-Effect Transistor Detection of Pathogenic Avian Influenza DNA

    Science.gov (United States)

    Lin, Chih-Heng; Chu, Chia-Jung; Teng, Kang-Ning; Su, Yi-Jr; Chen, Chii-Dong; Tsai, Li-Chu; Yang, Yuh-Shyong

    2012-02-01

    Fast and accurate diagnosis is critical in infectious disease surveillance and management. We proposed a DNA recovery system that can easily be adapted to DNA chip or DNA biosensor for fast identification and confirmation of target DNA. This method was based on the re-hybridization of DNA target with a recovery DNA to free the DNA probe. Functionalized silicon nanowire field-effect transistor (SiNW FET) was demonstrated to monitor such specific DNA-DNA interaction using high pathogenic strain virus hemagglutinin 1 (H1) DNA of avian influenza (AI) as target. Specific electric changes were observed in real-time for AI virus DNA sensing and device recovery when nanowire surface of SiNW FET was modified with complementary captured DNA probe. The recovery based SiNW FET biosensor can be further developed for fast identification and further confirmation of a variety of influenza virus strains and other infectious diseases.

  4. Development of an acoustic wave based biosensor for vapor phase detection of small molecules

    Science.gov (United States)

    Stubbs, Desmond

    For centuries scientific ingenuity and innovation have been influenced by Mother Nature's perfect design. One of her more elusive designs is that of the sensory olfactory system, an array of highly sensitive receptors responsible for chemical vapor recognition. In the animal kingdom this ability is magnified among canines where ppt (parts per trillion) sensitivity values have been reported. Today, detection dogs are considered an essential part of the US drug and explosives detection schemes. However, growing concerns about their susceptibility to extraneous odors have inspired the development of highly sensitive analytical detection tools or biosensors known as "electronic noses". In general, biosensors are distinguished from chemical sensors in that they use an entity of biological origin (e.g. antibody, cell, enzyme) immobilized onto a surface as the chemically-sensitive film on the device. The colloquial view is that the term "biosensors" refers to devices which detect the presence of entities of biological origin, such as proteins or single-stranded DNA and that this detection must take place in a liquid. Our biosensor utilizes biomolecules, specifically IgG monoclonal antibodies, to achieve molecular recognition of relatively small molecules in the vapor phase.

  5. Graphene, carbon nanotubes, zinc oxide and gold as elite nanomaterials for fabrication of biosensors for healthcare.

    Science.gov (United States)

    Kumar, Sandeep; Ahlawat, Wandit; Kumar, Rajesh; Dilbaghi, Neeraj

    2015-08-15

    Technological advancements worldwide at rapid pace in the area of materials science and nanotechnology have made it possible to synthesize nanoparticles with desirable properties not exhibited by the bulk material. Among variety of available nanomaterials, graphene, carbon nanotubes, zinc oxide and gold nanopartilces proved to be elite and offered amazing electrochemical biosensing. This encourages us to write a review which highlights the recent achievements in the construction of genosensor, immunosensor and enzymatic biosensor based on the above nanomaterials. Carbon based nanomaterials offers a direct electron transfer between the functionalized nanomaterials and active site of bioreceptor without involvement of any mediator which not only amplifies the signal but also provide label free sensing. Gold shows affinity towards immunological molecules and is most routinely used for immunological sensing. Zinc oxide can easily immobilize proteins and hence offers a large group of enzyme based biosensor. Modification of the working electrode by introduction of these nanomaterials or combination of two/three of above nanomaterials together and forming a nanocomposite reflected the best results with excellent stability, reproducibility and enhanced sensitivity. Highly attractive electrochemical properties and electrocatalytic activity of these elite nanomaterials have facilitated achievement of enhanced signal amplification needed for the construction of ultrasensitive electrochemical affinity biosensors for detection of glucose, cholesterol, Escherichia coli, influenza virus, cancer, human papillomavirus, dopamine, glutamic acid, IgG, IgE, uric acid, ascorbic acid, acetlycholine, cortisol, cytosome, sequence specific DNA and amino acids. Recent researches for bedside biosensors are also discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Poly(3,4-ethylenedioxythiophene)-based glucose biosensors

    NARCIS (Netherlands)

    Kros, A.; Hövell, W.F.M. van; Sommerdijk, N.A.J.M.; Nolte, R.J.M.

    2001-01-01

    Amperometric biosensors for the recognition of glucose oxidase (GOx) based on poly(3,4-ethylenedioxythiophene) (PEDOT) were fabricated for the first time. The resulting biosensor has potential applications for long-term glucose measurements.

  7. Nanopore biosensors for detection of proteins and nucleic acids

    NARCIS (Netherlands)

    Maglia, Giovanni; Soskine, Mikhael

    2014-01-01

    Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromoiecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.

  8. Optimization of Xenon Biosensors for Detection of Protein Interactions

    International Nuclear Information System (INIS)

    Lowery, Thomas J.; Garcia, Sandra; Chavez, Lana; Ruiz, E.Janette; Wu, Tom; Brotin, Thierry; Dutasta, Jean-Pierre; King, David S.; Schultz, Peter G.; Pines, Alex; Wemmer, David E.

    2005-08-01

    Hyperpolarized 129Xe NMR can detect the presence of specific low-concentration biomolecular analytes by means of the xenon biosensor, which consists of a water-soluble, targeted cryptophane-A cage that encapsulates xenon. In this work we use the prototypical biotinylated xenon biosensor to determine the relationship between the molecular composition of the xenon biosensor and the characteristics of protein-bound resonances. The effects of diastereomer overlap, dipole-dipole coupling, chemical shift anisotropy, xenon exchange, and biosensor conformational exchange on protein-bound biosensor signal were assessed. It was found that optimal protein-bound biosensor signal can be obtained by minimizing the number of biosensor diastereomers and using a flexible linker of appropriate length. Both the linewidth and sensitivity of chemical shift to protein binding of the xenon biosensor were found to be inversely proportional to linker length

  9. Mobility and height detection of particle labels in an optical evanescent wave biosensor with single-label resolution

    Energy Technology Data Exchange (ETDEWEB)

    Van Ommering, Kim; Koets, Marjo; Schleipen, Jean J H B; Prins, Menno W J [Philips Research Laboratories, 5656 AE Eindhoven (Netherlands); Somers, Philip A; Van IJzendoorn, Leo J, E-mail: menno.prins@philips.co [Department of Applied Physics, Eindhoven University of Technology, 5600 MB Eindhoven (Netherlands)

    2010-04-21

    Particle labels are used in biosensors to detect the presence and concentration of analyte molecules. In this paper we demonstrate an optical technique to measure the mobility and height of bound particle labels on a biosensor surface with single-label resolution. The technique is based on the detection of the particle-induced light scattering in an optical evanescent field. We show that the thermal particle motion in the optical evanescent field leads to intensity fluctuations that can accurately be detected. The technique is demonstrated using 290 bp (99 nm) DNA as an analyte and using polystyrene particles and magnetic particles with diameters between 500 and 1000 nm as labels. The particle intensity histograms show that quantitative height measurements are obtained for particles with uniform optical properties, and the intensity versus position plots reflect the analyte-antibody orientation and the analyte flexibility. The novel optical detection technique will lead to biosensors with very high sensitivity and specificity.

