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Sample records for protein whole-cell biosensor

  1. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    /mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS...

  2. Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2005-01-01

    Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N'-......Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N......-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies....

  3. Biosensors for Whole-Cell Bacterial Detection

    Science.gov (United States)

    Rushworth, Jo V.; Hirst, Natalie A.; Millner, Paul A.

    2014-01-01

    SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost. PMID:24982325

  4. Signal amelioration of electrophoretically deposited whole-cell biosensors using external electric fields

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Yoav, Hadar, E-mail: benyoav@post.tau.ac.il [Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel-Aviv 69978 (Israel); Amzel, Tal [Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel-Aviv 69978 (Israel); Sternheim, Marek [Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel-Aviv, 69978 (Israel); Belkin, Shimshon [Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, 91904 (Israel); Rubin, Adi [Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel-Aviv, 69978 (Israel); Shacham-Diamand, Yosi [Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel-Aviv 69978 (Israel); Freeman, Amihay [Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel-Aviv, 69978 (Israel)

    2011-11-01

    Highlights: > We present an electrochemical whole-cell biochip that can apply electric fields. > We examine the integration of cells on a biochip using electrophoretic deposition. > The effect of electric fields on the whole-cell biosensor has been demonstrated. > Relatively short DC electric pulse improves the performance of whole-cell biosensors. > Prolonged AC electric fields deteriorated the whole-cell biosensor performance. - Abstract: This paper presents an integrated whole-cell biochip system where functioning cells are deposited on the solid micro-machined surfaces while specially designed indium tin oxide electrodes that can be used to apply controllable electric fields during various stages; for example during cell deposition. The electrodes can be used also for sensing currents associated with the sensing mechanisms of electrochemical whole-cell biosensors. In this work a new approach integrating live bacterial cells on a biochip using electrophoretic deposition is presented. The biomaterial deposition technique was characterized under various driving potentials and chamber configurations. An analytical model of the electrophoretic deposition kinetics was developed and presented here. The deposited biomass included genetically engineered bacterial cells that may respond to toxic material exposure by expressing proteins that react with specific analytes generating electrochemically active byproducts. In this study the effect of external electric fields on the whole-cell biochips has been successfully developed and tested. The research hypothesis was that by applying electric fields on bacterial whole-cells, their permeability to the penetration of external analytes can be increased. This effect was tested and the results are shown here. The effect of prolonged and short external electric fields on the bioelectrochemical signal generated by sessile bacterial whole-cells in response to the presence of toxins was studied. It was demonstrated that relatively

  5. Signal amelioration of electrophoretically deposited whole-cell biosensors using external electric fields

    International Nuclear Information System (INIS)

    Ben-Yoav, Hadar; Amzel, Tal; Sternheim, Marek; Belkin, Shimshon; Rubin, Adi; Shacham-Diamand, Yosi; Freeman, Amihay

    2011-01-01

    Highlights: → We present an electrochemical whole-cell biochip that can apply electric fields. → We examine the integration of cells on a biochip using electrophoretic deposition. → The effect of electric fields on the whole-cell biosensor has been demonstrated. → Relatively short DC electric pulse improves the performance of whole-cell biosensors. → Prolonged AC electric fields deteriorated the whole-cell biosensor performance. - Abstract: This paper presents an integrated whole-cell biochip system where functioning cells are deposited on the solid micro-machined surfaces while specially designed indium tin oxide electrodes that can be used to apply controllable electric fields during various stages; for example during cell deposition. The electrodes can be used also for sensing currents associated with the sensing mechanisms of electrochemical whole-cell biosensors. In this work a new approach integrating live bacterial cells on a biochip using electrophoretic deposition is presented. The biomaterial deposition technique was characterized under various driving potentials and chamber configurations. An analytical model of the electrophoretic deposition kinetics was developed and presented here. The deposited biomass included genetically engineered bacterial cells that may respond to toxic material exposure by expressing proteins that react with specific analytes generating electrochemically active byproducts. In this study the effect of external electric fields on the whole-cell biochips has been successfully developed and tested. The research hypothesis was that by applying electric fields on bacterial whole-cells, their permeability to the penetration of external analytes can be increased. This effect was tested and the results are shown here. The effect of prolonged and short external electric fields on the bioelectrochemical signal generated by sessile bacterial whole-cells in response to the presence of toxins was studied. It was demonstrated that

  6. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production.

    Science.gov (United States)

    Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S; Polizzi, Karen M

    2017-06-01

    Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole-cell biosensors. Biotechnol. Bioeng. 2017;114: 1290-1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  7. Whole-Cell Fluorescent Biosensors for Bioavailability and Biodegradation of Polychlorinated Biphenyls

    Directory of Open Access Journals (Sweden)

    David Ryan

    2010-02-01

    Full Text Available Whole-cell microbial biosensors are one of the newest molecular tools used in environmental monitoring. Such biosensors are constructed through fusing a reporter gene such as lux, gfp or lacZ,to a responsive promoter. There have been many reports of the applications of biosensors, particularly their use in assaying pollutant toxicity and bioavailability. This paper reviews the basic concepts behind the construction of whole-cell microbial biosensors for pollutant monitoring, and describes the applications of two such biosensors for detecting the bioavailability and biodegradation of Polychlorinated Biphenyls (PCBs.

  8. Performance of a Cyanobacteria Whole Cell-Based Fluorescence Biosensor for Heavy Metal and Pesticide Detection

    Directory of Open Access Journals (Sweden)

    Salmijah Surif

    2013-05-01

    Full Text Available Whole cell biosensors always face the challenge of low stability of biological components and short storage life. This paper reports the effects of poly(2-hydroxyethyl methacrylate (pHEMA immobilization on a whole cell fluorescence biosensor for the detection of heavy metals (Cu, Pb, Cd, and pesticides (dichlorophenoxyacetic acid (2,4-D, and chlorpyrifos. The biosensor was produced by entrapping the cyanobacterium Anabaena torulosa on a cellulose membrane, followed by applying a layer of pHEMA, and attaching it to a well. The well was then fixed to an optical probe which was connected to a fluorescence spectrophotometer and an electronic reader. The optimization of the biosensor using several factors such as amount of HEMA and drying temperature were undertaken. The detection limits of biosensor without pHEMA for Cu, Cd, Pb, 2,4-D and chlorpyrifos were 1.195, 0.027, 0.0100, 0.025 and 0.025 µg/L respectively. The presence of pHEMA increased the limits of detection to 1.410, 0.250, 0.500, 0.235 and 0.117 µg/L respectively. pHEMA is known to enhance the reproducibility of the biosensor with average relative standard deviation (RSD of ±1.76% for all the pollutants tested, 48% better than the biosensor without pHEMA (RSD = ±3.73%. In storability test with Cu 5 µg/L, the biosensor with pHEMA performed 11.5% better than the test without pHEMA on day-10 and 5.2% better on day-25. pHEMA is therefore a good candidate to be used in whole cell biosensors as it increases reproducibility and enhances biosensor storability.

  9. Defining an additivity framework for mixture research in inducible whole-cell biosensors

    DEFF Research Database (Denmark)

    Martin-Betancor, K; Ritz, Christian; Fernández-Piñas, F

    2015-01-01

    A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differe......A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts...... for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability...... illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition...

  10. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    Science.gov (United States)

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible P ars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  11. Whole Cell Biosensor Using Anabaena torulosa with Optical Transduction for Environmental Toxicity Evaluation

    Directory of Open Access Journals (Sweden)

    Ling Shing Wong

    2013-01-01

    Full Text Available A whole cell-based biosensor using Anabaena torulosa for the detection of heavy metals (Cu, Pb, and Cd, 2,4-dichlorophenoxyacetate (2,4-D, and chlorpyrifos was constructed. The cyanobacteria were entrapped on a cellulose membrane through filtration. Then, the membrane was dried and fixed into a cylindrical well, which was designed to be attached to an optical probe. The probe was connected to fluorescence spectrometer with optical fibre. The presence of the toxicants was indicated by the change of fluorescence emission, before and after the exposure. The linear detection ranges for Cu, Pb, and Cd were 2.5–10.0 µg/L, 0.5–5.0 µg/L, and 0.5–10.0 µg/L, respectively, while 2,4-D and chlorpyrifos shared similar linear ranges of 0.05–0.75 µg/L. The biosensor showed good sensitivity with the lowest limits of detection (LLD for Cu, Pb, Cd, 2,4-D and chlorpyrifos determined at 1.195 µg/L, 0.100 µg/L, 0.027 µg/L, 0.025 µg/L, and 0.025 µg/L, respectively. The overall reproducibility of the biosensor (n=3 was <±6.35%. The biosensor had been tested with different combinations of toxicants, with the results showing predominantly antagonistic responses. The results confirmed that the biosensor constructed in this report is suitable to be used in quantitative and qualitative detections of heavy metals and pesticides.

  12. The Application of Whole Cell-Based Biosensors for Use in Environmental Analysis and in Medical Diagnostics

    Science.gov (United States)

    Gui, Qingyuan; Lawson, Tom; Shan, Suyan; Yan, Lu; Liu, Yong

    2017-01-01

    Various whole cell-based biosensors have been reported in the literature for the last 20 years and these reports have shown great potential for their use in the areas of pollution detection in environmental and in biomedical diagnostics. Unlike other reviews of this growing field, this mini-review argues that: (1) the selection of reporter genes and their regulatory proteins are directly linked to the performance of celllular biosensors; (2) broad enhancements in microelectronics and information technologies have also led to improvements in the performance of these sensors; (3) their future potential is most apparent in their use in the areas of medical diagnostics and in environmental monitoring; and (4) currently the most promising work is focused on the better integration of cellular sensors with nano and micro scaled integrated chips. With better integration it may become practical to see these cells used as (5) real-time portable devices for diagnostics at the bedside and for remote environmental toxin detection and this in situ application will make the technology commonplace and thus as unremarkable as other ubiquitous technologies. PMID:28703749

  13. HCN Producing Bacteria Enable Sensing Of Non-Bioavailable Hg Species by the Whole Cell Biosensor

    Science.gov (United States)

    Horvat, M.; Rijavec, T.; Koron, N.; Lapanje, A.

    2015-12-01

    Bacteria play an important role in Hg transformation reactions. The production of cyanide (HCN) and other secondary metabolites seems to be key elements involved in these transformations. Current hypotheses link the role of HCN production to growth inhibition of nonHCN producing competitor organisms (role of an antimicrobial agent). Our past investigations showed that HCN production did not correlate with antimicrobial activity and since pK value of HCN is very high (pK = 9,21), it can be expected that most of the produced HCN is removed from the microenvironment. This way, the expected inhibitory concentrations can hardly be reached. Accordingly, we proposed a new concept, where the ability of complexation of transient metals by HCN served as a regulation process for the accessibility of micro-elements. In our study, we focused on the presence of HCN producing bacteria and carried it out in the Hg contaminated environment connected to the Idrija Mercury Mine, Slovenia. We characterised the isolates according to the presence of Hg resistance (HgR), level of HCN production and genetic similarities. In laboratory setups, using our merR whole cell based biosensor, we determined the transformation of low bioavailable Hg0 and HgS forms into bioavailable Hg by these HCN producing bacteria. We observed that HgR strains producing HCN had the highest impact on increased Hg bioavailability. In the proposed ecological strategy HgR HCN producing bacteria increase their competitive edge over non-HgR competitors through the increase of Hg toxicity. Due to their activity, Hg is made available to other organisms as well and thus enters into the ecosystem. Finally, using some of the characteristics of bacteria (e.g. Hg resistance genetic elements), we developed a fully automated sensing approach, combining biosensorics and mechatronics, to measure the bioavailability of Hg in situ.

  14. A sensitive whole-cell biosensor for the simultaneous detection of a broad-spectrum of toxic heavy metal ions.

    Science.gov (United States)

    Cerminati, S; Soncini, F C; Checa, S K

    2015-04-07

    Bacterial biosensors are simple, cost-effective and efficient analytical tools for detecting bioavailable heavy metals in the environment. This work presents the design, construction and calibration of a novel whole-cell fluorescent biosensory device that, simultaneously and with high sensitivity, reports the presence of toxic mercury, lead, cadmium and/or gold ions in aqueous samples. This bio-reporter can be easily applied as an immediate alerting tool for detecting the presence of harmful pollutants in drinking water.

  15. Use of a Whole-Cell Biosensor and Flow Cytometry to Detect AHL Production by an Indigenous Soil Community During Decomposition

    DEFF Research Database (Denmark)

    Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2005-01-01

    originating from Vibrio fischeri. This resulted in a whole-cell biosensor, responding to the presence of AHL compounds. The biosensor was introduced to compost soil microcosms amended with nettle leaves. After 3 days of incubation, cells were extracted and analyzed by flow cytometry. All microcosms contained...

  16. 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.

  17. Ribotyping and whole-cell protein analysis of spirochetes isolated from arthropods in the Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Buňková, L.; Švec, P.; Halouzka, Jiří; Rudolf, Ivo; Němec, M.

    2008-01-01

    Roč. 15, č. 2 (2008), s. 225-230 ISSN 1232-1966 R&D Projects: GA AV ČR KJB600930613; GA ČR GA206/03/0726 Institutional research plan: CEZ:AV0Z60930519 Keywords : Borrelia burgdorferi sensu lato * Czech Republic * ribotyping * whole-cell protein analysis * taxonomy Subject RIV: EE - Microbiology, Virology Impact factor: 1.443, year: 2008 http://www.aaem.pl/pdf/15225.pdf

  18. Construction of an extended range whole-cell tetracycline biosensor by use of the tet(M) resistance gene

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2005-01-01

    protein gene. Tetracycline, oxytetracycline, chlortetracycline and minocycline all effectively induced the resulting Escherichia coli MC4100/pTGM biosensor and similar dose-response characteristics were recorded by flow cytometry for all four compounds. The novel tetracycline biosensor was responsive...

  19. In vivo detection and quantification of tetracycline by use of a whole-cell biosensor in the rat intestine

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars Hestbjerg; Licht, Tine Rask

    2004-01-01

    was observed. Reintroduction of the E. coli MC4100/pTGFP2 strain into animals already colonized by the plasmid-free E. coli strain the day before euthanasia made it possible to extract and analyze the biosensors from intestinal samples. The induction of GFP in the biosensor cells extracted from the animals...

  20. Application of a Bacillus subtilis Whole-Cell Biosensor (PliaI-lux) for the Identification of Cell Wall Active Antibacterial Compounds.

    Science.gov (United States)

    Kobras, Carolin Martina; Mascher, Thorsten; Gebhard, Susanne

    2017-01-01

    Whole-cell biosensors, based on the visualization of a reporter strain's response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, i.e., the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. We here describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances. The system is based on the well-characterized promoter P liaI , which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple "spot-on-lawn" assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.

  1. Bacterial Sunscreen: Layer-by-Layer Deposition of UV-Absorbing Polymers on Whole-Cell Biosensors (POSTPRINT)

    Science.gov (United States)

    2012-06-13

    Morozov, M. V.; Tazetdinova, D. I.; Alimova, F. K.; Hilmutdinov, A. K.; Zhdanov, R. I.; Kahraman, M.; Culha, M. Living Fungi Cells Encapsulated in...Riboswitches Based on Ligand-Dependent Modulation of Internal Ribosome Entry in Wheat Germ Extract and their Applications as Label-Free Biosensors. RNA 2011

  2. 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

  3. Application of a bacterial whole cell biosensor for the rapid detection of cytotoxicity in heavy metal contaminated seawater.