  10. Development of aptamers for in vivo and in vitro biosensor applications

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm

    block chemicals are now being sustainably produced in bacterial cell-factories. The development of new bacterial cell-factories is a difficult and expensive process, in part due to time required to screen for and optimize productions strains. A new promising way of reducing the development time...... is generating new and faster ways of screening and optimizing using biosensors. In this thesis we develop new functional biological recognition modules for biosensors. These DNA- and RNA-based recognition modules are called aptamers and are developed to interact with targets of choice. Aptamers are developed...... application) and small molecule food additives (for optimization production in cell factories). Additionally, the characterization an all-polymer physicochemical biosensor is presented for the detection of antibiotics in food products. These results have lead to the ongoing development of a high...

  11. Graphene-based field-effect transistor biosensors

    Science.gov (United States)

    Chen; , Junhong; Mao, Shun; Lu, Ganhua

    2017-06-14

    The disclosure provides a field-effect transistor (FET)-based biosensor and uses thereof. In particular, to FET-based biosensors using thermally reduced graphene-based sheets as a conducting channel decorated with nanoparticle-biomolecule conjugates. The present disclosure also relates to FET-based biosensors using metal nitride/graphene hybrid sheets. The disclosure provides a method for detecting a target biomolecule in a sample using the FET-based biosensor described herein.

  12. Fretting wear simulation of press-fitted shaft with finite element analysis and influence function method

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dong Hyong; Kwon, Seok Jin [Korea Railroad Research Institute, Uiwang (Korea, Republic of); Choi, Jae Boong; Kim, Young Jin [Sungkyunkwan University, Suwon (Korea, Republic of)

    2008-01-15

    In this paper the fretting wear of press-fitted specimens subjected to a cyclic bending load was simulated using finite element analysis and numerical method. The amount of microslip and contact variable at press-fitted and bending load condition in a press-fitted shaft was analysed by applying finite element method. With the finite element analysis result, a numerical approach was applied to predict fretting wear based on modified Archard's equation and updating the change of contact pressure caused by local wear with influence function method. The predicted wear profiles of press-fitted specimens at the contact edge wear compared with the experimental results obtained by rotating bending fatigue tests. It is shown that the depth of fretting wear by repeated slip between shaft and boss reaches the maximum value at the contact edge. The initial surface profile is continuously changed by the wear at the contact edge, and then the corresponding contact variables are redistributed. The work establishes a basis for numerical simulation of fretting wear on press fits.

  13. Fretting wear simulation of press-fitted shaft with finite element analysis and influence function method

    International Nuclear Information System (INIS)

    Lee, Dong Hyong; Kwon, Seok Jin; Choi, Jae Boong; Kim, Young Jin

    2008-01-01

    In this paper the fretting wear of press-fitted specimens subjected to a cyclic bending load was simulated using finite element analysis and numerical method. The amount of microslip and contact variable at press-fitted and bending load condition in a press-fitted shaft was analysed by applying finite element method. With the finite element analysis result, a numerical approach was applied to predict fretting wear based on modified Archard's equation and updating the change of contact pressure caused by local wear with influence function method. The predicted wear profiles of press-fitted specimens at the contact edge wear compared with the experimental results obtained by rotating bending fatigue tests. It is shown that the depth of fretting wear by repeated slip between shaft and boss reaches the maximum value at the contact edge. The initial surface profile is continuously changed by the wear at the contact edge, and then the corresponding contact variables are redistributed. The work establishes a basis for numerical simulation of fretting wear on press fits

  14. Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots.

    Science.gov (United States)

    Annio, Giacomo; Jennings, Travis; Tagit, Oya; Hildebrandt, Niko

    2018-05-23

    Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance.

  15. Chemical synthesis of dual labeled proteins via differently protected alkynes enables intramolecular FRET analysis.

    Science.gov (United States)

    Hayashi, Gosuke; Kamo, Naoki; Okamoto, Akimitsu

    2017-05-30

    We report a novel method for multisite protein conjugation by setting differently silyl-protected alkynes as conjugation handles, which can remain intact through the whole synthetic procedure and provide sequential and orthogonal conjugation. This strategy enables efficient preparation of a dual dye-labeled protein and structural analysis via an intramolecular FRET mechanism.

  16. On the geometry of the fuel rod supports concerning a fretting wear failure

    International Nuclear Information System (INIS)

    Kim, Hyung-Kyu; Lee, Young-Ho; Lee, Kang-Hee

    2008-01-01

    Geometrical conditions of spacer grid springs and dimples of a light water reactor fuel assembly are studied in this paper concerning a fuel rod's fretting wear failure. In this framework, the springs/dimples are categorized from the aspects of their orientation with respect to the fuel axis and the contact types. Possible motions on the contacts between the springs/dimples and fuel rods are estimated by conducting a flow-induced vibration test. Features of the wear scar and depth are investigated by independent fretting wear tests carried out with spring and dimple specimens of typical contact geometries. It is also attempted here to apply the contact mechanics theory to a fuel fretting wear analysis such as the prediction of a wear depth profile and its rate, which is influenced by the contact shape of the springs/dimples. It is shown that the theory can be applied to a dimensional control of a coining for the springs/dimples, which is usually carried out in a thin plate fabrication. From the results, the necessary conditions for a spring/dimple geometry for restraining a fretting wear failure are discussed

  17. An ad-hoc fretting wear tribotester design for thin steel wires

    Directory of Open Access Journals (Sweden)

    Llavori Iñigo

    2018-01-01

    Full Text Available Steel wire ropes experience fretting wear damage when the rope runs over a sheave promoting an oscillatory motion between the wires. Consequently, wear scars appear between the contacting wires leading to an increase of the stress field and the following rupture of the wires due to fatigue. That is why the understanding and prediction of the fretting wear phenomena of thin wires is fundamental in order to improve the performance of steel wire ropes. The present research deals with the design of an ad-hoc fretting wear test machine for thin wires. The test apparatus is designed for testing thin wires with a maximum diameter of 1.0 mm, at slip amplitudes ranging from 5 to 300 μm, crossing angle between 0-90°, and contacting force ranging from 0,5 to 5 N. The working principle of displacement amplitude and contacting force as well as the crossing angle between the wires are described. Preliminary studies for understanding the fretting wear characteristics are presented, analysing 0.45 mm diameter cold-drawn eutectoid carbon steel (0.8% C wires (tensile strength higher than 3000 MPa.

  18. Homo-FRET imaging as a tool to quantify protein and lipid clustering.

    Science.gov (United States)

    Bader, Arjen N; Hoetzl, Sandra; Hofman, Erik G; Voortman, Jarno; van Bergen en Henegouwen, Paul M P; van Meer, Gerrit; Gerritsen, Hans C

    2011-02-25

    Homo-FRET, Förster resonance energy transfer between identical fluorophores, can be conveniently measured by observing its effect on the fluorescence anisotropy. This review aims to summarize the possibilities of fluorescence anisotropy imaging techniques to investigate clustering of identical proteins and lipids. Homo-FRET imaging has the ability to determine distances between fluorophores. In addition it can be employed to quantify cluster sizes as well as cluster size distributions. The interpretation of homo-FRET signals is complicated by the fact that both the mutual orientations of the fluorophores and the number of fluorophores per cluster affect the fluorescence anisotropy in a similar way. The properties of the fluorescence probes are very important. Taking these properties into account is critical for the correct interpretation of homo-FRET signals in protein- and lipid-clustering studies. This is be exemplified by studies on the clustering of the lipid raft markers GPI and K-ras, as well as for EGF receptor clustering in the plasma membrane. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Homo-FRET Imaging as a tool to quantify protein and lipid clustering

    NARCIS (Netherlands)

    Bader, A.N.; Hoetzl, S.; Hofman, E.G.; Voortman, J.; van Bergen en Henegouwen, P.M.P.; van Meer, G.; Gerritsen, H.C.

    2010-01-01

    Homo-FRET, Förster resonance energy transfer between identical fluorophores, can be conveniently measured by observing its effect on the fluorescence anisotropy. This review aims to summarize the possibilities of fluorescence anisotropy imaging techniques to investigate clustering of identical

  20. Critical Shell Thickness of Core/Shell Upconversion Luminescence Nanoplatform for FRET Application

    NARCIS (Netherlands)

    Wang, Yu; Liu, Kai; Liu, Xiaomin; Dohnalova, Katerina; Gregorkiewicz, Tom; Kong, Xianggui; Aalders, Maurice C. G.; Buma, Wybren J.; Zhang, Hong

    2011-01-01

    Almost all the luminescence upconversion nanoparticles used for Forster resonant energy transfer (FRET) applications are bare cores based on the consideration that the energy transfer efficiency is optimized because the distance between energy donors and acceptors is minimized. On the other hand, it

  1. Pen-on-paper strategy for point-of-care testing: Rapid prototyping of fully written microfluidic biosensor.

    Science.gov (United States)

    Li, Zedong; Li, Fei; Xing, Yue; Liu, Zhi; You, Minli; Li, Yingchun; Wen, Ting; Qu, Zhiguo; Ling Li, Xiao; Xu, Feng

    2017-12-15

    Paper-based microfluidic biosensors have recently attracted increasing attentions in point-of-care testing (POCT) territories benefiting from their affordable, accessible and eco-friendly features, where technologies for fabricating such biosensors are preferred to be equipment free, easy-to-operate and capable of rapid prototyping. In this work, we developed a pen-on-paper (PoP) strategy based on two custom-made pens, i.e., a wax pen and a conductive-ink pen, to fully write paper-based microfluidic biosensors through directly writing both microfluidic channels and electrodes. Particularly, the proposed wax pen is competent to realize one-step fabrication of wax channels on paper, as the melted wax penetrates into paper during writing process without any post-treatments. The practical applications of the fabricated paper-based microfluidic biosensors are demonstrated by both colorimetric detection of Salmonella typhimurium DNA with detection limit of 1nM and electrochemical measurement of glucose with detection limit of 1mM. The developed PoP strategy for making microfluidic biosensors on paper characterized by true simplicity, prominent portability and excellent capability for rapid prototyping shows promising prospect in POCT applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A universal aptameric biosensor: Multiplexed detection of small analytes via aggregated perylene-based broad-spectrum quencher.

    Science.gov (United States)

    Hu, Rong; Zhang, Xi; Xu, Qiang; Lu, Dan-Qing; Yang, Yun-Hui; Xu, Quan-Qing; Ruan, Qiong; Mo, Liu-Ting; Zhang, Xiao-Bing

    2017-06-15

    A universal aptameric system based on the taking advantage of double-stranded DNA/perylene diimide (dsDNA/PDI) as the signal probe was developed for multiplexed detection of small molecules. Aptamers are single-stranded DNA or RNA oligonucleotides which are selected in vitro by a process known as systematic evolution of ligands by exponential enrichment. In this work, we synthesized a new kind of PDI and reported this aggregated PDI could quench the double-stranded DNA (dsDNA)-labeled fluorophores with a high quenching efficiency. The quenching efficiencies on the fluorescence of FAM, TAMRA and Cy5 could reach to 98.3%±0.9%, 97.2%±0.6% and 98.1%±1.1%, respectively. This broad-spectrum quencher was then adopted to construct a multicolor biosensor via a label-free approach. A structure-switching-triggered enzymatic recycling amplification was employed for signal amplification. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity towards small analytes. For other targets, changing the corresponding aptamer can achieve the goal. The quencher did not interfere with the catalytic activity of nuclease. The biosensor could be manipulated with similar sensitivity no matter in pre-addition or post-addition manner. Moreover, simultaneous and multiplexed analysis of several small molecules in homogeneous solution was achieved, demonstrating its potential application in the rapid screening of multiple biotargets. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. BioSentinel: Biosensors for Deep-Space Radiation Study

    Science.gov (United States)

    Lokugamage, Melissa P.; Santa Maria, Sergio R.; Marina, Diana B.; Bhattacharya, Sharmila

    2016-01-01

    The BioSentinel mission will be deployed on NASA's Exploration Mission 1 (EM-1) in 2018. We will use the budding yeast, Saccharomyces cerevisiae, as a biosensor to study the effect of deep-space radiation on living cells. The BioSentinel mission will be the first investigation of a biological response to space radiation outside Low Earth Orbit (LEO) in over 40 years. Radiation can cause damage such as double stand breaks (DSBs) on DNA. The yeast cell was chosen for this mission because it is genetically controllable, shares homology with human cells in its DNA repair pathways, and can be stored in a desiccated state for long durations. Three yeast strains will be stored dry in multiple microfluidic cards: a wild type control strain, a mutant defective strain that cannot repair DSBs, and a biosensor strain that can only grow if it gets DSB-and-repair events occurring near a specific gene. Growth and metabolic activity of each strain will be measured by a 3-color LED optical detection system. Parallel experiments will be done on the International Space Station and on Earth so that we can compare the results to that of deep space. One of our main objectives is to characterize the microfluidic card activation sequence before the mission. To increase the sensitivity of yeast cells as biosensors, desiccated yeast in each card will be resuspended in a rehydration buffer. After several weeks, the rehydration buffer will be exchanged with a growth medium in order to measure yeast growth and metabolic activity. We are currently working on a time-course experiment to better understand the effects of the rehydration buffer on the response to ionizing radiation. We will resuspend the dried yeast in our rehydration medium over a period of time; then each week, we will measure the viability and ionizing radiation sensitivity of different yeast strains taken from this rehydration buffer. The data obtained in this study will be useful in finalizing the card activation sequence for

  4. Recent Progress in Lectin-Based Biosensors

    Directory of Open Access Journals (Sweden)