    Science.gov (United States)

    Cui, Zhisong; Luan, Xiao; Jiang, Huichao; Li, Qian; Xu, Guangfei; Sun, Chengjun; Zheng, Li; Song, Yizhi; Davison, Paul A; Huang, Wei E

    2018-06-01

    A toxicity biosensor Acinetobacter baylyi Tox2 was constructed with the host strain A. baylyi ADP1 harboring a new and medium-copy-number plasmid pWH1274_lux, and was applied to detect the cytotoxicity of heavy metal contaminated seawater. The gene cassette luxCDABE was controlled by constitutively expressed promoter P tet on pWH1274_lux and the bioluminescence intensity of the biosensor reduces in proportional to the concentrations of toxic compounds. A. baylyi Tox2 exhibits tolerance to salinity, hence it is applicable to seawater samples. A. baylyi Tox2 and Mugilogobius chulae were exposed to different concentrations of heavy metals (Hg 2+ , Zn 2+ , Cu 2+ , and Cd 2+ ) in artificial seawater for performance comparison and Pearson correlation analysis showed a significant correlation (p heavy metal contaminated seawater. Furthermore, A. baylyi Tox2 was used to evaluate cytotoxicity of field-collected seawater samples. The results indicate that there was a significant correlation between the luminescence inhibition ratio (IR) of A. baylyi Tox2 and heavy metal concentrations detected by ICP-MS in the samples. Two seawater samples, which contained a high concentration of total heavy metals, exhibited stronger cytotoxicity than the samples containing low concentrations of heavy metals. In conclusion, A. baylyi Tox2 can be used as an alternative tool to aquatic animals for the evaluation of the cytotoxicity of heavy metal contamination in the marine environment. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

    Science.gov (United States)

    Parvez, Saba; Fu, Yuan; Li, Jiayang; Long, Marcus J C; Lin, Hong-Yu; Lee, Dustin K; Hu, Gene S; Aye, Yimon

    2015-01-14

    Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

  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. 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. Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

    DEFF Research Database (Denmark)

    Delvigne, Frank; Brognaux, Alison; Francis, Frédéric

    2011-01-01

    Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down......-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose...... fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great...

  9. Determination of Zinc, Cadmium and Lead Bioavailability in Contaminated Soils at the Single-Cell Level by a Combination of Whole-Cell Biosensors and Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Quentin Hurdebise

    2015-04-01

    Full Text Available Zinc, lead and cadmium are metallic trace elements (MTEs that are widespread in the environment and tend to accumulate in soils because of their low mobility and non-degradability. The purpose of this work is to evaluate the applicability of biosensors as tools able to provide data about the bioavailability of such MTEs in contaminated soils. Here, we tested the genetically-engineered strain Escherichia coli pPZntAgfp as a biosensor applicable to the detection of zinc, lead and cadmium by the biosynthesis of green fluorescent protein (GFP accumulating inside the cells. Flow cytometry was used to investigate the fluorescence induced by the MTEs. A curvilinear response to zinc between 0 and 25 mg/L and another curvilinear response to cadmium between 0 and 1.5 mg/L were highlighted in liquid media, while lead did not produce exploitable results. The response relating to a Zn2+/Cd2+ ratio of 10 was further investigated. In these conditions, E. coli pPZntAgfp responded to cadmium only. Several contaminated soils with a Zn2+/Cd2+ ratio of 10 were analyzed with the biosensor, and the metallic concentrations were also measured by atomic absorption spectroscopy. Our results showed that E. coli pPZntAgfp could be used as a monitoring tool for contaminated soils being processed.

  10. 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

  11. Typing of Typhoidal Salmonella Using Extraction of Water Soluble Whole Cell Proteins and Analysing by SDS-PAGE

    Directory of Open Access Journals (Sweden)

    R. Yousefi Mashouf

    2005-10-01

    Full Text Available Introduction & Objective : Salmonella is one of the most important genus of Enterobacteriacea family. The aim of this study was typing of typhoidal Salmonella by SDS-PAGE and comparing the results with those of serotyping method.Materials and Methods: In this study, 4 reference strains of Salmonella species, 5 reference strains of Enterobacteriacea family and 100 clinical isolates of Salmonella that were previously collected from laboratories of Hamadan medical centers were studied. Serotyping of strains were performed by Biomereux and Difco monovalent antisera. Whole-cell proteins of strains were also separated on 10% poly acrylamide gel. Gels were stained by Coomassie Brilliant Blue and analyzed by densitometry. Results: Of 100 cases of Salmonella species, 43 cases (43% were S. typhi, 20 cases (20% were S. typhymurium, 12 cases (12% were S. para typhi B, 10 cases (10% were S. para typhi C, S. para typhi A 1 case (1% and other cases were non-typhoidal Salmonella. The results of serotyping were compared with the results obtained by SDS-PAGE. Many protein bands from 220 KDa to 18.5 KDa were detected by SDS-PAGE and they were used to differentiate the strains. S. typhi serotypes were divided into 5 sub-species and S. para typhi B and C were divided each into 3 sub-species. Protein profiles of the reference strains of Salmonella were compared with protein profiles of Enterobacteriaceae species and showed some differences in major protein bands, however, they had a very similar protein band in 43 KDa area. Conclusion: Since our data was able to divide Salmonella species to sub-types and differentiate them from Enterobacteriacea species, we concluded that analsying SDS-PAGE profile of water soluble whole-cell proteins can be used for typing of these organisms and it is comparble with serotyping, nevertheless, further researches are needed to establish SDS-PAGE method and to replace it with serotyping method.

  12. Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering

    Directory of Open Access Journals (Sweden)

    Mitsuyoshi Ueda

    2013-09-01

    Full Text Available Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called “arming technology”, can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.

  13. Arming Technology in Yeast-Novel Strategy for Whole-cell Biocatalyst and Protein Engineering.

    Science.gov (United States)

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2013-09-09

    Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called "arming technology", can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.

  14. Avoiding acidic region streaking in two-dimensional gel electrophoresis: case study with two bacterial whole cell protein extracts.

    Science.gov (United States)

    Roy, Arnab; Varshney, Umesh; Pal, Debnath

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  15. Applications of whole-cell bacterial sensors in biotechnology and environmental science

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, Kiyohito [Osaka Univ., Suita (Japan). Graduate School of Pharmaceutical Sciences

    2007-01-15

    Biosensors have major advantages over chemical or physical analyses with regard to specificity, sensitivity, and portability. Recently, many types of whole-cell bacterial biosensors have been developed using recombinant DNA technology. The bacteria are genetically engineered to respond to the presence of chemicals or physiological stresses by synthesizing a reporter protein, such as luciferase, {beta}-galactosidase, or green fluorescent protein. In addition to an overview of conventional biosensors, this minireview discusses a novel type of biosensor using a photosynthetic bacterium as the sensor strain and the crtA gene, which is responsible for carotenoid synthesis, as the reporter. Since bacteria possess a wide variety of stress-response mechanisms, including antioxidation, heat-shock responses, nutrient-starvation, and membrane-damage responses, DNA response elements for several stress-response proteins can be fused with various reporter genes to construct a versatile set of bacterial biosensors for a variety of analytes. Portable biosensors for on-site monitoring have been developed using a freeze-dried biosensing strain, and cell array biosensors have been designed for high-throughput analysis. Moreover, in the future, the use of single-cell biosensors will permit detailed analyses of samples. Signals from such sensors could be detected with digital imaging, epifluorescence microscopy, and/or flow cytometry. (orig.)

  16. 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)

  17. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Jørgensen, K.; Christensen, H.

    1997-01-01

    Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing...... methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole- cell protein profiling and ribotyping......, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently...

  18. 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

  19. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

    Science.gov (United States)

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-07-18

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process.

  20. Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.

    Science.gov (United States)

    Bodenberger, Nicholas; Kubiczek, Dennis; Paul, Patrick; Preising, Nico; Weber, Lukas; Bosch, Ramona; Hausmann, Rudolf; Gottschalk, Kay-Eberhard; Rosenau, Frank

    2017-03-01

    Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100 μm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150 μM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.

  1. 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.

  2. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    Science.gov (United States)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  3. Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder

    NARCIS (Netherlands)

    Haasnoot, W.; Olieman, K.; Cazemier, G.; Verheijen, R.

    2001-01-01

    The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders.

  4. Protein-modified nanocrystalline diamond thin films for biosensor applications.

    Science.gov (United States)

    Härtl, Andreas; Schmich, Evelyn; Garrido, Jose A; Hernando, Jorge; Catharino, Silvia C R; Walter, Stefan; Feulner, Peter; Kromka, Alexander; Steinmüller, Doris; Stutzmann, Martin

    2004-10-01

    Diamond exhibits several special properties, for example good biocompatibility and a large electrochemical potential window, that make it particularly suitable for biofunctionalization and biosensing. Here we show that proteins can be attached covalently to nanocrystalline diamond thin films. Moreover, we show that, although the biomolecules are immobilized at the surface, they are still fully functional and active. Hydrogen-terminated nanocrystalline diamond films were modified by using a photochemical process to generate a surface layer of amino groups, to which proteins were covalently attached. We used green fluorescent protein to reveal the successful coupling directly. After functionalization of nanocrystalline diamond electrodes with the enzyme catalase, a direct electron transfer between the enzyme's redox centre and the diamond electrode was detected. Moreover, the modified electrode was found to be sensitive to hydrogen peroxide. Because of its dual role as a substrate for biofunctionalization and as an electrode, nanocrystalline diamond is a very promising candidate for future biosensor applications.

  5. Self-Assembly of Protein Nanostructures to Enhance Biosensor Sensitivity

    Science.gov (United States)

    Olsen, Bradley; Dong, Xuehui; Obermeyer, Allie

    The Langmuir adsorption isotherm predicts that the number of bound species on a surface at a given concentration will be directly proportional to the number of binding sites on the surface. Therefore, the number of binding events in a biosensor may be increased at a given analyte concentration if the surface density of binding domains is increased. Here, we demonstrate the formation of block copolymers where one block is a human IgG antibody or a nanobody and self-assemble these molecules into nanostructured films with a high density of binding sites. The type of nanostructure formed and the rate of transport through the protein-polymer layers are explored as a function of coil fraction of the protein-polymer conjugate block copolymers, showing optima for transport and assembly that depend upon the identity of the protein. For small enough analytes, binding to the antibodies and nanobodies is linear with film thickness, indicating that the entire film is accessible. Consistent with the enhanced number of binding sites and the prediction of the Langmuir isotherm, the films improve sensitivity by several orders of magnitude relative to chemisorbed protein layers used in current sensor designs. Current research is integrating this new material technology into prototype sensors. Work supported by the Air Force Office of Scientific Reesearch (AFOSR).

  6. 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...

  7. Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

    Science.gov (United States)

    Ast, Cindy; Foret, Jessica; Oltrogge, Luke M; De Michele, Roberto; Kleist, Thomas J; Ho, Cheng-Hsun; Frommer, Wolf B

    2017-09-05

    Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

  8. Combination of pneumococcal surface protein A (PspA with whole cell pertussis vaccine increases protection against pneumococcal challenge in mice.

    Directory of Open Access Journals (Sweden)

    Maria Leonor S Oliveira

    Full Text Available Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP that is currently part of the DTP (diphtheria-tetanus-pertussis vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wP(low--a new generation vaccine that contains low levels of B. pertussis LPS--conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wP(low vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTP(low protected mice against challenge with two

  9. Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

    Science.gov (United States)

    Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

    2016-01-01

    We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

  10. NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis.

    Science.gov (United States)

    Megger, Dominik A; Pott, Leona L; Rosowski, Kristin; Zülch, Birgit; Tautges, Stephanie; Bracht, Thilo; Sitek, Barbara

    2017-01-01

    Tandem mass tags (TMT) are usually introduced at the levels of isolated proteins or peptides. Here, for the first time, we report the labeling of whole cells and a critical evaluation of its performance in comparison to conventional labeling approaches. The obtained results indicated that TMT protein labeling using intact cells is generally possible, if it is coupled to a subsequent enrichment using anti-TMT antibody. The quantitative results were similar to those obtained after labeling of isolated proteins and both were found to be slightly complementary to peptide labeling. Furthermore, when using NHS-based TMT, no specificity towards cell surface proteins was observed in the case of cell labeling. In summary, the conducted study revealed first evidence for the general possibility of TMT cell labeling and highlighted limitations of NHS-based labeling reagents. Future studies should therefore focus on the synthesis and investigation of membrane impermeable TMTs to increase specificity towards cell surface proteins.

  11. Tailoring odorant-binding protein coatings characteristics for surface acoustic wave biosensor development

    Energy Technology Data Exchange (ETDEWEB)

    Di Pietrantonio, F., E-mail: fabio.dp@idasc.cnr.it [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Benetti, M. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Dinca, V. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania); Cannatà, D. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Verona, E. [Institute for Photonics and Nanotechnologies, National Research Council of Italy, Via del Cineto Romano 42, 00156 Rome (Italy); D’Auria, S. [Institute of Protein Biochemistry, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples (Italy); Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania)

    2014-05-01

    In this study, wild type bovine odorant-binding proteins (wtbOBPs) were deposited by matrix-assisted pulsed laser evaporation (MAPLE) and utilized as active material on surface acoustic wave (SAW) biosensors. Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) were used to determine the chemical, morphological characteristics of the protein thin films. The FTIR data demonstrates that the functional groups of wtbOBPs do not suffer significant changes in the MAPLE-deposited films when compared to the reference one. The topographical studies show that the homogeneity, density and the roughness of the coatings are related mainly to the laser parameters (fluence and number of pulses). SAW biosensor responses to different concentrations of R-(–)-1-octen-3-ol (octenol) and R-(–)-carvone (carvone) were evaluated. The obtained sensitivities, achieved through the optimization of deposition parameters, demonstrated that MAPLE is a promising deposition technique for SAW biosensor implementation.

  12. Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

    Science.gov (United States)

    Meng Zhang; Peh, Jessie; Hergenrother, Paul J; Cunningham, Brian T

    2014-01-01

    High throughput screening of protein-small molecule binding interactions using label-free optical biosensors is challenging, as the detected signals are often similar in magnitude to experimental noise. Here, we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with sub-picometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets with binding affinities or inhibition constants in the sub-nanomolar to low micromolar range. The demonstrated ability to perform detection in the presence of several interfering compounds opens the potential for increasing the throughput of the approach. As an example application, we performed a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II (CA II), in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

  13. Protein Biosensors Based on Polymer Nanowires, Carbon Nanotubes and Zinc Oxide Nanorods

    Directory of Open Access Journals (Sweden)

    Taeksoo Ji

    2011-05-01

    Full Text Available The development of biosensors using electrochemical methods is a promising application in the field of biotechnology. High sensitivity sensors for the bio-detection of proteins have been developed using several kinds of nanomaterials. The performance of the sensors depends on the type of nanostructures with which the biomaterials interact. One dimensional (1-D structures such as nanowires, nanotubes and nanorods are proven to have high potential for bio-applications. In this paper we review these three different kinds of nanostructures that have attracted much attention at recent times with their great performance as biosensors. Materials such as polymers, carbon and zinc oxide have been widely used for the fabrication of nanostructures because of their enhanced performance in terms of sensitivity, biocompatibility, and ease of preparation. Thus we consider polymer nanowires, carbon nanotubes and zinc oxide nanorods for discussion in this paper. We consider three stages in the development of biosensors: (a fabrication of biomaterials into nanostructures, (b alignment of the nanostructures and (c immobilization of proteins. Two different methods by which the biosensors can be developed at each stage for all the three nanostructures are examined. Finally, we conclude by mentioning some of the major challenges faced by many researchers who seek to fabricate biosensors for real time applications.