    Baozhen Wang

    2015-12-01

    Full Text Available This article reviews recent progress in the development of lectin-based biosensors used for the determination of glucose, pathogenic bacteria and toxins, cancer cells, and lectins. Lectin proteins have been widely used for the construction of optical and electrochemical biosensors by exploiting the specific binding affinity to carbohydrates. Among lectin proteins, concanavalin A (Con A is most frequently used for this purpose as glucose- and mannose-selective lectin. Con A is useful for immobilizing enzymes including glucose oxidase (GOx and horseradish peroxidase (HRP on the surface of a solid support to construct glucose and hydrogen peroxide sensors, because these enzymes are covered with intrinsic hydrocarbon chains. Con A-modified electrodes can be used as biosensors sensitive to glucose, cancer cells, and pathogenic bacteria covered with hydrocarbon chains. The target substrates are selectively adsorbed to the surface of Con A-modified electrodes through strong affinity of Con A to hydrocarbon chains. A recent topic in the development of lectin-based biosensors is a successful use of nanomaterials, such as metal nanoparticles and carbon nanotubes, for amplifying output signals of the sensors. In addition, lectin-based biosensors are useful for studying glycan expression on living cells.

  5. S-Layer Protein-Based Biosensors

    Directory of Open Access Journals (Sweden)

    Bernhard Schuster

    2018-04-01

    Full Text Available The present paper highlights the application of bacterial surface (S- layer proteins as versatile components for the fabrication of biosensors. One technologically relevant feature of S-layer proteins is their ability to self-assemble on many surfaces and interfaces to form a crystalline two-dimensional (2D protein lattice. The S-layer lattice on the surface of a biosensor becomes part of the interface architecture linking the bioreceptor to the transducer interface, which may cause signal amplification. The S-layer lattice as ultrathin, highly porous structure with functional groups in a well-defined special distribution and orientation and an overall anti-fouling characteristics can significantly raise the limit in terms of variety and the ease of bioreceptor immobilization, compactness of bioreceptor molecule arrangement, sensitivity, specificity, and detection limit for many types of biosensors. The present paper discusses and summarizes examples for the successful implementation of S-layer lattices on biosensor surfaces in order to give a comprehensive overview on the application potential of these bioinspired S-layer protein-based biosensors.

  6. S-Layer Protein-Based Biosensors.

    Science.gov (United States)

    Schuster, Bernhard

    2018-04-11

    The present paper highlights the application of bacterial surface (S-) layer proteins as versatile components for the fabrication of biosensors. One technologically relevant feature of S-layer proteins is their ability to self-assemble on many surfaces and interfaces to form a crystalline two-dimensional (2D) protein lattice. The S-layer lattice on the surface of a biosensor becomes part of the interface architecture linking the bioreceptor to the transducer interface, which may cause signal amplification. The S-layer lattice as ultrathin, highly porous structure with functional groups in a well-defined special distribution and orientation and an overall anti-fouling characteristics can significantly raise the limit in terms of variety and the ease of bioreceptor immobilization, compactness of bioreceptor molecule arrangement, sensitivity, specificity, and detection limit for many types of biosensors. The present paper discusses and summarizes examples for the successful implementation of S-layer lattices on biosensor surfaces in order to give a comprehensive overview on the application potential of these bioinspired S-layer protein-based biosensors.

  7. Functional design of electrolytic biosensor

    Science.gov (United States)

    Gamage Preethichandra, D. M.; Mala Ekanayake, E. M. I.; Onoda, M.

    2017-11-01

    A novel amperometric biosensbased on conjugated polypyrrole (PPy) deposited on a Pt modified ITO (indium tin oxide) conductive glass substrate and their performances are described. We have presented a method of developing a highly sensitive and low-cost nano-biosensor for blood glucose measurements. The fabrication method proposed decreases the cost of production significantly as the amount of noble metals used is minimized. A nano-corrugated PPy substrate was developed through pulsed electrochemical deposition. The sensitivity achieved was 325 mA/(Mcm2) and the linear range of the developed sensor was 50-60 mmol/l. Then the application of the electrophoresis helps the glucose oxidase (GOx) on the PPy substrate. The main reason behind this high enzyme loading is the high electric field applied across the sensor surface (working electrode) and the counter electrode where that pushes the nano-scale enzyme particles floating in the phosphate buffer solution towards the substrate. The novel technique used has provided an extremely high sensitivities and very high linear ranges for enzyme (GOx) and therefore can be concluded that this is a very good technique to load enzyme onto the conducting polymer substrates.

  8. Magnetoresistive biosensors for quantitative proteomics

    Science.gov (United States)

    Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.

    2017-08-01

    Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.

  9. An aptamer-based fluorescence bio-sensor for chiral recognition of arginine enantiomers.

    Science.gov (United States)

    Yuan, Haiyan; Huang, Yunmei; Yang, Jidong; Guo, Yuan; Zeng, Xiaoqing; Zhou, Shang; Cheng, Jiawei; Zhang, Yuhui

    2018-07-05

    In this study, a novel aptamer - based fluorescence bio-sensor (aptamer-AuNps) was developed for chiral recognition of arginine (Arg) enantiomers based on aptamer and gold nanoparticles (AuNps). Carboxyfluorescein (FAM) labeled aptamers (Apt) were absorbed on AuNps and their fluorescence intensity could be significantly quenched by AuNps based on fluorescence resonance energy transfer (FRET). Once d-Arg or l-Arg were added into the above solution, the aptamer specifically bind to Arg enantiomers and released from AuNps, so the fluorescence intensity of d-Arg system and l-Arg system were all enhanced. The affinity of Apt to l-Arg is tighter to d-Arg, so the enhanced fluorescence signals of l-Arg system was stronger than d-Arg system. What's more, the enhanced fluorescence were directly proportional to the concentration of d-Arg and l-Arg ranging from 0-300 nM and 0-400 nM with related coefficients of 0.9939 and 0.9952, respectively. Furthermore, the method was successfully applied to detection l-Arg in human urine samples with satisfactory results. Eventually, a simple "OR" logic gate with d-Arg &l-Arg as inputs and AuNps aggregation state as outputs was fabricated, which can help us understand the chiral recognition process deeply. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Simple estimation of Förster Resonance Energy Transfer (FRET) orientation factor distribution in membranes.

    Science.gov (United States)

    Loura, Luís M S

    2012-11-19

    Because of its acute sensitivity to distance in the nanometer scale, Förster resonance energy transfer (FRET) has found a large variety of applications in many fields of chemistry, physics, and biology. One important issue regarding the correct usage of FRET is its dependence on the donor-acceptor relative orientation, expressed as the orientation factor k(2). Different donor/acceptor conformations can lead to k(2) values in the 0 ≤ k(2) ≤ 4 range. Because the characteristic distance for FRET, R(0), is proportional to (k(2))1/6, uncertainties in the orientation factor are reflected in the quality of information that can be retrieved from a FRET experiment. In most cases, the average value of k(2) corresponding to the dynamic isotropic limit ( = 2/3) is used for computation of R(0) and hence donor-acceptor distances and acceptor concentrations. However, this can lead to significant error in unfavorable cases. This issue is more critical in membrane systems, because of their intrinsically anisotropic nature and their reduced fluidity in comparison to most common solvents. Here, a simple numerical simulation method for estimation of the probability density function of k(2) for membrane-embedded donor and acceptor fluorophores in the dynamic regime is presented. In the simplest form, the proposed procedure uses as input the most probable orientations of the donor and acceptor transition dipoles, obtained by experimental (including linear dichroism) or theoretical (such as molecular dynamics simulation) techniques. Optionally, information about the widths of the donor and/or acceptor angular distributions may be incorporated. The methodology is illustrated for special limiting cases and common membrane FRET pairs.

  11. Detection of protease activity by fluorescent protein FRET sensors: from computer simulation to live cells

    Science.gov (United States)

    Goryashchenko, Alexander S.; Khrenova, Maria G.; Savitsky, Alexander P.

    2018-04-01

    Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ 2 can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb3+- TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions—fluorescence lifetime up to milliseconds—to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.

  12. Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions.

    Science.gov (United States)

    Götz, Markus; Wortmann, Philipp; Schmid, Sonja; Hugel, Thorsten

    2018-01-30

    Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail. Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates. By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

  13. Label-free detection of sex determining region Y (SRY) via capacitive biosensor

    KAUST Repository

    Sivashankar, Shilpa

    2016-10-20

    In this work, we present for the first time, the use of a simple fractal capacitive biosensor for the quantification and detection of sex-determining region Y (SRY) genes. This section of genetic code, which is found on the Y chromosome, finds importance for study as it causes fetuses to develop characteristics of male sex-like gonads when a mutation occurs. It is also an important genetic code in men, and disorders involving the SRY gene can cause infertility and sexual malfunction that lead to a variety of gene mutational disorders. We have therefore designed silicon-based, label-free fractal capacitive biosensors to quantify various proteins and genes. We take advantage of a good dielectric material, Parylene C for enhancing the performance of the sensors. We have integrated these sensors with a simple microchannel for easy handling of fluids on the detection area. The read-out value of an Agilent LCR meter used to measure capacitance of the sensor at a frequency of 1 MHz determined gene specificity and gene quantification. These data revealed that the capacitance measurement of the capacitive biosensor for the SRY gene depended on both the target and the concentration of DNA. The experimental outcomes in the present study can be used to detect DNA and its variations in crucial fields that have a great impact on our daily lives, such as clinical and veterinary diagnostics, industrial and environmental testing and forensic sciences.

  14. Impedimetric Dengue Biosensor based on Functionalized Graphene Oxide Wrapped Silica Particles

    International Nuclear Information System (INIS)

    Jin, Seon-Ah; Poudyal, Shishir; Marinero, Ernesto E.; Kuhn, Richard J.; Stanciu, Lia A.

    2016-01-01

    Highlights: • 3D graphene oxide based material design. • Fabrication of a label-free dengue DNA and RNA impedimetric biosensor. • Design of a surface-based dengue sensor with good selectivity and detection limit. - Abstract: A composite of 3-Aminopropyltriethoxysilane (APTES) functionalized graphene oxide (APTES-GO) wrapped on SiO 2 particles (SiO 2 @APTES-GO) was prepared via self-assembly. Transmission electron microscopy (TEM) and ATR-Fourier Transform Infrared spectroscopy (ATR-FTIR) confirmed wrapping of the SiO 2 particles by the APTES-GO sheets. An impedimetric biosensor was constructed and used to sensitively detect dengue DNA and dengue RNA via primer hybridization using different oligonucleotide sequences. The results demonstrated that the SiO 2 @APTES-GO electrode material led to enhanced dengue RNA detection sensitivity with selectivity and detection limit (1 femto-Molar), compared to both APTES-GO and APTES-SiO 2 . The three-dimensional structure, higher contact area, electrical properties and the ability for rapid hybridization offered by the SiO 2 @APTES-GO led to the successful design of a dengue biosensor with the lowest detection limit reported to date.

  15. Electrochemical impedance-based DNA sensor using a modified single walled carbon nanotube electrode

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Jessica E. [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Pillai, Shreekumar [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States); Ram, Manoj Kumar, E-mail: mkram@usf.edu [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Kumar, Ashok [Department of Mechanical Engineering, University of South Florida, Tampa, FL (United States); Nanomaterials and Nanomanufacturing Research Center, University of South Florida, Tampa, FL (United States); Singh, Shree R. [Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL (United States)

    2011-07-20

    Carbon nanotubes have become promising functional materials for the development of advanced electrochemical biosensors with novel features which could promote electron-transfer with various redox active biomolecules. This paper presents the detection of Salmonella enterica serovar Typhimurium using chemically modified single walled carbon nanotubes (SWNTs) with single stranded DNA (ssDNA) on a polished glassy carbon electrode. Hybridization with the corresponding complementary ssDNA has shown a shift in the impedance studies due to a higher charge transfer in ssDNA. The developed biosensor has revealed an excellent specificity for the appropriate targeted DNA strand. The methodologies to prepare and functionalize the electrode could be adopted in the development of DNA hybridization biosensor.

  16. Detection of DNA of genetically modified maize by a silicon nanowire field-effect transistor

    International Nuclear Information System (INIS)

    Pham, Van Binh; Tung Pham, Xuan Thanh; Duong Dang, Ngoc Thuy; Tuyen Le, Thi Thanh; Tran, Phu Duy; Nguyen, Thanh Chien; Nguyen, Van Quoc; Dang, Mau Chien; Tong, Duy Hien; Van Rijn, Cees J M

    2011-01-01

    A silicon nanowire field-effect transistor based sensor (SiNW-FET) has been proved to be the most sensitive and powerful device for bio-detection applications. In this paper, SiNWs were first fabricated by using our recently developed deposition and etching under angle technique (DEA), then used to build up the complete SiNW device based biosensor. The fabricated SiNW biosensor was used to detect DNA of genetically modified maize. As the DNA of the genetically modified maize has particular DNA sequences of 35S promoter, we therefore designed 21 mer DNA oligonucleotides, which are used as a receptor to capture the transferred DNA of maize. In our work, the SiNW biosensor could detect DNA of genetically modified maize with concentrations down to about 200 pM

  17. Hydrogen peroxide biosensor based on titanium oxide

    Science.gov (United States)

    Halim, Nur Hamidah Abdul; Heng, Lee Yook; Hashim, Uda

    2015-09-01

    In this work, a biosensor utilizing modified titania, TiO2 particles using aminopropyl-triethoxy-silane, (APTS) for developing hydrogen peroxide biosensor is presented. The surface of Ti-APTS particles is used as a support for hemoglobin immobilization via covalent bonding. The performance of the biosensor is determined by differential pulse voltammetry. The linear response was observed at the reduction current of redox mediator probe [FeCN6]3-/4- at potential between 0.22 V to 0.24 V. The preliminary result for electrochemistry study on this modified electrode is reported. The preliminary linear range is obtained from 1×10-2 M to 1×10-8 M.