  14. Non-specific protein-surface interactions in the context of particle based biosensors

    NARCIS (Netherlands)

    Kemper, M.

    2013-01-01

    Biosensors are compact devices that can be used at the point-of-care to measure low quantities of biomarkers in a complex body fluid like blood or saliva. Many biomarkers in the human body are protein molecules, for example the biomarkers that are used for the diagnosis of myocardial infarction.

  15. Making bio-sense of toxicity: new developments in whole-cell biosencors

    DEFF Research Database (Denmark)

    Sørensen, Søren Johannes; Burmølle, Mette; Hansen, Lars Hestbjerg

    2006-01-01

    Bacterial whole-cell biosensors are very useful for toxicity measurements of various samples. Semi-specific biosensors, containing fusions of stress-regulated promoters and reporter genes, have several advantages over the traditional, general biosensors that are based on constitutively expressed ....... The application of in situ inoculation and single-cell detection, combined with the introduction of new reporter genes and refined detection equipment, could lead to the extensive use of semi-specific, stress-responsive biosensors for toxicity estimations in the future....... reporter genes. Furthermore, semi-specific biosensors are constantly being refined to lower their sensitivity and, in combination, are able to detect a wide range of toxic agents. However, the requirement for a positive response of these biosensors to toxicants can result in false-negative responses...

  16. Protein Adsorption onto Nanomaterials for the Development of Biosensors and Analytical Devices: A Review

    Science.gov (United States)

    Bhakta, Samir A.; Evans, Elizabeth; Benavidez, Tomás E.; Garcia, Carlos D.

    2014-01-01

    An important consideration for the development of biosensors is the adsorption of the bio recognition element to the surface of a substrate. As the first step in the immobilization process, adsorption affects most immobilization routes and much attention is given into the research of this process to maximize the overall activity of the bio sensor. The use of nanomaterials, specifically nanoparticles and nanostructured films, offers advantageous properties that can be fine-tuned for interaction with specific proteins to maximize activity, minimize structural changes, and enhance the catalytic step. In the biosensor field, protein-nanomaterial interactions are an emerging trend that span across many disciplines. This review addresses recent publications about the proteins most frequently used, their most relevant characteristics, and the conditions required to adsorb them to nanomaterials. When relevant and available, subsequent analytical figures of merits are discussed for selected biosensors. The general trend amongst the research papers allows concluding that the use of nanomaterials has already provided significant improvements in the analytical performance of many biosensors and that this research field will continue to grow. PMID:25892065

  17. Protein-Based Graphene Biosensors: Optimizing Artificial Chemoreception in Bilayer Lipid Membranes

    Directory of Open Access Journals (Sweden)

    Christina G. Siontorou

    2016-09-01

    Full Text Available Proteinaceous moieties are critical elements in most detection systems, including biosensing platforms. Their potential is undoubtedly vast, yet many issues regarding their full exploitation remain unsolved. On the other hand, the biosensor formats with the higher marketability probabilities are enzyme in nature and electrochemical in concept. To no surprise, alternative materials for hosting catalysis within an electrode casing have received much attention lately to demonstrate a catalysis-coated device. Graphene and ZnO are presented as ideal materials to modify electrodes and biosensor platforms, especially in protein-based detection. Our group developed electrochemical sensors based on these nanomaterials for the sensitive detection of cholesterol using cholesterol oxidase incorporated in stabilized lipid films. A comparison between the two platforms is provided and discussed. In a broader sense, the not-so-remote prospect of quickly assembling a protein-based flexible biosensing detector to fulfill site-specific requirements is appealing to both university researchers and industry developers.

  18. Construction and Potential Applications of Biosensors for Proteins in Clinical Laboratory Diagnosis.

    Science.gov (United States)

    Liu, Xuan; Jiang, Hui

    2017-12-04

    Biosensors for proteins have shown attractive advantages compared to traditional techniques in clinical laboratory diagnosis. In virtue of modern fabrication modes and detection techniques, various immunosensing platforms have been reported on basis of the specific recognition between antigen-antibody pairs. In addition to profit from the development of nanotechnology and molecular biology, diverse fabrication and signal amplification strategies have been designed for detection of protein antigens, which has led to great achievements in fast quantitative and simultaneous testing with extremely high sensitivity and specificity. Besides antigens, determination of antibodies also possesses great significance for clinical laboratory diagnosis. In this review, we will categorize recent immunosensors for proteins by different detection techniques. The basic conception of detection techniques, sensing mechanisms, and the relevant signal amplification strategies are introduced. Since antibodies and antigens have an equal position to each other in immunosensing, all biosensing strategies for antigens can be extended to antibodies under appropriate optimizations. Biosensors for antibodies are summarized, focusing on potential applications in clinical laboratory diagnosis, such as a series of biomarkers for infectious diseases and autoimmune diseases, and an evaluation of vaccine immunity. The excellent performances of these biosensors provide a prospective space for future antibody-detection-based disease serodiagnosis.

  19. Network single-walled carbon nanotube biosensors for fast and highly sensitive detection of proteins

    International Nuclear Information System (INIS)

    Hu Pingan; Zhang Jia; Wen Zhenzhong; Zhang Can

    2011-01-01

    Detection of proteins is powerfully assayed in the diagnosis of diseases. A strategy for the development of an ultrahigh sensitivity biosensor based on a network single-walled carbon nanotube (SWNT) field-effect transistor (FET) has been demonstrated. Metallic SWNTs (m-SWNTs) in the network nanotube FET were selectively removed or cut via a carefully controlled procedure of electrical break-down (BD), and left non-conducting m-SWNTs which magnified the Schottky barrier (SB) area. This nanotube FET exhibited ultrahigh sensitivity and fast response to biomolecules. The lowest detection limit of 0.5 pM was achieved by exploiting streptavidin (SA) or a biotin/SA pair as the research model, and BD-treated nanotube biosensors had a 2 x 10 4 -fold lower minimum detectable concentration than the device without BD treatment. The response time is in the range of 0.3-3 min.

  20. Whole-cell protein profiles are useful for distinguishing enterococcal species recovered from clinical specimens Los perfiles de proteínas totales son útiles para distinguir especies de enterococos recuperados de muestras clínicas

    Directory of Open Access Journals (Sweden)

    R. Massa

    2007-12-01

    Full Text Available Whole-cell protein analysis was performed for differentiating 150 enterococcal isolates to the species level, which had previously been identified by extended phenotypic conventional tests. Whole-cell protein profile (WCPP showed a high degree of similarity within species and comparison between species revealed important differences in band profiles. All Enterococcus faecalis and Enterococcus faecium isolates were properly located into their corresponding species, regardless of their clinical source and susceptibility pattern. Moreover, WCPP allowed relocation of some isolates that had erroneously been identified by the usual conventional scheme (i.e. two atypical arginine-negative E. faecalis isolates. WCPP proved to be a simple method to ascertain the various enterococcal species, especially those other than E. faecalis, and may be a suitable tool for high-complexity or reference clinical laboratories.La comparación del perfil de proteínas totales permitió agrupar 150 aislamientos de enterococos dentro de la especie en la que habían sido ubicados por el esquema convencional de pruebas bioquímicas. Los patrones de proteínas totales, comparados visualmente, se mantuvieron con alto grado de similitud intraespecie y revelaron diferencias notorias en la comparación interespecie. Todos los aislamientos de Enterococcus faecalis y Enterococcus faecium, independientemente de los sitios de aislamiento, cuadro clínico del paciente, biotipo o antibiotipo, fueron fácilmente encuadrados en su especie. Asimismo, el estudio del perfil de proteínas totales de enterococos permitió reubicar taxonómicamente aislamientos que habían sido incorrectamente identificados por los métodos bioquímicos convencionales, como por ejemplo dos aislamientos atípicos de E. faecalis arginina negativos. Dado que la metodología empleada es económica y rápida, la comparación de perfiles de proteínas totales en SDS-PAGE podría ser considerada una herramienta

  1. Biosensor immunoassay for traces of hazelnut protein in olive oil

    NARCIS (Netherlands)

    Bremer, M.G.E.G.; Smits, N.G.E.; Haasnoot, W.

    2009-01-01

    The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct

  2. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

    Science.gov (United States)

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

    2014-10-01

    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Renu; Hong, Seongkyeol [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Jang, Jaesung, E-mail: jjang@unist.ac.kr [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); School of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of)

    2015-01-01

    Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO{sub 2} aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO{sub 2}) aerosols (a mixture of solid and gaseous CO{sub 2}), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL{sup −1}) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO{sub 2} aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors.

  4. Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

    International Nuclear Information System (INIS)

    Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

    2015-01-01

    Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO 2 aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO 2 ) aerosols (a mixture of solid and gaseous CO 2 ), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL −1 ) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO 2 aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors

  5. Post hoc support vector machine learning for impedimetric biosensors based on weak protein-ligand interactions.

    Science.gov (United States)

    Rong, Y; Padron, A V; Hagerty, K J; Nelson, N; Chi, S; Keyhani, N O; Katz, J; Datta, S P A; Gomes, C; McLamore, E S

    2018-04-30

    Impedimetric biosensors for measuring small molecules based on weak/transient interactions between bioreceptors and target analytes are a challenge for detection electronics, particularly in field studies or in the analysis of complex matrices. Protein-ligand binding sensors have enormous potential for biosensing, but achieving accuracy in complex solutions is a major challenge. There is a need for simple post hoc analytical tools that are not computationally expensive, yet provide near real time feedback on data derived from impedance spectra. Here, we show the use of a simple, open source support vector machine learning algorithm for analyzing impedimetric data in lieu of using equivalent circuit analysis. We demonstrate two different protein-based biosensors to show that the tool can be used for various applications. We conclude with a mobile phone-based demonstration focused on the measurement of acetone, an important biomarker related to the onset of diabetic ketoacidosis. In all conditions tested, the open source classifier was capable of performing as well as, or better, than the equivalent circuit analysis for characterizing weak/transient interactions between a model ligand (acetone) and a small chemosensory protein derived from the tsetse fly. In addition, the tool has a low computational requirement, facilitating use for mobile acquisition systems such as mobile phones. The protocol is deployed through Jupyter notebook (an open source computing environment available for mobile phone, tablet or computer use) and the code was written in Python. For each of the applications, we provide step-by-step instructions in English, Spanish, Mandarin and Portuguese to facilitate widespread use. All codes were based on scikit-learn, an open source software machine learning library in the Python language, and were processed in Jupyter notebook, an open-source web application for Python. The tool can easily be integrated with the mobile biosensor equipment for rapid

  6. Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

    Directory of Open Access Journals (Sweden)

    Juan A. González-Vera

    2015-11-01

    Full Text Available Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

  7. 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.

  8. Clinical validation of integrated nucleic acid and protein detection on an electrochemical biosensor array for urinary tract infection diagnosis.

    Directory of Open Access Journals (Sweden)

    Ruchika Mohan

    Full Text Available BACKGROUND: Urinary tract infection (UTI is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. METHODOLOGY/PRINCIPAL FINDINGS: The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both. CONCLUSION/SIGNIFICANCE: We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for

  9. CFD Modeling of Chamber Filling in a Micro-Biosensor for Protein Detection.

    Science.gov (United States)

    Islamov, Meiirbek; Sypabekova, Marzhan; Kanayeva, Damira; Rojas-Solórzano, Luis

    2017-10-03

    Tuberculosis (TB) remains one of the main causes of human death around the globe. The mortality rate for patients infected with active TB goes beyond 50% when not diagnosed. Rapid and accurate diagnostics coupled with further prompt treatment of the disease is the cornerstone for controlling TB outbreaks. To reduce this burden, the existing gap between detection and treatment must be addressed, and dedicated diagnostic tools such as biosensors should be developed. A biosensor is a sensing micro-device that consists of a biological sensing element and a transducer part to produce signals in proportion to quantitative information about the binding event. The micro-biosensor cell considered in this investigation is designed to operate based on aptamers as recognition elements against Mycobacterium tuberculosis secreted protein MPT64, combined in a microfluidic-chamber with inlet and outlet connections. The microfluidic cell is a miniaturized platform with valuable advantages such as low cost of analysis with low reagent consumption, reduced sample volume, and shortened processing time with enhanced analytical capability. The main purpose of this study is to assess the flooding characteristics of the encapsulated microfluidic cell of an existing micro-biosensor using Computational Fluid Dynamics (CFD) techniques. The main challenge in the design of the microfluidic cell lies in the extraction of entrained air bubbles, which may remain after the filling process is completed, dramatically affecting the performance of the sensing element. In this work, a CFD model was developed on the platform ANSYS-CFX using the finite volume method to discretize the domain and solving the Navier-Stokes equations for both air and water in a Eulerian framework. Second-order space discretization scheme and second-order Euler Backward time discretization were used in the numerical treatment of the equations. For a given inlet-outlet diameter and dimensions of an in-house built cell chamber

  10. Self-cleaned electrochemical protein imprinting biosensor basing on a thermo-responsive memory hydrogel.

    Science.gov (United States)

    Wei, Yubo; Zeng, Qiang; Hu, Qiong; Wang, Min; Tao, Jia; Wang, Lishi

    2018-01-15

    Herein, the self-cleaned electrochemical protein imprinting biosensor basing on a thermo-responsive memory hydrogel was constructed on a glassy carbon electrode (GCE) with a free radical polymerization method. Combining the advantages of thermo-responsive molecular imprinted polymers and electrochemistry, the resulted biosensor presents a novel self-cleaned ability for bovine serum albumin (BSA) in aqueous media. As a temperature controlled gate, the hydrogel film undergoes the adsorption and desorption of BSA basing on a reversible structure change with the external temperature stimuli. In particular, these processes have been revealed by the response of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of electroactive [Fe(CN) 6 ] 3-/4- . The results have been supported by the evidences of scanning electron microscopy (SEM) and contact angles measurements. Under the optimal conditions, a wide detection range from 0.02μmolL -1 to 10μmolL -1 with a detection limit of 0.012 μmolL -1 (S/N = 3) was obtained for BSA. This proposed BSA sensor also possesses high selectivity, excellent stability, acceptable recovery and good reproducibility in its practical applications. Copyright © 2017. Published by Elsevier B.V.

  11. 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

  12. 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.

  13. Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

    International Nuclear Information System (INIS)

    Bernard, C; Leduc, A; Barbeau, J; Saoudi, B; Yahia, L'H; Crescenzo, G De

    2006-01-01

    Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen-oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty

  14. Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

    Science.gov (United States)

    Bernard, C.; Leduc, A.; Barbeau, J.; Saoudi, B.; Yahia, L'H.; DeCrescenzo, G.

    2006-08-01

    Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen-oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.

  15. Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors

    International Nuclear Information System (INIS)

    Liao, V.H.-C.; Chien, M.-T.; Tseng, Y.-Y.; Ou, K.-L.

    2006-01-01

    A green fluorescent protein (GFP)-based bacterial biosensor Escherichia coli DH5α (pVLCD1) was developed based on the expression of gfp under the control of the cad promoter and the cadC gene of Staphylococcus aureus plasmid pI258. DH5α (pVLCD1) mainly responded to Cd(II), Pb(II), and Sb(III), the lowest detectable concentrations being 0.1 nmol L -1 , 10 nmol L -1 , and 0.1 nmol L -1 , respectively, with 2 h exposure. The biosensor was field-tested to measure the relative bioavailability of the heavy metals in contaminated sediments and soil samples. The results showed that the majority of heavy metals remained adsorbed to soil particles: Cd(II)/Pb(II) was only partially available to the biosensor in soil-water extracts. Our results demonstrate that the GFP-based bacterial biosensor is useful and applicable in determining the bioavailability of heavy metals with high sensitivity in contaminated sediment and soil samples and suggests a potential for its inexpensive application in environmentally relevant sample tests. - Nonpathogenic GFP-based bacterial biosensor is applicable in determining the bioavailability of heavy metals in environmental samples

  16. 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.

  17. Signal amplification in electrochemical detection of buckwheat allergenic protein using field effect transistor biosensor by introduction of anionic surfactant

    Directory of Open Access Journals (Sweden)

    Sho Hideshima

    2016-03-01

    Full Text Available Food allergens, especially buckwheat proteins, sometimes induce anaphylactic shock in patients after ingestion. Development of a simple and rapid screening method based on a field effect transistor (FET biosensor for food allergens in food facilities or products is in demand. In this study, we achieved the FET detection of a buckwheat allergenic protein (BWp16, which is not charged enough to be electrically detected by FET biosensors, by introducing additional negative charges from anionic surfactants to the target proteins. A change in the FET characteristics reflecting surface potential caused by the adsorption of target charged proteins was observed when the target sample was coupled with the anionic surfactant (sodium dodecyl sulfate; SDS, while no significant response was detected without any surfactant treatment. It was suggested that the surfactant conjugated with the protein could be useful for the charge amplification of the target proteins. The surface plasmon resonance analysis revealed that the SDS-coupled proteins were successfully captured by the receptors immobilized on the sensing surface. Additionally, we obtained the FET responses at various concentrations of BWp16 ranging from 1 ng/mL to 10 μg/mL. These results suggest that a signal amplification method for FET biosensing is useful for allergen detection in the food industry. Keywords: Field effect transistor biosensor, Food allergen, Signal amplification, Ionic surfactant, Intrinsic charge

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

    OpenAIRE

    Ramesh K. Jha; Jeremy M. Bingen; Christopher W. Johnson; Theresa L. Kern; Payal Khanna; Daniel S. Trettel; Charlie E.M. Strauss; Gregg T. Beckham; Taraka Dale

    2018-01-01

    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 sens...

  19. Handheld highly selective plasmonic chem/biosensor using engineered binding proteins for extreme conformational changes

    Science.gov (United States)

    Kosciolek, Derek J.; Sonar, Ajay; Lepak, Lori A.; Schnatz, Peter; Bendoym, Igor; Brown, Mia C.; Koder, Ronald L.; Crouse, David T.

    2017-08-01

    In this project we develop a handheld, portable, highly selective and sensitive chem/biosensor that has potential applications in both airborne and water-based environmental sensing. The device relies on a plasmonic chip of subwavelength-scale periodic gold rods engineered to resonate in the near infrared. The chip is functionalized with a novel class of proteins that exhibit large conformational changes upon binding to a specific target analyte. The subsequent change in local refractive index near the surface of the gold is one to two orders of magnitude greater than current conventional methods, which produces a readily measurable 5 to 10 percent difference in light transmission. This allows us to forgo traditional, bulky tabletop setups in favor of a compact form factor. Using commercially available optics to construct a transmission-based optical train, measured changes in bulk refractive index are presented here. While synthesis of binding protein efforts are focused on heme as analyte for proof of concept validation, the functionalized protein can be engineered to pair with a wide variety of analytes with minimal alterations to the plasmonic chip or device design. Such flexibility allows for this device to potentially meet the needs of first responders and health care professionals in a multitude of scenarios.

  20. Matrix-insensitive protein assays push the limits of biosensors in medicine

    DEFF Research Database (Denmark)

    Gaster, Richard S; Hall, Drew A; Nielsen, Carsten Haagen

    2009-01-01

    Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse...... magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense....

  1. 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....

  2. Label-free SnO2 nanowire FET biosensor for protein detection

    Science.gov (United States)

    Jakob, Markus H.; Dong, Bo; Gutsch, Sebastian; Chatelle, Claire; Krishnaraja, Abinaya; Weber, Wilfried; Zacharias, Margit

    2017-06-01

    Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 μl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.

  3. Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform | Office of Cancer Genomics

    Science.gov (United States)

    The vast datasets generated by next generation gene sequencing and expression profiling have transformed biological and translational research. However, technologies to produce large-scale functional genomics datasets, such as high-throughput detection of protein-protein interactions (PPIs), are still in early development. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform featured with both high sensitivity and robustness in a mammalian cell environment remains to be established.

  4. Strategies for "wiring" redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology.

    Science.gov (United States)

    Nöll, Tanja; Nöll, Gilbert

    2011-07-01

    In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.

  5. Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers.

    Science.gov (United States)

    Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

    2015-04-15

    Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

  6. Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

    Directory of Open Access Journals (Sweden)

    Jihea Moon

    2015-04-01

    Full Text Available Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

  7. Dual-gate polysilicon nanoribbon biosensors enable high sensitivity detection of proteins

    International Nuclear Information System (INIS)

    Zeimpekis, I; Sun, K; Hu, C; Ditshego, N M J; De Planque, M R R; Chong, H M H; Morgan, H; Ashburn, P; Thomas, O

    2016-01-01

    We demonstrate the advantages of dual-gate polysilicon nanoribbon biosensors with a comprehensive evaluation of different measurement schemes for pH and protein sensing. In particular, we compare the detection of voltage and current changes when top- and bottom-gate bias is applied. Measurements of pH show that a large voltage shift of 491 mV pH"−"1 is obtained in the subthreshold region when the top-gate is kept at a fixed potential and the bottom-gate is varied (voltage sweep). This is an improvement of 16 times over the 30 mV pH"−"1 measured using a top-gate sweep with the bottom-gate at a fixed potential. A similar large voltage shift of 175 mV is obtained when the protein avidin is sensed using a bottom-gate sweep. This is an improvement of 20 times compared with the 8.8 mV achieved from a top-gate sweep. Current measurements using bottom-gate sweeps do not deliver the same signal amplification as when using bottom-gate sweeps to measure voltage shifts. Thus, for detecting a small signal change on protein binding, it is advantageous to employ a double-gate transistor and to measure a voltage shift using a bottom-gate sweep. For top-gate sweeps, the use of a dual-gate transistor enables the current sensitivity to be enhanced by applying a negative bias to the bottom-gate to reduce the carrier concentration in the nanoribbon. For pH measurements, the current sensitivity increases from 65% to 149% and for avidin sensing it increases from 1.4% to 2.5%. (paper)

  8. 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.

  9. The ITO-capped WO3 nanowires biosensor based on field-effect transistor in label-free protein sensing

    International Nuclear Information System (INIS)

    Shariati, Mohsen

    2017-01-01

    The fabrication of ITO-capped WO 3 nanowires associated with their bio-sensing properties in field-effect transistor diagnostics basis as a biosensor has been reported. The bio-sensing property for manipulated nanowires elucidated that the grown nanostructures were very sensitive to protein. The ITO-capped WO 3 nanowires biosensor showed an intensive bio-sensing activity against reliable protein. Polylysine strongly charged bio-molecule was applied as model system to demonstrate the implementation of materialized biosensor. The employed sensing mechanism was 'label-free' and depended on bio-molecule's intrinsic charge. For nanowires synthesis, the vapor-liquid-solid mechanism was used. Nanowires were beyond a few hundred nanometers in lengths and around 15-20 nm in diameter, while the globe cap's size on the nanowires was around 15-25 nm. The indium tin oxide (ITO) played as catalyst in nanofabrication for WO 3 nanowires growth and had outstanding role in bio-sensing especially for bio-molecule adherence. In applied electric field presence, the fabricated device showed the great potential to enhance medical diagnostics. (orig.)

  10. Development of a biosensor protein bullet as a fluorescent method for fast detection of Escherichia coli in drinking water.

    Directory of Open Access Journals (Sweden)

    Ignacio Gutiérrez-Del-Río

    Full Text Available Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking

  11. Development of a biosensor protein bullet as a fluorescent method for fast detection of Escherichia coli in drinking water.

    Science.gov (United States)

    Gutiérrez-Del-Río, Ignacio; Marín, Laura; Fernández, Javier; Álvarez San Millán, María; Ferrero, Francisco Javier; Valledor, Marta; Campo, Juan Carlos; Cobián, Natalia; Méndez, Ignacio; Lombó, Felipe

    2018-01-01

    Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins) failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking water.

  12. Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

    Science.gov (United States)

    Chen, Mei-ling; Guo, Qin; Wang, Rui-zhi; Xu, Juan; Zhou, Chen-wei; Ruan, Hui; He, Guo-qing

    2011-07-01

    Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

  13. Manganese modified CdTe/CdS quantum dots as an immunoassay biosensor for the detection of Golgi protein-73.

    Science.gov (United States)

    Liu, Wei; Zhang, Aixia; Xu, Guanhong; Wei, Fangdi; Yang, Jing; Hu, Qin

    2016-01-05

    In this paper, a new fluorescence bioassay for Golgi protein-73 (GP73), a promising marker for monitoring liver tumor, was developed by using anti-GP73 antibody (GP73 Ab) capped quantum dots (QDs) coupled with protein A/G agarose beads in an attempt to improve the analysis time, cost and operation. First, carboxylic-functionalized Mn modified CdTe/CdS QDs were synthesized and covalently conjugated with GP73 Ab, then protein A/G agarose beads were specifically combined with the QDs-conjugated Ab to form the QDs-Ab-beads conjugate, which could capture and separate GP73 from the sample through simple centrifugation. It was found that the fluorescence intensity of the above QDs-Ab-beads biosensor could be specifically quenched by GP73 added. A simple, rapid and specific quantitative method for GP73 protein was proposed using the as-prepared QDs-Ab-beads as a biosensor. Under the optimized conditions, the calibration curve of the proposed assay showed good linearity with a correlation coefficient of 0.9935 in the concentration range of 20-150 ng/mL of GP73 protein. The limit of detection (defined as 3σ/K) was 10 ng/mL. The method built exhibited a great potential in the clinic test of GP73. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Fabrication and characterization of a chemically oxidized-nanostructured porous silicon based biosensor implementing orienting protein A.

    Science.gov (United States)

    Naveas, Nelson; Hernandez-Montelongo, Jacobo; Pulido, Ruth; Torres-Costa, Vicente; Villanueva-Guerrero, Raúl; Predestinación García Ruiz, Josefa; Manso-Silván, Miguel

    2014-03-01

    Nanostructured porous silicon (PSi) elicits as a very attractive material for future biosensing systems due to its high surface area, biocompatibility and well-established fabrication methods. In order to engineer its performance as a biosensor transducer platform, the density of immunoglobulins properly immobilized and oriented onto the surface needs to be optimized. In this work we fabricated and characterized a novel biosensing system focusing on the improvement of the biofunctionalization cascade. The system consists on a chemically oxidized PSi platform derivatized with 3-aminopropyltriethoxysilane (APTS) that is coupled to Staphylococcus protein A (SpA). The chemical oxidation has previously demonstrated to enhance the biofunctionalization process and here "by implementing SpA" a molecularly oriented immunosensor is achieved. The biosensor system is characterized in terms of its chemical composition, wettability and optical reflectance. Finally, this system is successfully exploited to develop a biosensor for detecting asymmetric dimethylarginine (ADMA), an endogenous molecule involved in cardiovascular diseases. Therefore, this work is relevant from the point of view of design and optimization of the biomolecular immobilization cascade on PSi surfaces with the added value of contribution to the development of new assays for detecting ADMA with a view on prevention of cardiovascular diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. WholeCellSimDB: a hybrid relational/HDF database for whole-cell model predictions.

    Science.gov (United States)

    Karr, Jonathan R; Phillips, Nolan C; Covert, Markus W

    2014-01-01

    Mechanistic 'whole-cell' models are needed to develop a complete understanding of cell physiology. However, extracting biological insights from whole-cell models requires running and analyzing large numbers of simulations. We developed WholeCellSimDB, a database for organizing whole-cell simulations. WholeCellSimDB was designed to enable researchers to search simulation metadata to identify simulations for further analysis, and quickly slice and aggregate simulation results data. In addition, WholeCellSimDB enables users to share simulations with the broader research community. The database uses a hybrid relational/hierarchical data format architecture to efficiently store and retrieve both simulation setup metadata and results data. WholeCellSimDB provides a graphical Web-based interface to search, browse, plot and export simulations; a JavaScript Object Notation (JSON) Web service to retrieve data for Web-based visualizations; a command-line interface to deposit simulations; and a Python API to retrieve data for advanced analysis. Overall, we believe WholeCellSimDB will help researchers use whole-cell models to advance basic biological science and bioengineering. http://www.wholecellsimdb.org SOURCE CODE REPOSITORY: URL: http://github.com/CovertLab/WholeCellSimDB. © The Author(s) 2014. Published by Oxford University Press.

  16. 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...

  17. 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.

  18. Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Hondroulis, Evangelia; Liu Chang; Li Chenzhong, E-mail: licz@fiu.edu [Nanobioengineering/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, 10555 West Flagler Street, Miami, FL 33174 (United States)

    2010-08-06

    A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

  19. Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

    Science.gov (United States)

    Hondroulis, Evangelia; Liu, Chang; Li, Chen-Zhong

    2010-08-01

    A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

  20. Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

    International Nuclear Information System (INIS)

    Hondroulis, Evangelia; Liu Chang; Li Chenzhong

    2010-01-01

    A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

  1. ZnO nanorod biosensor for highly sensitive detection of specific protein binding

    International Nuclear Information System (INIS)

    Kim, Jin Suk; Park, Won Il; Lee, Chul Ho; Yi, Gyu Chul

    2006-01-01

    We report on the fabrication of electrical biosensors based on functionalized ZnO nanorod surfaces with biotin for highly sensitive detection of biological molecules. Due to the clean interface and easy surface modification, the ZnO nanorod sensors can easily detect streptavidin binding down to a concentration of 25 nM, which is more sensitive than previously reported one-dimensional (1D) nanostructure electrical biosensors. In addition, the unique device structure with a micrometer-scale hole at the center of the ZnO nanorod's conducting channel reduces the leakage current from the aqueous solution, hence enhancing device sensitivity. Moreover, ZnO nanorod field-effect-transistor (FET) sensors may open up opportunities to create many other oxide nanorod electrical sensors for highly sensitive and selective real-time detection of a wide variety of biomolecules.

  2. 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.

  3. AlGaN/GaN High Electron Mobility Transistor-Based Biosensor for the Detection of C-Reactive Protein.

    Science.gov (United States)

    Lee, Hee Ho; Bae, Myunghan; Jo, Sung-Hyun; Shin, Jang-Kyoo; Son, Dong Hyeok; Won, Chul-Ho; Jeong, Hyun-Min; Lee, Jung-Hee; Kang, Shin-Won

    2015-07-28

    In this paper, we propose an AlGaN/GaN high electron mobility transistor (HEMT)-based biosensor for the detection of C-reactive protein (CRP) using a null-balancing circuit. A null-balancing circuit was used to measure the output voltage of the sensor directly. The output voltage of the proposed biosensor was varied by antigen-antibody interactions on the gate surface due to CRP charges. The AlGaN/GaN HFET-based biosensor with null-balancing circuit applied shows that CRP can be detected in a wide range of concentrations, varying from 10 ng/mL to 1000 ng/mL. X-ray photoelectron spectroscopy was carried out to verify the immobilization of self-assembled monolayer with Au on the gated region.