  18. Thermoresponsive Magnetic Nano-Biosensors for Rapid Measurements of Inorganic Arsenic and Cadmium

    Directory of Open Access Journals (Sweden)

    Isamu Maeda

    2012-10-01

    Full Text Available Green fluorescent protein-tagged sensor proteins, ArsR-GFP and CadC-GFP, have been produced as biosensors for simple and low-cost quantification of As(III or Cd(II. In this study, the sensor protein-promoter DNA complexes were reconstructed on the surfaces of magnetic particles of different sizes. After the surface modification all the particles could be attracted by magnets, and released different amounts of GFP-tagged protein, according to the metal concentrations within 5 min, which caused significant increases in fluorescence. A detection limit of 1 µg/L for As(III and Cd(II in purified water was obtained only with the nanoparticles exhibiting enough magnetization after heat treatment for 1 min. Therefore, thermoresponsive magnetic nano-biosensors offer great advantages of rapidity and sensitivity for the measurement of the toxic metals in drinking water.

  19. Thermoresponsive magnetic nano-biosensors for rapid measurements of inorganic arsenic and cadmium.

    Science.gov (United States)

    Siddiki, Mohammad Shohel Rana; Shimoaoki, Shun; Ueda, Shunsaku; Maeda, Isamu

    2012-10-18

    Green fluorescent protein-tagged sensor proteins, ArsR-GFP and CadC-GFP, have been produced as biosensors for simple and low-cost quantification of As(III) or Cd(II). In this study, the sensor protein-promoter DNA complexes were reconstructed on the surfaces of magnetic particles of different sizes. After the surface modification all the particles could be attracted by magnets, and released different amounts of GFP-tagged protein, according to the metal concentrations within 5 min, which caused significant increases in fluorescence. A detection limit of 1 µg/L for As(III) and Cd(II) in purified water was obtained only with the nanoparticles exhibiting enough magnetization after heat treatment for 1 min. Therefore, thermoresponsive magnetic nano-biosensors offer great advantages of rapidity and sensitivity for the measurement of the toxic metals in drinking water.

  20. Biosensors for breast cancer diagnosis: A review of bioreceptors, biotransducers and signal amplification strategies.

    Science.gov (United States)

    Mittal, Sunil; Kaur, Hardeep; Gautam, Nandini; Mantha, Anil K

    2017-02-15

    Breast cancer is highly prevalent in females and accounts for second highest number of deaths, worldwide. Cumbersome, expensive and time consuming detection techniques presently available for detection of breast cancer potentiates the need for development of novel, specific and ultrasensitive devices. Biosensors are the promising and selective detection devices which hold immense potential as point of care (POC) tools. Present review comprehensively scrutinizes various breast cancer biosensors developed so far and their technical evaluation with respect to efficiency and potency of selected bioreceptors and biotransducers. Use of glycoproteins, DNA biomarkers, micro-RNA, circulatory tumor cells (CTC) and some potential biomarkers are introduced briefly. The review also discusses various strategies used in signal amplification such as nanomaterials, redox mediators, p19 protein, duplex specific nucleases (DSN) and redox cycling. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Design & fabrication of cantilever array biosensors

    DEFF Research Database (Denmark)

    Boisen, Anja; Thundat, T

    2009-01-01

    Surface immobilization of functional receptors on microfabricated cantilever arrays offers a new paradigm for the development of biosensors based on nanomechanics. Microcantilever-based systems are capable of real-time, multiplexed detection of unlabeled disease markers in extremely small volumes......, electronic processing, and even local telemetry on a single chip have the potential of satisfying the need for highly sensitive and selective multiple-target detection in very small samples. Here we will review the design and fabrication process of cantilever-based biosensors....

  2. Biosensor technology for pesticides--a review.

    Science.gov (United States)

    Verma, Neelam; Bhardwaj, Atul

    2015-03-01

    Pesticides, due to their lucrative outcomes, are majorly implicated in agricultural fields for crop production enhancement. Due to their pest removal properties, pesticides of various classes have been designed to persist in the environment over a longer duration after their application to achieve maximum effectiveness. Apart from their recalcitrant structure and agricultural benefits, pesticides also impose acute toxicological effects onto the other various life forms. Their accumulation in the living system may prove to be detrimental if established in higher concentrations. Thus, their prompt and accurate analysis is a crucial matter of concern. Conventional techniques like chromatographic techniques (HPLC, GC, etc.) used for pesticides detection are associated with various limitations like stumpy sensitivity and efficiency, time consumption, laboriousity, requirement of expensive equipments and highly trained technicians, and many more. So there is a need to recruit the methods which can detect these neurotoxic compounds sensitively, selectively, rapidly, and easily in the field. Present work is a brief review of the pesticide effects, their current usage scenario, permissible limits in various food stuffs and 21st century advancements of biosensor technology for pesticide detection. Due to their exceptional performance capabilities, easiness in operation and on-site working, numerous biosensors have been developed for bio-monitoring of various environmental samples for pesticide evaluation immensely throughout the globe. Till date, based on sensing element (enzyme based, antibody based, etc.) and type of detection method used (Electrochemical, optical, and piezoelectric, etc.), a number of biosensors have been developed for pesticide detection. In present communication, authors have summarized 21st century's approaches of biosensor technology for pesticide detection such as enzyme-based biosensors, immunosensors, aptamers, molecularly imprinted polymers, and

  3. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang; Xiao, Shaobo; Chen, Huanchun

    2014-01-01

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis

  4. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang, E-mail: wangdang511@126.com; Xiao, Shaobo; Chen, Huanchun

    2014-10-03

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.

  5. Development of electrochemical biosensors with various types of zeolites

    Science.gov (United States)

    Soldatkina, O. V.; Kucherenko, I. S.; Soldatkin, O. O.; Pyeshkova, V. M.; Dudchenko, O. Y.; Akata Kurç, B.; Dzyadevych, S. V.

    2018-03-01

    In the work, different types of zeolites were used for the development of enzyme-based electrochemical biosensors. Zeolites were added to the biorecognition elements of the biosensors and served as additional components of the biomembranes or adsorbents for enzymes. Three types of biosensors (conductometric, amperometric and potentiometric) were studied. The developed biosensors were compared with the similar biosensors without zeolites. The biosensors contained the following enzymes: urease, glucose oxidase, glutamate oxidase, and acetylcholinesterase and were intended for the detection of urea, glucose, glutamate, and acetylcholine, respectively. Construction of the biosensors using the adsorption of enzymes on zeolites has several advantages: simplicity, good reproducibility, quickness, absence of toxic compounds. These benefits are particularly important for the standardization and further mass production of the biosensors. Furthermore, a biosensor for the sucrose determination contained a three-enzyme system (invertase/mutatorase/glucose oxidase), immobilized by a combination of adsorption on silicalite and cross-linking via glutaraldehyde; such combined immobilization demonstrated better results as compared with adsorption or cross-linking separately. The analysis of urea and sucrose concentrations in the real samples was carried out. The results, obtained with biosensors, had high correlation with the results of traditional analytical methods, thus the developed biosensors are promising for practical applications.