  4. AlGaN/GaN High Electron Mobility Transistor-Based Biosensor for the Detection of C-Reactive Protein

    Directory of Open Access Journals (Sweden)

    Hee Ho Lee

    2015-07-01

    Full Text Available In this paper, we propose an AlGaN/GaN high electron mobility transistor (HEMT-based biosensor for the detection of C-reactive protein (CRP using a null-balancing circuit. A null-balancing circuit was used to measure the output voltage of the sensor directly. The output voltage of the proposed biosensor was varied by antigen-antibody interactions on the gate surface due to CRP charges. The AlGaN/GaN HFET-based biosensor with null-balancing circuit applied shows that CRP can be detected in a wide range of concentrations, varying from 10 ng/mL to 1000 ng/mL. X-ray photoelectron spectroscopy was carried out to verify the immobilization of self-assembled monolayer with Au on the gated region.

  5. Yeast cell surface display for lipase whole cell catalyst and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  6. Phosphoglycerate kinase enhanced immunity of the whole cell of Streptococcus agalactiae in tilapia, Oreochromis niloticus.

    Science.gov (United States)

    Wang, Yi-Ting; Huang, Hsing-Yen; Tsai, Ming-An; Wang, Pei-Chi; Jiang, Bo-Huang; Chen, Shih-Chu

    2014-12-01

    Streptococcus agalactiae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of warmwater fish species. The outer-surface proteins in bacterial pathogens play an important role in pathogenesis. We evaluated the immunogenicity of two of the identified surface proteins namely phosphoglycerate kinase (PGK) and ornithine carbamoyl-transferase (OCT). PGK and OCT were over-expressed and purified from Escherichia coli and used as the subunit vaccines in tilapia. Tilapia immunized with the S. agalactiae modified bacteria vaccine (whole cell preparations with recombinant PGK and OCT proteins) individually were tested for the efficacy. OCT and PGK combined with WC had a higher survival rate. A high-level protection and significant specific antibody responses against S. agalactiae challenge was observed upon the vaccinated tilapia with the purified PGK protein and S. agalactiae whole cells. The specific antibody titer against S. agalactiae antigen suggested that increased antibody titers were correlated with post-challenge survival rate. Il-1β expression profile was higher in PGK + WC-treated group. Tnf-α expression in the PGK + WC group was significantly increased. Taken together, our results suggested the combinations of recombinant protein and whole cell may elicit immune responses that reach greater protection than that of individual S. agalactiae components. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. 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.

  8. Poly(oligoethylene glycol methacrylate) dip-coating: turning cellulose paper into a protein-repellent platform for biosensors.

    Science.gov (United States)

    Deng, Xudong; Smeets, Niels M B; Sicard, Clémence; Wang, Jingyun; Brennan, John D; Filipe, Carlos D M; Hoare, Todd

    2014-09-17

    The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

  9. Analyte induced water adsorbability in gas phase biosensors: the influence of ethinylestradiol on the water binding protein capacity.

    Science.gov (United States)

    Snopok, Borys; Kruglenko, Ivanna

    2015-05-07

    An ultra-sensitive gas phase biosensor/tracer/bio-sniffer is an emerging technology platform designed to provide real-time information on air-borne analytes, or those in liquids, through classical headspace analysis. The desired bio-sniffer measures gaseous 17α- ethinylestradiol (ETED) as frequency changes on a quartz crystal microbalance (QCM), which is a result of the interactions of liquid sample components in the headspace (ETED and water) with a biorecognition layer. The latter was constructed by immobilization of polyclonal antiserum against a phenolic A-ring of estrogenic receptors through protein A. The QCM response exhibited stretched exponential kinetics of negative frequency shifts with reversible and "irreversible" components of mass uptake onto the sensor surface in static headspace conditions when exposed to water solutions of ETED over the sensor working range, from 10(-10) to 10(-17) g L(-1). It was shown that the variations in the QCM response characteristics are due to the change of the water-binding capacity of the sensing layer induced by protein transformations initiated by the binding of ETED molecules. This result is well correlated with the natural physiological function of estrogens in controlling the homeostasis of body fluids in living beings.

  10. Functional Polymers in Protein Detection Platforms: Optical, Electrochemical, Electrical, Mass-Sensitive, and Magnetic Biosensors

    Directory of Open Access Journals (Sweden)

    Jong-in Hahm

    2011-03-01

    Full Text Available The rapidly growing field of proteomics and related applied sectors in the life sciences demands convenient methodologies for detecting and measuring the levels of specific proteins as well as for screening and analyzing for interacting protein systems. Materials utilized for such protein detection and measurement platforms should meet particular specifications which include ease-of-mass manufacture, biological stability, chemical functionality, cost effectiveness, and portability. Polymers can satisfy many of these requirements and are often considered as choice materials in various biological detection platforms. Therefore, tremendous research efforts have been made for developing new polymers both in macroscopic and nanoscopic length scales as well as applying existing polymeric materials for protein measurements. In this review article, both conventional and alternative techniques for protein detection are overviewed while focusing on the use of various polymeric materials in different protein sensing technologies. Among many available detection mechanisms, most common approaches such as optical, electrochemical, electrical, mass-sensitive, and magnetic methods are comprehensively discussed in this article. Desired properties of polymers exploited for each type of protein detection approach are summarized. Current challenges associated with the application of polymeric materials are examined in each protein detection category. Difficulties facing both quantitative and qualitative protein measurements are also identified. The latest efforts on the development and evaluation of nanoscale polymeric systems for improved protein detection are also discussed from the standpoint of quantitative and qualitative measurements. Finally, future research directions towards further advancements in the field are considered.

  11. 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

  12. The ITO-capped WO{sub 3} nanowires biosensor based on field-effect transistor in label-free protein sensing

    Energy Technology Data Exchange (ETDEWEB)

    Shariati, Mohsen [Sharif University of Technology, Institute for Nanoscience and Nanotechnology, Tehran (Iran, Islamic Republic of)

    2017-05-15

    The fabrication of ITO-capped WO{sub 3} nanowires associated with their bio-sensing properties in field-effect transistor diagnostics basis as a biosensor has been reported. The bio-sensing property for manipulated nanowires elucidated that the grown nanostructures were very sensitive to protein. The ITO-capped WO{sub 3} nanowires biosensor showed an intensive bio-sensing activity against reliable protein. Polylysine strongly charged bio-molecule was applied as model system to demonstrate the implementation of materialized biosensor. The employed sensing mechanism was 'label-free' and depended on bio-molecule's intrinsic charge. For nanowires synthesis, the vapor-liquid-solid mechanism was used. Nanowires were beyond a few hundred nanometers in lengths and around 15-20 nm in diameter, while the globe cap's size on the nanowires was around 15-25 nm. The indium tin oxide (ITO) played as catalyst in nanofabrication for WO{sub 3} nanowires growth and had outstanding role in bio-sensing especially for bio-molecule adherence. In applied electric field presence, the fabricated device showed the great potential to enhance medical diagnostics. (orig.)

  13. Whole Cell Biosensor for Polychlorinated Biphenyl Analysis Based on Optical Detection

    Czech Academy of Sciences Publication Activity Database

    Gavlasová, Pavla; Kuncová, Gabriela; Kochankova, L.; Macková, M.

    2008-01-01

    Roč. 62, č. 3 (2008), s. 304-312 ISSN 0964-8305 R&D Projects: GA ČR(CZ) GA104/05/2637; GA ČR(CZ) GA203/06/1244; GA MŠk OC 121 Institutional research plan: CEZ:AV0Z40720504 Keywords : pseudomonas species P2 * immobilization of bacteria * sol-gel Subject RIV: EE - Microbiology, Virology Impact factor: 1.375, year: 2008

  14. Whole-Cell Optical Biosensor for Mercury – Operational Conditions in Saline Water

    Czech Academy of Sciences Publication Activity Database

    Solovyev, Andrey; Kuncová, Gabriela; Demnerová, K.

    2015-01-01

    Roč. 69, č. 1 (2015), s. 183-191 ISSN 0366-6352 R&D Projects: GA TA ČR TA03010544 Institutional support: RVO:67985858 Keywords : mercury detection assay * bioluminescent bioreporter * sea water Subject RIV: CA - Inorganic Chemistry Impact factor: 1.326, year: 2015

  15. Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor

    DEFF Research Database (Denmark)

    Hansen, L. H.; Aarestrup, Frank Møller; Sørensen, S. J.

    2002-01-01

    and maintenance of fecal coliform bacteria resistant to tetracycline. Initially, large quantities of water-extractable CTC were excreted from the pigs and measurable amounts were detected even at 30 days after treatment cessation. This led to a sharp rise in the number of tetracycline resistant coliform bacteria...... in the feces, to within the same order of magnitude as the total coliform count. The high level of tetracycline resistance was maintained in spite of the declining concentration of tetracycline. (C) 2002 Elsevier Science B.V. All rights reserved....

  16. 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...

  17. A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Seunghyun; Karim, Muhammad Nazmul [Texas Tech Univ., Lubbock, TX (United States). Dept. of Chemical Engineering

    2011-08-15

    In this research, a recombinant whole cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum - endoglucanase (Cel5A), exoglucanase (Cel9E), and {beta}-glucosidase - on the surface of the Escherichia coli LY01. The modified strain is identified as LY01/pRE1H-AEB. The cellulases were displayed on the surface of the cell by fusing with an anchor protein, PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid-swollen cellulose (PASC) and the dilute acid-pretreated corn stover. Ethanol production was 3.59 {+-} 0.15 g/L using 10 g/L of PASC, which corresponds to a theoretical yield of 95.4 {+-} 0.15%. Ethanol production was 0.30 {+-} 0.02 g/L when 1 g/L equivalent of glucose in the cellulosic fraction of the dilute sulfuric acid-pretreated corn stover (PCS) was fermented for 84 h. A total of 0.71 {+-} 0.12 g/L ethanol was produced in 48 h when the PCS was fermented in the simultaneous saccharification and co-fermentation mode using the hemicellulosic (1 g/L of total soluble sugar) and as well as the cellulosic (1 g/L of glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrates into ethanol in a single reactor. The developed C. cellulolyticum-E. coli whole cell biocatalyst also overcame the incompatible temperature problem of the frequently reported fungal-yeast systems. (orig.)

  18. Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma

    Czech Academy of Sciences Publication Activity Database

    Piliarik, Marek; Bocková, Markéta; Homola, Jiří

    2010-01-01

    Roč. 26, č. 4 (2010), s. 1656-1661 ISSN 0956-5663 R&D Projects: GA AV ČR KAN200670701 Institutional research plan: CEZ:AV0Z20670512 Keywords : Surface plasmon resonance * Protein array * Cancer marker Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 5.361, year: 2010

  19. A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

    Directory of Open Access Journals (Sweden)

    Carlos R. Arias-Barreiro

    2010-06-01

    Full Text Available A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2. Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm were determined as 1.0 x 10−7 (arsenite, 1.0 x 10−4 (naphthalene, 1.0 x 10−4 (Cu2+, 3.8 x 10−4 (H2O2, 1.0 x 10−3 (Cd2+, 1.0 x 10−3 (Zn2+, 1.0 x 10−2 (menadione, 1.0 (triphenyltin, 1.56 (zinc pyrithione, 3.1 (selenite and 6.3 (Pb2+, respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.

  20. Sensitive electrochemiluminescence biosensor based on Au-ITO hybrid bipolar electrode amplification system for cell surface protein detection.

    Science.gov (United States)

    Wu, Mei-Sheng; Yuan, Da-Jing; Xu, Jing-Juan; Chen, Hong-Yuan

    2013-12-17

    Here we developed a novel hybrid bipolar electrode (BPE)-electrochemiluminescence (ECL) biosensor based on hybrid bipolar electrode (BPE) for the measurement of cancer cell surface protein using ferrocence (Fc) labeled aptamer as signal recognition and amplification probe. According to the electric neutrality of BPE, the cathode of U-shaped ITO BPE was electrochemically deposited by Au nanoparticles (NPs) to enhance its conductivity and surface area, decrease the overpotential of O2 reduction, which would correspondingly increase the oxidation current of Ru(bpy)3(2+)/tripropylamine (TPA) on the anode of BPE and resulting a ∼4-fold enhancement of ECL intensity. Then a signal amplification strategy was designed by introducing Fc modified aptamer on the anode surface of BPE through hybridization for detecting the amount of mucin-1 on MCF-7 cells. The presence of Fc could not only inhibit the oxidation of Ru(bpy)3(2+) because of its lower oxidation potential, its oxidation product Fc(+) could also quench the ECL of Ru(bpy)3(2+)/TPA by efficient energy-transfer from the excited-state Ru(bpy)3(2+)* to Fc(+), making the ECL intensity greatly quenched. On the basis of the cathodic Au NPs induced ECL enhancing coupled with anodic Fc induced signal quenching amplification, the approach allowed detection of mucin-1 aptamer at a concentration down to 0.5 fM and was capable of detecting a minimum of 20 MCF-7 cells. Besides, the amount of mucin-1 on MCF-7 cells was calculated to be 9041 ± 388 molecules/cell. This approach therefore shows great promise in bioanalysis.

  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. High performance dendrimer functionalized single-walled carbon nanotubes field effect transistor biosensor for protein detection

    Science.gov (United States)

    Rajesh, Sharma, Vikash; Puri, Nitin K.; Mulchandani, Ashok; Kotnala, Ravinder K.

    2016-12-01

    We report a single-walled carbon nanotube (SWNT) field-effect transistor (FET) functionalized with Polyamidoamine (PAMAM) dendrimer with 128 carboxyl groups as anchors for site specific biomolecular immobilization of protein antibody for C-reactive protein (CRP) detection. The FET device was characterized by scanning electron microscopy and current-gate voltage (I-Vg) characteristic studies. A concentration-dependent decrease in the source-drain current was observed in the regime of clinical significance, with a detection limit of ˜85 pM and a high sensitivity of 20% change in current (ΔI/I) per decade CRP concentration, showing SWNT being locally gated by the binding of CRP to antibody (anti-CRP) on the FET device. The low value of the dissociation constant (Kd = 0.31 ± 0.13 μg ml-1) indicated a high affinity of the device towards CRP analyte arising due to high anti-CRP loading with a better probe orientation on the 3-dimensional PAMAM structure.

  4. Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

    Science.gov (United States)

    Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

    2015-01-01

    The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.

  5. Engineering Rugged Field Assays to Detect Hazardous Chemicals Using Spore-Based Bacterial Biosensors.

    Science.gov (United States)

    Wynn, Daniel; Deo, Sapna; Daunert, Sylvia

    2017-01-01

    Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited. These problems can be circumvented by developing biosensors in Bacillus spores, which can be engineered to address all of these concerns. In its sporulated state, a whole cell-based biosensor has a remarkably long life span and is exceptionally resistant to environmental insult. When these spores are germinated for use in analytical techniques, they show no loss in performance, even after long periods of storage under harsh conditions. In this chapter, we will discuss the development and use of whole cell-based sensors, their adaptation to spore-based biosensors, their current applications, and future directions in the field. © 2017 Elsevier Inc. All rights reserved.

  6. Yeast surface displaying glucose oxidase as whole-cell biocatalyst: construction, characterization, and its electrochemical glucose sensing application.

    Science.gov (United States)

    Wang, Hongwei; Lang, Qiaolin; Li, Liang; Liang, Bo; Tang, Xiangjiang; Kong, Lingrang; Mascini, Marco; Liu, Aihua

    2013-06-18

    The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

  7. 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.

  8. 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

  9. Surface plasmon resonance biosensor based on engineered proteins for direct detection of interferon-gamma in diluted blood plasma

    Czech Academy of Sciences Publication Activity Database

    Šípová, Hana; Ševců, Veronika; Kuchař, Milan; Ahmad, Jawid Nazir; Mikulecký, Pavel; Osičková, Adriana; Malý, Petr; Homola, Jiří

    2012-01-01

    Roč. 174, č. 11 (2012), s. 306-311 ISSN 0925-4005 R&D Projects: GA AV ČR KAN200670701 Institutional support: RVO:67985882 ; RVO:61388971 ; RVO:86652036 Keywords : Interferon gamma * Surface plasmon resonance * Biosensor Subject RIV: JB - Sensors, Measurment, Regulation Impact factor: 3.535, year: 2012

  10. Biosensors : basic features and application for fatty acid-binding protein, an early plasma marker of myocardial injury

    NARCIS (Netherlands)

    van der Voort, D; McNeil, CA; Renneberg, R; Korf, J; Hermens, WT; Glatz, JFC

    2005-01-01

    Biosensors, especially immunosensors, are of great value for use in a clinical setting, since these are based on antigen-antibody reactions which are highly sensitive and specific. Furthermore, it becomes more and more important to measure specific compounds in biological matrices, such as blood or

  11. 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)

  12. Detection of organic compounds with whole-cell bioluminescent bioassays.

    Science.gov (United States)

    Xu, Tingting; Close, Dan; Smartt, Abby; Ripp, Steven; Sayler, Gary

    2014-01-01

    Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices.

  13. Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly.

    Science.gov (United States)

    Patra, Digambara

    2010-01-15

    A synchronous fluorescence probe based biosensor for estimation of albumin with high sensitivity and selectivity was developed. Unlike conventional fluorescence emission or excitation spectral measurements, synchronous fluorescence measurement offered exclusively a new synchronous fluorescence peak in the shorter wavelength range upon binding of chrysene with protein making it an easy identification tool for albumin determination. The cooperative binding of a fluorescence probe, chrysene, in a supramolecular host-protein assembly during various albumin assessments was investigated. The presence of supramolecular host molecules such as beta-cyclodextrin, curucurbit[6]uril or curucurbit[7]uril have little influence on sensitivity or limit of detection during albumin determination but reduced dramatically interference from various coexisting metal ion quenchers/enhancers. Using the present method the limit of detection for BSA and gamma-Globulin was found to be 0.005 microM which is more sensitive than reported values. Copyright 2009 Elsevier B.V. All rights reserved.

  14. Extended Solution Gate OFET-based Biosensor for Label-free Glial Fibrillary Acidic Protein Detection with Polyethylene Glycol-Containing Bioreceptor Layer.

    Science.gov (United States)

    Song, Jian; Dailey, Jennifer; Li, Hui; Jang, Hyun-June; Zhang, Pengfei; Wang, Jeff Tza-Huei; Everett, Allen D; Katz, Howard E

    2017-05-25

    A novel organic field effect transistor (OFET) -based biosensor is described for label-free glial fibrillary acidic protein (GFAP) detection. We report the first use of an extended solution gate structure where the sensing area and the organic semiconductor are separated, and a reference electrode is not needed. Different molecular weight polyethylene glycols (PEGs) are mixed into the bio-receptor layer to help extend the Debye screening length. The drain current change was significantly increased with the help of higher molecular weight PEGs, as they are known to reduce the dielectric constant. We also investigated the sensing performance under different gate voltage (V g ). The sensitivity increased after we decreased V g from -5 V to -2 V, because the lower V g is much closer to the OFET threshold voltage and the influence of attached negatively charged proteins become more apparent. Finally, the selectivity experiments toward different interferents were performed. The stability and selectivity are promising for clinical applications.

  15. 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

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Whole-cell fungal transformation of precursors into dyes

    Directory of Open Access Journals (Sweden)

    Jarosz-Wilkołazka Anna

    2010-07-01

    Full Text Available Abstract Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25. Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other

  18. Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

    OpenAIRE

    Alderete, J F

    1984-01-01

    An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses durin...

  19. Development of a continuous bioconversion system using a thermophilic whole-cell biocatalyst.

    Science.gov (United States)

    Ninh, Pham Huynh; Honda, Kohsuke; Yokohigashi, Yukako; Okano, Kenji; Omasa, Takeshi; Ohtake, Hisao

    2013-03-01

    The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.

  20. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors

    Science.gov (United States)

    Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans-Peter; Panke, Sven; Held, Martin

    2015-08-01

    Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

  1. Identification and use of an alkane transporter plug-in for application in biocatalysis and whole-cell biosensing of alkanes

    DEFF Research Database (Denmark)

    Grant, Chris; Deszcz, Dawid; Wei, Yu-Chia

    2014-01-01

    Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially rele...

  2. 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...

  3. A global benchmark study using affinity-based biosensors

    DEFF Research Database (Denmark)

    Rich, Rebecca L; Papalia, Giuseppe A; Flynn, Peter J

    2009-01-01

    To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users...... the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used....

  4. 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

  5. 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.

  6. Novel automated blood separations validate whole cell biomarkers.

    Directory of Open Access Journals (Sweden)

    Douglas E Burger

    Full Text Available Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs. Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs of fresh blood samples.To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes.Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.

  7. Novel automated blood separations validate whole cell biomarkers.

    Science.gov (United States)

    Burger, Douglas E; Wang, Limei; Ban, Liqin; Okubo, Yoshiaki; Kühtreiber, Willem M; Leichliter, Ashley K; Faustman, Denise L

    2011-01-01

    Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples. To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes. Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.

  8. Biosensors and environmental health

    National Research Council Canada - National Science Library

    Preedy, Victor R; Patel, Vinood B

    2012-01-01

    .... Coverage includes personal toxicity testing, soil and risk assessment, pesticide, insecticides, parasites, nitrate, endocrine disruptors, heavy metals, food contamination, whole cell bioreporters...

  9. 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

  10. A global benchmark study using affinity-based biosensors

    NARCIS (Netherlands)

    Rich, Rebecca L.; Papalia, Giusseppe A.; Krishnamoorthy, G.; Beusink, J.B.; Pak, Brian J.; Myszka, David G.; more, more

    2009-01-01

    To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

    International Nuclear Information System (INIS)

    Rebelo, Tânia S.C.R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M.H.; Noronha, João P.; Sales, M. Goreti F.

    2014-01-01

    Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10 −11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

  17. 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...

  18. 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)

  19. 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

  20. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

    Directory of Open Access Journals (Sweden)

    Kwon Seok-Joon

    2006-04-01

    Full Text Available Abstract Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC. The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

  1. Whole-cell bioreporters and risk assessment of environmental pollution: A proof-of-concept study using lead

    International Nuclear Information System (INIS)

    Zhang, Xiaokai; Qin, Boqiang; Deng, Jianming; Wells, Mona

    2017-01-01

    As the world burden of environmental contamination increases, it is of the utmost importance to develop streamlined approaches to environmental risk assessment in order to prioritize mitigation measures. Whole-cell biosensors or bioreporters and speciation modeling have both become of increasing interest to determine the bioavailability of pollutants, as bioavailability is increasingly in use as an indicator of risk. Herein, we examine whether bioreporter results are able to reflect expectations based on chemical reactivity and speciation modeling, with the hope to extend the research into a wider framework of risk assessment. We study a specific test case concerning the bioavailability of lead (Pb) in aqueous environments containing Pb-complexing ligands. Ligands studied include ethylene diamine tetra-acetic acid (EDTA), meso-2,3 dimercaptosuccinic acid (DMSA), leucine, methionine, cysteine, glutathione, and humic acid (HA), and we also performed experiments using natural water samples from Lake Tai (Taihu), the third largest lake in China. We find that EDTA, DMSA, cysteine, glutathione, and HA amendment significantly reduced Pb bioavailability with increasing ligand concentration according to a log-sigmoid trend. Increasing dissolved organic carbon in Taihu water also had the same effect, whereas leucine and methionine had no notable effect on bioavailability at the concentrations tested. We find that bioreporter results are in accord with the reduction of aqueous Pb 2+ that we expect from the relative complexation affinities of the different ligands tested. For EDTA and HA, for which reasonably accurate ionization and complexation constants are known, speciation modeling is in agreement with bioreporter response to within the level of uncertainty recognised as reasonable by the United States Environmental Protection Agency for speciation-based risk assessment applications. These findings represent a first step toward using bioreporter technology to streamline

  2. Summary of the DREAM8 Parameter Estimation Challenge: Toward Parameter Identification for Whole-Cell Models.

    Directory of Open Access Journals (Sweden)

    Jonathan R Karr

    2015-05-01

    Full Text Available Whole-cell models that explicitly represent all cellular components at the molecular level have the potential to predict phenotype from genotype. However, even for simple bacteria, whole-cell models will contain thousands of parameters, many of which are poorly characterized or unknown. New algorithms are needed to estimate these parameters and enable researchers to build increasingly comprehensive models. We organized the Dialogue for Reverse Engineering Assessments and Methods (DREAM 8 Whole-Cell Parameter Estimation Challenge to develop new parameter estimation algorithms for whole-cell models. We asked participants to identify a subset of parameters of a whole-cell model given the model's structure and in silico "experimental" data. Here we describe the challenge, the best performing methods, and new insights into the identifiability of whole-cell models. We also describe several valuable lessons we learned toward improving future challenges. Going forward, we believe that collaborative efforts supported by inexpensive cloud computing have the potential to solve whole-cell model parameter estimation.

  3. A general strategy to construct small molecule biosensors in eukaryotes.

    Science.gov (United States)

    Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

    2015-12-29

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

  4. Recent Advances in Optical Biosensors for Environmental Monitoring and Early Warning

    Directory of Open Access Journals (Sweden)

    Anna Zhu

    2013-10-01

    Full Text Available The growing number of pollutants requires the development of innovative analytical devices that are precise, sensitive, specific, rapid, and easy-to-use to meet the increasing demand for legislative actions on environmental pollution control and early warning. Optical biosensors, as a powerful alternative to conventional analytical techniques, enable the highly sensitive, real-time, and high-frequency monitoring of pollutants without extensive sample preparation. This article reviews important advances in functional biorecognition materials (e.g., enzymes, aptamers, DNAzymes, antibodies and whole cells that facilitate the increasing application of optical biosensors. This work further examines the significant improvements in optical biosensor instrumentation and their environmental applications. Innovative developments of optical biosensors for environmental pollution control and early warning are also discussed.

  5. Translating University Biosensor Research to a High School Laboratory Experience

    Science.gov (United States)

    Heldt, Caryn L.; Bank, Alex; Turpeinen, Dylan; King, Julia A.

    2016-01-01

    The need to increase science, technology, engineering, and mathematics (STEM) graduates is great. To interest more students into STEM degrees, we made our graphene biosensor research portable, inexpensive, and safe to demonstrate technology development to high school students. The students increased their knowledge of biosensors and proteins, and…

  6. 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

  7. Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine

    NARCIS (Netherlands)

    Kraan, H.; Have, Ten R.; Maas, van der L.; Kersten, G.F.A.; Amorij, J.P.

    2016-01-01

    A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick

  8. Risk of Brain Damage Following Pertussis Immunization with Whole-Cell cf Acellular Vaccines

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-06-01

    Full Text Available Serious neurological disorders reported following whole-cell (WC in comparison to acellular (AC pertussis vaccines (PV were evaluated by the Genetic Centers of America, Silver Spring, MD.

  9. 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

  10. Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling

    Directory of Open Access Journals (Sweden)

    Glick Benjamin S

    2009-04-01

    Full Text Available Abstract Background Whole-cell labeling is a common application of fluorescent proteins (FPs, but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties. Results Building on previous studies of DsRed mutants, we created two DsRed-Express2 derivatives: E2-Orange, an orange FP, and E2-Red/Green, a dual-color FP with both red and green emission. We show that these new FPs retain the low cytotoxicity of DsRed-Express2. In addition, we show that these new FPs are useful as second or third colors for flow cytometry and fluorescence microscopy. Conclusion E2-Orange and E2-Red/Green will facilitate the production of healthy, stably fluorescent cell lines and transgenic organisms for multi-color labeling studies.

  11. Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Zhaojuan Zheng

    Full Text Available Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h. No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose.

  12. Use of Tunable Whole-Cell Bioreporters to Assess Bioavailable Cadmium and Remediation Performance in Soils.

    Directory of Open Access Journals (Sweden)

    Youngdae Yoon

    Full Text Available It is important to have tools to measure the bioavailability to assess the risks of pollutants because the bioavailability is defined as the portions of pollutants showing the biological effects on living organisms. This study described the construction of tunable Escherichia coli whole-cell bioreporter (WCB using the promoter region of zinc-inducible operon and its application on contaminated soils. It was verified that this WCB system showed specific and sensitive responses to cadmium rather than zinc in the experimental conditions. It was inferred that Cd(II associates stronger with ZntR, a regulatory protein of zinc-inducible operon, than other metal ions. Moreover, the expression of reporter genes, egfp and mcherry, were proportional to the concentration of cadmium, thereby being a quantitative sensor to monitor bioavailable cadmium. The capability to determine bioavailable cadmium was verified with Cd(II amended LUFA soils, and then the applicability on environmental systems was investigated with field soils collected from smelter area in Korea before and after soil-washing. The total amount of cadmium was decreased after soil washing, while the bioavailability was increased. Consequently, it would be valuable to have tools to assess bioavailability and the effectiveness of soil remediation should be evaluated in the aspect of bioavailability as well as removal efficiency.

  13. Use of Tunable Whole-Cell Bioreporters to Assess Bioavailable Cadmium and Remediation Performance in Soils.

    Science.gov (United States)

    Yoon, Youngdae; Kim, Sunghoon; Chae, Yooeun; Kang, Yerin; Lee, Youngshim; Jeong, Seung-Woo; An, Youn-Joo

    2016-01-01

    It is important to have tools to measure the bioavailability to assess the risks of pollutants because the bioavailability is defined as the portions of pollutants showing the biological effects on living organisms. This study described the construction of tunable Escherichia coli whole-cell bioreporter (WCB) using the promoter region of zinc-inducible operon and its application on contaminated soils. It was verified that this WCB system showed specific and sensitive responses to cadmium rather than zinc in the experimental conditions. It was inferred that Cd(II) associates stronger with ZntR, a regulatory protein of zinc-inducible operon, than other metal ions. Moreover, the expression of reporter genes, egfp and mcherry, were proportional to the concentration of cadmium, thereby being a quantitative sensor to monitor bioavailable cadmium. The capability to determine bioavailable cadmium was verified with Cd(II) amended LUFA soils, and then the applicability on environmental systems was investigated with field soils collected from smelter area in Korea before and after soil-washing. The total amount of cadmium was decreased after soil washing, while the bioavailability was increased. Consequently, it would be valuable to have tools to assess bioavailability and the effectiveness of soil remediation should be evaluated in the aspect of bioavailability as well as removal efficiency.

  14. Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

    OpenAIRE

    Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

    2016-01-01

    C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1?3?mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammator...

  15. 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.

  16. 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.

  17. 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)

  18. Identification of protective pneumococcal T(H17 antigens from the soluble fraction of a killed whole cell vaccine.

    Directory of Open Access Journals (Sweden)

    Kristin L Moffitt

    Full Text Available Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA with an adjuvant protects mice from colonization by a T(H17 CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine.