  6. Improved electrochemical nucleic acid biosensor based on polyaniline-polyvinyl sulphonate

    Energy Technology Data Exchange (ETDEWEB)

    Prabhakar, Nirmal [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi-110012 (India); Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi-110016 (India); Sumana, G.; Arora, Kavita [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi-110012 (India); Singh, Harpal [Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi-110016 (India); Malhotra, B.D. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi-110012 (India)], E-mail: bansi.malhotra@gmail.com

    2008-05-01

    DNA biosensor based on polyaniline (PANI)-polyvinyl sulphonate (PVS) has been fabricated using electrochemical entrapment technique for detection of organophosphorus pesticides (chlorpyrifos and malathion). These double stranded calf thymus DNA (dsCT-DNA) entrapped PANI-PVS/indium-tin-oxide (ITO) bioelectrodes have been characterized using square wave voltammetry (SWV), Fourier transform infra-red spectroscopy (FT-IR), scanning electron microscopy (SEM) and electrochemical impedance techniques, respectively. These dsCT-DNA entrapped PANI-PVS/ITO bioelectrodes have been found to have response time of 30 s, stability of about 6 months and detection limit for chlorpyrifos and malathion as 0.5 ppb and 0.01 ppm, respectively.

  7. Improved electrochemical nucleic acid biosensor based on polyaniline-polyvinyl sulphonate

    International Nuclear Information System (INIS)

    Prabhakar, Nirmal; Sumana, G.; Arora, Kavita; Singh, Harpal; Malhotra, B.D.

    2008-01-01

    DNA biosensor based on polyaniline (PANI)-polyvinyl sulphonate (PVS) has been fabricated using electrochemical entrapment technique for detection of organophosphorus pesticides (chlorpyrifos and malathion). These double stranded calf thymus DNA (dsCT-DNA) entrapped PANI-PVS/indium-tin-oxide (ITO) bioelectrodes have been characterized using square wave voltammetry (SWV), Fourier transform infra-red spectroscopy (FT-IR), scanning electron microscopy (SEM) and electrochemical impedance techniques, respectively. These dsCT-DNA entrapped PANI-PVS/ITO bioelectrodes have been found to have response time of 30 s, stability of about 6 months and detection limit for chlorpyrifos and malathion as 0.5 ppb and 0.01 ppm, respectively

  8. Functional oligonucleotide recognition nanomodules for electrochemical DNA biosensors

    OpenAIRE

    Campàs i Homs, Mònica

    2002-01-01

    El objetivo de esta tesis ha sido diseñar, caracterizar y optimizar un array de sensores de ADN electroquímico. Para el estudio de la inmovilización de las sondas de oligonucleótidos y la detección de la hibridación se realizaron experimentos preliminares con un sistema simplificado. Dicho sistema demostró que las monocapas auto-ensambladas (SAMs) en superficies de oro eran apropiadas como método de inmovilización. Debido al rápido desarrollo de los sensores de ADN hacia los arrays de ADN, se...

  9. Derivation of Elastic Stress Concentration Factor Equations for Debris Fretting Flaws in Pressure Tubes of Pressurized Heavy Water Reactors

    International Nuclear Information System (INIS)

    Kim, Jong Sung; Oh, Young Jin

    2014-01-01

    If volumetric flaws such as bearing pad fretting flaws and debris fretting flaws are detected in the pressure tubes of pressurized heavy water reactors during in-service inspection, the initiation of fatigue cracks and delayed hydrogen cracking from the detected volumetric flaws shall be assessed by using elastic stress concentration factors in accordance with CSA N285.8-05. The CSA N285.8-05 presents only an approximate formula based on linear elastic fracture mechanics for the debris fretting flaw. In this study, an engineering formula considering the geometric characteristics of the debris fretting flaw in detail was derived using two-dimensional finite element analysis and Kinectrics, Inc.'s engineering procedure with slight modifications. Comparing the application results obtained using the derived formula with the three-dimensional finite element analysis results, it is found that the results obtained using the derived formula agree well with the results of the finite element analysis

  10. Optimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Roots

    KAUST Repository

    Long, Yuchen; Stahl, Yvonne; Weidtkamp-Peters, Stefanie; Smet, Wouter; Du, Yujuan; Gadella, Theodorus W. J.; Goedhart, Joachim; Scheres, Ben; Blilou, Ikram

    2018-01-01

    Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living

  11. Graphene and graphene-like two-denominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications.

    Science.gov (United States)

    Tian, Feng; Lyu, Jing; Shi, Jingyu; Yang, Mo

    2017-03-15

    In the past decades, Förster resonance energy transfer (FRET) has been applied in many biological applications to reveal the biological information at the nanoscale. Recently, graphene and graphene-like two-dimensional (2D) nanomaterials started to be used in FRET assays as donors or acceptors including graphene oxide (GO), graphene quantum dot (GQD), graphitic-carbon nitride nanosheets (g-C 3 N 4 ) and transition metal dichalcogenides (e.g. MoS 2 , MnO 2, and WS 2 ). Due to the remarkable properties such as large surface to volume ratio, tunable energy band, photoluminescence and excellent biocompatibility, these 2D nanomaterials based FRET assays have shown great potential in various biological applications. This review summarizes the recent development of graphene and graphene-like 2D nanomaterials based FRET assays in applications of biosensing, bioimaging, and drug delivery monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Unlubricated Gross Slip Fretting Wear of Metallic Plasma Sprayed Coatings for Ti6A14V Surfaces

    National Research Council Canada - National Science Library

    Hager, Jr., Carl H; Sanders, Jeffrey H; Sharma, Shashi K

    2006-01-01

    ... to simulate cold engine startup. Alternative coatings such as plasma sprayed molybdenum and nickel were also evaluated because of their potential for reducing fretting wear under certain simulated engine conditions...

  13. Electrochemical detection of specific DNA sequences from PCR amplicons on carbon and mercury electrodes using Meldola's Blue as an intercalator

    Czech Academy of Sciences Publication Activity Database

    Kerman, K.; Özkan, D.; Kara, P.; Karadeniz, H.; Özkan, Z.; Erdem, A.; Jelen, František; Özsöz, M.