  19. Calcium phosphate–gold nanoparticles nanocomposite for protein adsorption and mediator-free H2O2 biosensor construction

    International Nuclear Information System (INIS)

    Xu Qin; Lu Guiju; Bian XiaoJun; Jin Gendi; Wang Wei; Hu Xiaoya; Wang Yang; Yang Zhanjun

    2012-01-01

    This work reports a new method for the preparation and application of a kind of biocompatible calcium phosphate–gold nanoparticles (Ca 3 (PO 4 ) 2 –AuNPs) nanocomposite. UV–vis spectroscopy and transmittance electron microscopy (TEM) have been used to monitor the formation process of the nanocomposite and to examine the interaction between calcium phosphate and gold nanoparticles (AuNPs). The nanocomposite has multiple sites and improved conductivity which make it suitable for the binding of proteins to construct electrochemical sensors. Myoglobin (Mb) adsorbed on the nanocomposite retained its native structure which was proved by Fourier transform infrared spectroscopy (FTIR). Direct electron transfer between the adsorbed Mb and the electrode was observed. Further results demonstrated that the adsorbed Mb has good electrocatalytic activity towards the reduction of H 2 O 2 in the absence of any mediator. Highlights: ► Using gelatin modified gold nanoparticles to prepare needle-like calcium phosphate. ► Calcium phosphate provides multiple sites for protein adsorption. ► Gold nanoparticles act as electron tunneling. ► Myoglobin adsorbed on the material showed direct electrochemistry and good catalysis.

  20. Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

    KAUST Repository

    Gajos, Katarzyna

    2018-03-05

    Time-of-flight secondary ion mass spectrometry has been employed to examine, with biomolecular discrimination, sensing arm areas (20 μm x 600 μm) of integrated onto silicon chips Mach-Zehnder interferometers aiming to optimize their biofunctionalization with regard to indirect immunochemical (competitive) detection of ochratoxin A. Sensing areas are examined after: modification with (3-aminopropyl)triethoxysilane, spotting of OTA-ovalbumin conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin, reaction with OTA-specific mouse monoclonal antibody followed by goat anti-mouse IgG secondary antibody. Component mass loadings of all proteins involved in immunodetection are determined from TOF-SIMS micro-analysis combined with ellipsometry of planar surfaces. These data show that partial desorption of surface-bound probe and blocking protein takes place upon primary immunoreaction to a degree that depends on probe concentration in spotting solution. Taking into account this desorption, apparent binding stoichiometry of both antibodies in immune complexes formed onto chip surface is determined more accurately than the respective evaluation based on real-time sensor response. In addition, mass loadings for probe and secondary antibody is observed to saturate for optimum probe concentrations. Also, principal component analysis of TOF-SIMS data could resolve both immunoreactions and biofunctionalization and discriminate surfaces prepared with optimum probe concentrations from those prepared using suboptimum ones.

  1. Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

    KAUST Repository

    Gajos, Katarzyna; Budkowski, Andrzej; Petrou, Panagiota; Pagkali, Varvara; Awsiuk, Kamil; Rysz, Jakub; Bernasik, Andrzej; Misiakos, Konstantinos; Raptis, Ioannis; Kakabakos, Sotirios

    2018-01-01

    Time-of-flight secondary ion mass spectrometry has been employed to examine, with biomolecular discrimination, sensing arm areas (20 μm x 600 μm) of integrated onto silicon chips Mach-Zehnder interferometers aiming to optimize their biofunctionalization with regard to indirect immunochemical (competitive) detection of ochratoxin A. Sensing areas are examined after: modification with (3-aminopropyl)triethoxysilane, spotting of OTA-ovalbumin conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin, reaction with OTA-specific mouse monoclonal antibody followed by goat anti-mouse IgG secondary antibody. Component mass loadings of all proteins involved in immunodetection are determined from TOF-SIMS micro-analysis combined with ellipsometry of planar surfaces. These data show that partial desorption of surface-bound probe and blocking protein takes place upon primary immunoreaction to a degree that depends on probe concentration in spotting solution. Taking into account this desorption, apparent binding stoichiometry of both antibodies in immune complexes formed onto chip surface is determined more accurately than the respective evaluation based on real-time sensor response. In addition, mass loadings for probe and secondary antibody is observed to saturate for optimum probe concentrations. Also, principal component analysis of TOF-SIMS data could resolve both immunoreactions and biofunctionalization and discriminate surfaces prepared with optimum probe concentrations from those prepared using suboptimum ones.

  2. Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

    Science.gov (United States)

    Gajos, Katarzyna; Budkowski, Andrzej; Petrou, Panagiota; Pagkali, Varvara; Awsiuk, Kamil; Rysz, Jakub; Bernasik, Andrzej; Misiakos, Konstantinos; Raptis, Ioannis; Kakabakos, Sotirios

    2018-06-01

    Time-of-flight secondary ion mass spectrometry has been employed to examine, with biomolecular discrimination, sensing arm areas (20 μm × 600 μm) of integrated onto silicon chips Mach-Zehnder interferometers aiming to optimize their biofunctionalization with regard to indirect immunochemical (competitive) detection of ochratoxin A. Sensing areas are examined after: modification with (3-aminopropyl)triethoxysilane, spotting of OTA-ovalbumin conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin, reaction with OTA-specific mouse monoclonal antibody followed by goat anti-mouse IgG secondary antibody. Component mass loadings of all proteins involved in immunodetection are determined from TOF-SIMS micro-analysis combined with ellipsometry of planar surfaces. These data show that partial desorption of surface-bound probe and blocking protein takes place upon primary immunoreaction to a degree that depends on probe concentration in spotting solution. Taking into account this desorption, apparent binding stoichiometry of both antibodies in immune complexes formed onto chip surface is determined more accurately than the respective evaluation based on real-time sensor response. In addition, mass loadings for probe and secondary antibody is observed to saturate for optimum probe concentrations. Also, principal component analysis of TOF-SIMS data could resolve both immunoreactions and biofunctionalization and discriminate surfaces prepared with optimum probe concentrations from those prepared using suboptimum ones.

  3. 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.

  4. Integration of Fractal Biosensor in a Digital Microfluidic Platform

    KAUST Repository

    Mashraei, Yousof; Sivashankar, Shilpa; Buttner, Ulrich; Salama, Khaled N.

    2016-01-01

    fractal electrode biosensor that is used for both droplet actuation and sensing C-reactive protein (CRP) concentration levels to assess cardiac disease risk. Our proposed electrode is the first two-terminal electrode design to be integrated into DMF

  5. 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

  6. Whole-cell bioreporters and risk assessment of environmental pollution: A proof-of-concept study using lead.

    Science.gov (United States)

    Zhang, Xiaokai; Qin, Boqiang; Deng, Jianming; Wells, Mona

    2017-10-01

    As the world burden of environmental contamination increases, it is of the utmost importance to develop streamlined approaches to environmental risk assessment in order to prioritize mitigation measures. Whole-cell biosensors or bioreporters and speciation modeling have both become of increasing interest to determine the bioavailability of pollutants, as bioavailability is increasingly in use as an indicator of risk. Herein, we examine whether bioreporter results are able to reflect expectations based on chemical reactivity and speciation modeling, with the hope to extend the research into a wider framework of risk assessment. We study a specific test case concerning the bioavailability of lead (Pb) in aqueous environments containing Pb-complexing ligands. Ligands studied include ethylene diamine tetra-acetic acid (EDTA), meso-2,3 dimercaptosuccinic acid (DMSA), leucine, methionine, cysteine, glutathione, and humic acid (HA), and we also performed experiments using natural water samples from Lake Tai (Taihu), the third largest lake in China. We find that EDTA, DMSA, cysteine, glutathione, and HA amendment significantly reduced Pb bioavailability with increasing ligand concentration according to a log-sigmoid trend. Increasing dissolved organic carbon in Taihu water also had the same effect, whereas leucine and methionine had no notable effect on bioavailability at the concentrations tested. We find that bioreporter results are in accord with the reduction of aqueous Pb 2+ that we expect from the relative complexation affinities of the different ligands tested. For EDTA and HA, for which reasonably accurate ionization and complexation constants are known, speciation modeling is in agreement with bioreporter response to within the level of uncertainty recognised as reasonable by the United States Environmental Protection Agency for speciation-based risk assessment applications. These findings represent a first step toward using bioreporter technology to streamline

  7. 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.

  8. Biosensors for detection of mercury in contaminated soils

    International Nuclear Information System (INIS)

    Bontidean, Ibolya; Mortari, Alessia; Leth, Suzanne; Brown, Nigel L.; Karlson, Ulrich; Larsen, Martin M.; Vangronsveld, Jaco; Corbisier, Philippe; Csoeregi, Elisabeth

    2004-01-01

    Biosensors based on whole bacterial cells and on bacterial heavy metal binding protein were used to determine the mercury concentration in soil. The soil samples were collected in a vegetable garden accidentally contaminated with elemental mercury 25 years earlier. Bioavailable mercury was measured using different sensors: a protein-based biosensor, a whole bacterial cell based biosensor, and a plant sensor, i.e. morphological and biochemical responses in primary leaves and roots of bean seedlings grown in the mercury-contaminated soil. For comparison the total mercury concentration of the soil samples was determined by AAS. Whole bacterial cell and protein-based biosensors gave accurate responses proportional to the total amount of mercury in the soil samples. On the contrary, plant sensors were found to be less useful indicators of soil mercury contamination, as determined by plant biomass, mercury content of primary leaves and enzyme activities

  9. Semi-autonomous inline water analyzer: design of a common light detector for bacterial, phage, and immunological biosensors.

    Science.gov (United States)

    Descamps, Elodie C T; Meunier, Damien; Brutesco, Catherine; Prévéral, Sandra; Franche, Nathalie; Bazin, Ingrid; Miclot, Bertrand; Larosa, Philippe; Escoffier, Camille; Fantino, Jean-Raphael; Garcia, Daniel; Ansaldi, Mireille; Rodrigue, Agnès; Pignol, David; Cholat, Pierre; Ginet, Nicolas

    2017-01-01

    The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.

  10. Construction of a highly efficient Bacillus subtilis 168 whole-cell biocatalyst and its application in the production of L-ornithine.

    Science.gov (United States)

    Wang, Meizhou; Xu, Meijuan; Rao, Zhiming; Yang, Taowei; Zhang, Xian

    2015-11-01

    L-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of L-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 °C, respectively. In addition, the recombinant arginase exhibited a strong Mn(2+) preference. When using whole-cell biocatalyst-based bioconversion, a hyper L-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for L-ornithine production in industry.

  11. 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...

  12. 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.

  13. Oriented immobilization of His-tagged kinase RIO1 protein on redox active N-(IDA-like)-Cu(II) monolayer deposited on gold electrode—The base of electrochemical biosensor

    International Nuclear Information System (INIS)

    Mielecki, Marcin; Wojtasik, Justyn; Zborowska, Magdalena; Kurzątkowska, Katarzyna; Grzelak, Krystyna; Dehaen, Wim; Radecki, Jerzy; Radecka, Hanna

    2013-01-01

    Highlights: ► The redox active N-(IDA-like)-Cu(II) monolayer is suitable for oriented and stable immobilization of His-tagged kinase Rio1. ► Cu(II) deposited onto the electrode surface play double role: immobilization sites for His-tagged proteins and transduction centres tracking the protein–small molecule interactions. ► The base of biosensor response towards target compound is the change of Rio1 conformation lading to alternation of the permeability of counter ions to Cu(II) redox centres. -- Abstract: The fabrication of electrochemical biosensor consists of the following successive steps: formation of thiol derivative of iminodiacetic acid (IDA-like/N-heterocyclic donor) and N-acetylcysteamine (NAC) self-assembled monolayer on the Au electrode, complexation of Cu(II) by N(IDA-like) attached to the surface of the Au electrode and immobilization of kinase protein Rio1 through N(IDA-like)-Cu(II)-histidine-tag covalent bond formation. Each step of modification was controlled by cyclic voltammetry, electrochemical impedance spectrometry and atomic force microscopy. The interactions between rHis 6 -Rio1 attached to the surface of the electrode and tyrphostin inhibitor (2E)-N-Benzyl-2-cyano-3-(3,4-dihydroxyphenyl)-acrylamide (AG-490) and its analogue (2-cyano-N-(4-methoxyphenyl)-3-(pyridin-3-yl)prop-2-enamide) (CPE), present in aqueous solution were monitored with Osteryoung square wave voltammetry. The basis of the biosensor response was the change in the electrochemical properties of Cu(II) redox centres upon formation of the rHis 6 -Rio1-inhibitor complex. A linear responses with high reproducibility and stability were observed between 0.10 and 0.40 μM of AG-490 as well as of CPE. The interaction between rHis 6 -Rio1 and AG-490 was stronger than the interaction with its analogue CPE. Cu(II) redox current decrease of 37.9 ± 1.6% and 23.3 ± 1.0% were observed in the presence of 0.40 μM of AG-490 and CPE, respectively. The presented biosensor could be

  14. 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.

  15. Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

    OpenAIRE

    Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

    2015-01-01

    Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, s...

  16. Quantitative analysis of dual whole-cell voltage-clamp determination of gap junctional conductance

    NARCIS (Netherlands)

    van Rijen, H. V.; Wilders, R.; van Ginneken, A. C.; Jongsma, H. J.

    1998-01-01

    The dual whole-cell voltage-clamp technique is used widely for determination of kinetics and conductance of gap junctions. The use of this technique may, however, occasion to considerable errors. We have analysed the errors in steady state junctional conductance measurements under different

  17. Progress in emerging techniques for characterization of immobilized viable whole-cell biocatalysts

    Czech Academy of Sciences Publication Activity Database

    Bučko, M.; Vikartovská, A.; Schenkmayerová, A.; Tkáč, J.; Filip, J.; Chorvát Jr., D.; Neděla, Vilém; Ansorge-Schumacher, M.B.; Gemeiner, P.

    2017-01-01

    Roč. 71, č. 11 (2017), s. 2309-2324 ISSN 0366-6352 Institutional support: RVO:68081731 Keywords : bioelectrocatalysis * imaging techniques * immobilized whole-cell biocatalyst * multienzyme cascade reactions * online kinetics Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering OBOR OECD: Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation Impact factor: 1.258, year: 2016

  18. Does whole-cell pertussis vaccine protect black South African infants?

    African Journals Online (AJOL)

    The whole-cell pertussis vaccine currently used in South Africa has not been adequately evaluated for post-vaccination events and immunogenicity. A trial of this vaccine combined with diphtheria and tetanus toxoids (DTP) was undertaken in 115 black babies who received primary vaccination at 2, 4 and 6 months of age.

  19. 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...

  20. 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.

  1. Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization.

    Science.gov (United States)

    Schrewe, Manfred; Julsing, Mattijs K; Lange, Kerstin; Czarnotta, Eik; Schmid, Andreas; Bühler, Bruno

    2014-09-01

    The oxyfunctionalization of unactivated C−H bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis-Menten-type kinetics for each reaction step. In two-liquid phase biotransformations, the product formation pattern was found to be controlled by DAME availability. Supplying DAME as bulk organic phase led to accumulation of the terminal alcohol as the predominant product. Limiting DAME availability via application of bis(2-ethylhexyl)phthalate (BEHP) as organic carrier solvent enabled almost exclusive acid accumulation. Furthermore, utilization of BEHP enhanced catalyst stability by reducing toxic effects of substrate and products. A further shift towards the overoxidized products was achieved by co-expression of the gene encoding the alcohol dehydrogenase AlkJ, which was shown to catalyze efficient and irreversible alcohol to aldehyde oxidation in vivo. With DAME as organic phase, the aldehyde accumulated as main product using resting cells containing AlkBGT, AlkL, as well as AlkJ. This study highlights the versatility of whole-cell biocatalysis for synthesis of industrially relevant bifunctional building blocks and demonstrates how integrated reaction and catalyst engineering can be implemented to control product formation patterns in biocatalytic multistep reactions. © 2014 Wiley Periodicals, Inc.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Nano technologies for Biosensor and Bio chip

    International Nuclear Information System (INIS)

    Kim, I.M.; Park, T.J.; Paskaleva, E.E.; Sun, F.; Seo, J.W.; Mehta, K.K.