    2004-01-01

    Roč. 28, č. 5 (2004), s. 523-533 ISSN 1010-7614 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA * biosensor * Meldola's Blue Subject RIV: BO - Biophysics Impact factor: 0.579, year: 2004

  14. Investigation of fretting behaviour in pressure armour layers of flexible pipes

    Science.gov (United States)

    Don Rasika Perera, Solangarachchige

    The incidence of fretting damage in the pressure armour wires of flexible pipes used in offshore oil explorations has been investigated. A novel experimental facility which is capable of simulating nub and valley contact conditions of interlocking wire winding with dynamic slip, representative of actual pipe loading, has been developed. The test set-up is equipped with a state of the art data acquisition system and a controller with transducers to measure and control the normal load, slip amplitude and friction force at the contact, in addition to the hoop stress in the wire. Tests were performed with selected loading and the fretted regions were examined using optical microscopy techniques. Results show that the magnitude of contact loading and the slip amplitude have a distinct influence on surface damage. Surface cracks originated from a fretting scar were observed at high contact loads in mixed slip sliding while surface damage predominantly due to wear was observed under gross slip. The position of surface cracks and the wear profile have been related to the contact pressure distribution. The evolution of friction force and surface damage under different slip and normal pressure conditions has been analysed. A fracture mechanics based numerical procedure has been developed to analyse the fretting damage behaviour. A severity parameter is proposed in order to ascertain whether the crack growth is in mode I or mode II cracking. The analysis show the influence of mode II cracking in the early stages of crack growth following which the crack deviates in the mode I direction making mode I the dominant crack propagation mechanism. The crack path determined by the numerical procedure correlates well with the experimental results. A numerical analysis was carried out for the fretting fatigue condition where a cyclic bulk stress superimposes with the friction force. The analysis correlates well with short crack growth behaviour. The analysis confirms that fretting is a

  15. Fretting-corrosion behavior in hip implant modular junctions: The influence of friction energy and pH variation.

    Science.gov (United States)

    Royhman, Dmitry; Patel, Megha; Runa, Maria J; Wimmer, Markus A; Jacobs, Joshua J; Hallab, Nadim J; Mathew, Mathew T

    2016-09-01

    Recently, there has been increasing concern in the orthopedic community over the use of hip implant modular devices due to an increasing number of reports of early failure, failure that has been attributed to fretting-corrosion at modular interfaces. Much is still unknown about the electrochemical and mechanical degradation mechanisms associated with the use of such devices. Accordingly, the purpose of our study was to develop a methodology for testing the fretting-corrosion behavior of modular junctions. A fretting-corrosion apparatus was used to simulate the fretting-corrosion conditions of a CoCrMo hip implant head on a Ti6Al4V hip implant stem. The device features two perpendicularly-loaded CoCrMo pins that articulated against a Ti6Al4V rod. A sinusoidal fretting motion was applied to the rod at various displacement amplitudes (25, 50, 100, 150 and 200μm) at a constant load of 200N. Bovine calf serum at two different pH levels (3.0 and 7.6) was used to simulate the fluid environment around the joint. Experiments were conducted in two modes of electrochemical control - free-potential and potentiostatic. Electrochemical impedance spectroscopy tests were done before and after the fretting motion to assess changes in corrosion kinetics. In free potential mode, differences were seen in change in potential as a function of displacement amplitude. In general, VDrop (the drop in potential at the onset of fretting), VFretting, (the average potential during fretting), ΔVFretting (the change in potential from the onset of fretting to its termination) and VRecovery (the change in potential from the termination of fretting until stabilization) appeared linear at both pH levels, but showed drastic deviation from linearity at 100μm displacement amplitude. Subsequent EDS analysis revealed a large number of Ti deposits on the CoCrMo pin surfaces. Potentiostatic tests at both pH levels generally showed increasing current with increasing displacement amplitude. Electrochemical

  16. Hybridization assay of insect antifreezing protein gene by novel multilayered porous silicon nucleic acid biosensor.

    Science.gov (United States)

    Lv, Xiaoyi; Chen, Liangliang; Zhang, Hongyan; Mo, Jiaqing; Zhong, Furu; Lv, Changwu; Ma, Ji; Jia, Zhenhong

    2013-01-15

    A fabrication of a novel simple porous silicon polybasic photonic crystal with symmetrical structure has been reported as a nucleic acid biosensor for detecting antifreeze protein gene in insects (Microdera puntipennis dzhungarica), which would be helpful in the development of some new transgenic plants with tolerance of freezing stress. Compared to various porous silicon-based photonic configurations, porous silicon polytype layered structure is quite easy to prepare and shows more stability; moreover, polybasic photonic crystals with symmetrical structure exhibit interesting optical properties with a sharp resonance in the reflectance spectrum, giving a higher Q factor which causes higher sensitivity for sensing performance. In this experiment, DNA oligonucleotides were immobilized into the porous silicon pores using a standard crosslink chemistry method. The porous silicon polybasic symmetrical structure sensor possesses high specificity in performing controlled experiments with non-complementary DNA. The detection limit was found to be 21.3nM for DNA oligonucleotides. The fabricated multilayered porous silicon-based DNA biosensor has potential commercial applications in clinical chemistry for determination of an antifreeze protein gene or other genes. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. In vitro simulation of fretting-corrosion in hip implant modular junctions: The influence of pH.

    Science.gov (United States)

    Royhman, Dmitry; Patel, Megha; Jacobs, Joshua J; Wimmer, Markus A; Hallab, Nadim J; Mathew, Mathew T

    2018-02-01

    The fretting-corrosion behavior of mixed metal contacts is affected by various mechanical and electrochemical parameters. Crevice conditions at the junction and patient-specific pathologies can affect the pH of the prosthetic environment. The main objective of this study is to understand the effect of pH variation at the stem/head junction of the hip implant under fretting corrosion exposure. We hypothesized that pH will have a significant influence on the fretting-corrosion behavior hip implant modular junctions. A custom-made setup was used to evaluate the fretting corrosion behavior of hip implant modular junctions. A Newborn calf serum solution (30 g/L protein content) was used to simulate the synovial fluid environment. A sinusoidal fretting motion, with a displacement amplitude of +50 µm, was applied to the Ti alloy rod. The effects of pathology driven, periprosthetic pH variation were simulated at four different pH levels (3.0, 4.5, 6.0 and 7.6). Electrochemical and mechanical properties were evaluated before, during, and after the applied fretting motion. The impedance of the system was increased in response to the fretting motion. The hysteresis tangential load/displacement behavior was not affected by pH level. The worn surfaces of CoCrMo pins exhibited the presence of tribolayer or organic deposits, in the pH 4.5 group, which may explain the lower drop in potential and mass loss observed in that group. Mechanically dominated wear mechanisms, namely, adhesive wear was shown in the pH 7.6 group, which may account for a higher potential drop and metal content loss. This study suggests that the fretting-corrosion mechanisms in hip implant are affected by the pH levels of the surrounding environment and patient-specific factors. Copyright © 2017. Published by Elsevier Ltd.

  18. Chemo-mechanical pushing of proteins along single-stranded DNA.

    Science.gov (United States)

    Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

    2016-05-31

    Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

  19. Spreeta-based biosensor for endocrine disruptors

    NARCIS (Netherlands)

    Marchesini, G.R.; Koopal, K.; Meulenberg, E.; Haasnoot, W.; Irth, H.

    2007-01-01

    The construction and performance of an automated low-cost Spreeta¿-based prototype biosensor system for the detection of endocrine disrupting chemicals (EDCs) is described. The system consists primarily of a Spreeta miniature liquid sensor incorporated into an aluminum flow cell holder, dedicated to

  20. Amperometric biosensors based on conducting nanotubes

    NARCIS (Netherlands)

    Kros, Alexander

    2000-01-01

    This thesis describes a multidisciplinary study towards the development of a glucose biosensor that in the future can be used for in vivo implantations. The research focuses on three major topics, viz. the construction of the glucose sensor, the development of a biocompatible coating and a study of