    2015-01-01

    The bio sensing devices are characterized by their biological receptors, which have specificity to their corresponding analytes. These analytes are a vast and diverse group of biological molecules, DNAs, proteins (such as antibodies), fatty acids, or entire biological systems, such as pathogenic bacteria, viruses, cancerous cells, or other living organisms. A main challenge in the development of biosensor applications is the efficient recognition of a biological signal in a low signal-to-noise ratio environment, and its transduction into an electrochemical, optical, or other signals. The advent of nano material technology greatly increased the potential for achieving exquisite sensitivity of such devises, due to the innate high surface-to-volume ratio and high reactivity of the nano material. The second major challenge facing the biosensor application, that of sca lability, is addressed by multiplexing and miniaturizing of the biosensor devises into a bio chip. In recent years, biosensor and bio chip technologies have made significant progress by taking advantages of diverse kinds of nano materials that are derived from nano technology

  7. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale.

    Science.gov (United States)

    Wachtmeister, Jochen; Rother, Dörte

    2016-12-01

    Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalyst's costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  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. A High-Throughput Oxidative Stress Biosensor Based on Escherichia coli roGFP2 Cells Immobilized in a k-Carrageenan Matrix

    Directory of Open Access Journals (Sweden)

    Lia Ooi

    2015-01-01

    Full Text Available Biosensors fabricated with whole-cell bacteria appear to be suitable for detecting bioavailability and toxicity effects of the chemical(s of concern, but they are usually reported to have drawbacks like long response times (ranging from hours to days, narrow dynamic range and instability during long term storage. Our aim is to fabricate a sensitive whole-cell oxidative stress biosensor which has improved properties that address the mentioned weaknesses. In this paper, we report a novel high-throughput whole-cell biosensor fabricated by immobilizing roGFP2 expressing Escherichia coli cells in a k-carrageenan matrix, for the detection of oxidative stress challenged by metalloid compounds. The E. coli roGFP2 oxidative stress biosensor shows high sensitivity towards arsenite and selenite, with wide linear range and low detection limit (arsenite: 1.0 × 10−3–1.0 × 101 mg·L−1, LOD: 2.0 × 10−4 mg·L−1; selenite: 1.0 × 10−5–1.0 × 102 mg·L−1, LOD: 5.8 × 10−6 mg·L−1, short response times (0–9 min, high stability and reproducibility. This research is expected to provide a new direction in performing high-throughput environmental toxicity screening with living bacterial cells which is capable of measuring the bioavailability and toxicity of environmental stressors in a friction of a second.

  10. Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications

    Czech Academy of Sciences Publication Activity Database

    Polakovič, M.; Švitel, J.; Bučko, M.; Filip, J.; Neděla, Vilém; Ansorge-Schumacher, M.B.; Gemeiner, P.

    2017-01-01

    Roč. 39, č. 5 (2017), s. 667-683 ISSN 0141-5492 Institutional support: RVO:68081731 Keywords : biocatalysis * immobilization methods * immobilized whole-cell biocatalyst * multienzyme cascade reactions * process economics * reaction engineering Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering OBOR OECD: Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation Impact factor: 1.730, year: 2016

  11. 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.

  12. 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.

  13. Applications and Mechanisms of Ionic Liquids in Whole-Cell Biotransformation

    Science.gov (United States)

    Fan, Lin-Lin; Li, Hong-Ji; Chen, Qi-He

    2014-01-01

    Ionic liquids (ILs), entirely composed of cations and anions, are liquid solvents at room temperature. They are interesting due to their low vapor pressure, high polarity and thermostability, and also for the possibility to fine-tune their physicochemical properties through modification of the chemical structures of their cations or anions. In recent years, ILs have been widely used in biotechnological fields involving whole-cell biotransformations of biodiesel or biomass, and organic compound synthesis with cells. Research studies in these fields have increased from the past decades and compared to the typical solvents, ILs are the most promising alternative solvents for cell biotransformations. However, there are increasing limitations and new challenges in whole-cell biotransformations with ILs. There is little understanding of the mechanisms of ILs’ interactions with cells, and much remains to be clarified. Further investigations are required to overcome the drawbacks of their applications and to broaden their application spectrum. This work mainly reviews the applications of ILs in whole-cell biotransformations, and the possible mechanisms of ILs in microbial cell biotransformation are proposed and discussed. PMID:25007820

  14. Applications and mechanisms of ionic liquids in whole-cell biotransformation.

    Science.gov (United States)

    Fan, Lin-Lin; Li, Hong-Ji; Chen, Qi-He

    2014-07-09

    Ionic liquids (ILs), entirely composed of cations and anions, are liquid solvents at room temperature. They are interesting due to their low vapor pressure, high polarity and thermostability, and also for the possibility to fine-tune their physicochemical properties through modification of the chemical structures of their cations or anions. In recent years, ILs have been widely used in biotechnological fields involving whole-cell biotransformations of biodiesel or biomass, and organic compound synthesis with cells. Research studies in these fields have increased from the past decades and compared to the typical solvents, ILs are the most promising alternative solvents for cell biotransformations. However, there are increasing limitations and new challenges in whole-cell biotransformations with ILs. There is little understanding of the mechanisms of ILs' interactions with cells, and much remains to be clarified. Further investigations are required to overcome the drawbacks of their applications and to broaden their application spectrum. This work mainly reviews the applications of ILs in whole-cell biotransformations, and the possible mechanisms of ILs in microbial cell biotransformation are proposed and discussed.

  15. Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis.

    Science.gov (United States)

    Werner, Sean R; Morgan, John A

    2009-07-15

    Glycosyltransferases are promising biocatalysts for the synthesis of small molecule glycosides. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation) was investigated as a whole-cell biocatalyst. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin-7-O-glucoside was formed and a higher specific glucosyl transfer activity was found using a galactose inducible expression system compared to a constitutive expression system. However, S. cerevisiae expressing the GT constitutively was significantly more productive than the galactose inducible system under in vivo conditions. Interestingly, the glycosides were recovered directly from the culture broth and did not accumulate intracellularly. A previously uncharacterized naringenin glycoside formed using the D. caryophyllus GT was identified as naringenin-4'-O-glucoside. It was found that S. cerevisiae cells hydrolyze naringenin-7-O-glucoside during whole-cell biocatalysis, resulting in a low final glycoside titer. When phloretin was added as a substrate to the yeast strain expressing the GT constitutively, the natural product phlorizin was formed. This study demonstrates S. cerevisiae is a promising whole-cell biocatalyst host for the production of valuable glycosides.

  16. Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases ? and ? for nonhomologous end joining in human whole-cell extracts

    OpenAIRE

    Akopiants, Konstantin; Zhou, Rui-Zhe; Mohapatra, Susovan; Valerie, Kristoffer; Lees-Miller, Susan P.; Lee, Kyung-Jong; Chen, David J.; Revy, Patrick; de Villartay, Jean-Pierre; Povirk, Lawrence F.

    2009-01-01

    XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5? or 3? overhangs, and no joining at all of partially complementary 3? overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped us...

  17. Optical Biosensors to Explore Biological Systems

    DEFF Research Database (Denmark)

    Palanco, Marta Espina; Mogensen, Klaus Bo; Andersen, Nils H. Skovgaard

    2016-01-01

    their capability to work in biosensor devices. For example, Raman spectroscopy can be non-invasive and can provide 1 μm of spatial resolution in 1 second of collection time, well suited for sensing. Moreover, it may give information at the single cell and even approaching the single molecule scale. Here we present...... protein may be used as an efficient sensor in an organic environment via a biomimetic membrane model. The combination of both biomimetic membranes and protein membranes as a signal transduction medium has interesting applications in biology and medicine. It is crucial that the matrix where a protein...

  18. 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.

  19. Direct electron transfer: an approach for electrochemical biosensors with higher selectivity and sensitivity

    Directory of Open Access Journals (Sweden)

    Freire Renato S.

    2003-01-01

    Full Text Available The most promising approach for the development of electrochemical biosensors is to establish a direct electrical communication between the biomolecules and the electrode surface. This review focuses on advances, directions and strategies in the development of third generation electrochemical biosensors. Subjects covered include a brief description of the fundamentals of the electron transfer phenomenon and amperometric biosensor development (different types and new oriented enzyme immobilization techniques. Special attention is given to different redox enzymes and proteins capable of electrocatalyzing reactions via direct electron transfer. The analytical applications and future trends for third generation biosensors are also presented and discussed.

  20. Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction.

    Science.gov (United States)

    VerHeul, Ross; Sweet, Craig; Thompson, David H

    2018-03-26

    Elastin-like polypeptides (ELP) are a well-known class of proteins that are being increasingly utilized in a variety of biomedical applications, due to their beneficial physicochemical properties. A unifying feature of ELP is their demonstration of a sequence tunable inverse transition temperature (Tt) that enables purification using a simple, straightforward process called inverse transition cycling (ITC). Despite the utility of ITC, the process is inherently limited to ELP with an experimentally accessible Tt. Since the underlying basis for the ELP Tt is related to its high overall hydrophobicity, we anticipated that ELP would be excellent candidates for purification by organic extraction. We report the first method for rapidly purifying ELP directly from whole E. coli cells or clarified lysates using pure organic solvents and solvent mixtures, followed by aqueous back extraction. Our results show that small ELP and a large ELP-fusion protein can be isolated in high yield from whole cells or cell lysates with greater than 95% purity in less than 30 min and with very low levels of LPS and DNA contamination.

  1. Investigation of Halohydrins Degradation by Whole Cells and Cell-free Extract of Pseudomonas putida DSM 437: A Kinetic Approach

    Directory of Open Access Journals (Sweden)

    A. Konti

    2017-10-01

    Full Text Available The biodegradation of two halohydrins (1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol by P. putida DSM 437 was investigated. Intact cells of previously acclimatized P. putida DSM 437 as well as cell-free extracts were used in order to study the degradation kinetics. When whole cells were used, a maximum biodegradation rate of 3-CPD (vmax = 1.28.10–5 mmol mg–1 DCW h–1 was determined, which was more than 4 times higher than that of 1,3-DCP. However, the affinity towards both halohydrins (Km was practically the same. When using cell-free extract, the apparent vmax and Km values for 1,3-DCP were estimated at 9.61.10–6 mmol mg–1 protein h–1 and 8.00 mM, respectively, while for 3-CPD the corresponding values were 2.42.10–5 mmol mg–1 protein h–1 and 9.07 mM. GC-MS analysis of cell-free extracts samples spiked with 1,3-DCP revealed the presence of 3-CPD and glycerol, intermediates of 1,3-DCP degradation pathway. 3-CPD degradation was strongly inhibited by the presence of epichlorohydrin and to a lesser extent by glycidol, intermediates of dehalogenation pathway.

  2. The Role of Lipid Droplets in Mortierella alpina Aging Revealed by Integrative Subcellular and Whole-Cell Proteome Analysis.

    Science.gov (United States)

    Yu, Yadong; Li, Tao; Wu, Na; Jiang, Ling; Ji, Xiaojun; Huang, He

    2017-03-07

    Lipid droplets (LDs) participate in many cellular processes in oleaginous microorganisms. However, the exact function of LDs in the Mortierella alpina aging process remains elusive. Herein, subcellular proteomics was employed to unveil the composition and dynamics of the LD proteome in the aging M. alpina for the first time. More than 400 proteins were detected in LDs and 62 of them changed expression significantly during aging. By combining the LD proteomic data with whole-cell data, we found that the carbohydrate metabolism and de novo lipid biosynthesis were all inhibited during aging of M. alpina mycelia. The up-regulation of fructose metabolism-related enzymes in LDs might imply that LDs facilitated the fructose metabolism, which in turn might cause pyruvate to accumulate and enter malate-pyruvate cycle, and ultimately, provide additional NADPH for the synthesis of arachidonic acid (ARA). Lysophospholipase and lecithinase were up-regulated in LDs during the aging process, suggesting that the phospholipids and lecithin were starting to be hydrolyzed, in order to release fatty acids for the cells. The impairment of the anti-oxidant system might lead to the accumulation of ROS and consequently cause the up-regulation of autophagy-related proteins in LDs, which further induces the M. alpina mycelia to activate the autophagy process.

  3. Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lu; Aryal, Uma K.; Dai, Ziyu; Mason, Alisa C.; Monroe, Matthew E.; Tian, Zhixin; Zhou, Jianying; Su, Dian; Weitz, Karl K.; Liu, Tao; Camp, David G.; Smith, Richard D.; Baker, Scott E.; Qian, Weijun

    2012-01-01

    Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 unique N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.

  4. Biosensor discovery of thyroxine transport disrupting chemicals

    International Nuclear Information System (INIS)

    Marchesini, Gerardo R.; Meimaridou, Anastasia; Haasnoot, Willem; Meulenberg, Eline; Albertus, Faywell; Mizuguchi, Mineyuki; Takeuchi, Makoto; Irth, Hubertus; Murk, Albertinka J.

    2008-01-01

    Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two inhibition assays using the main thyroid hormone transport proteins, T4 binding globulin (TBG) and transthyretin (TTR), in combination with a T4-coated biosensor chip were optimized and automated for screening chemical libraries. The transport protein-based biosensor assays were rapid, high throughput and bioeffect-related. A library of 62 chemicals including the natural hormones, polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals was tested with the two assays. We discovered ten new active compounds with moderate to high affinity for TBG with the TBG assay. Strikingly, the most potent binding was observed with hydroxylated metabolites of the brominated diphenyl ethers (BDEs) BDE 47, BDE 49 and BDE 99, that are commonly found in human plasma. The TTR assay confirmed the activity of previously identified hydroxylated metabolites of PCBs and PBDEs, halogenated BPA and genistein. These results show that the hydroxylated metabolites of the ubiquitous PBDEs not only target the T4 transport at the TTR level, but also, and to a great extent, at the TBG level where most of the T4 in humans is circulating. The optimized SPR biosensor-based transport protein assay is a suitable method for high throughput screening of large libraries for potential thyroid hormone disrupting compounds

  5. Biosensor discovery of thyroxine transport disrupting chemicals.

    Science.gov (United States)

    Marchesini, Gerardo R; Meimaridou, Anastasia; Haasnoot, Willem; Meulenberg, Eline; Albertus, Faywell; Mizuguchi, Mineyuki; Takeuchi, Makoto; Irth, Hubertus; Murk, Albertinka J

    2008-10-01

    Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two inhibition assays using the main thyroid hormone transport proteins, T4 binding globulin (TBG) and transthyretin (TTR), in combination with a T4-coated biosensor chip were optimized and automated for screening chemical libraries. The transport protein-based biosensor assays were rapid, high throughput and bioeffect-related. A library of 62 chemicals including the natural hormones, polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals was tested with the two assays. We discovered ten new active compounds with moderate to high affinity for TBG with the TBG assay. Strikingly, the most potent binding was observed with hydroxylated metabolites of the brominated diphenyl ethers (BDEs) BDE 47, BDE 49 and BDE 99, that are commonly found in human plasma. The TTR assay confirmed the activity of previously identified hydroxylated metabolites of PCBs and PBDEs, halogenated BPA and genistein. These results show that the hydroxylated metabolites of the ubiquitous PBDEs not only target the T4 transport at the TTR level, but also, and to a great extent, at the TBG level where most of the T4 in humans is circulating. The optimized SPR biosensor-based transport protein assay is a suitable method for high throughput screening of large libraries for potential thyroid hormone disrupting compounds.

  6. Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture

    International Nuclear Information System (INIS)

    Dold, K.M.; Greenlee, W.F.

    1990-01-01

    A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells

  7. 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.

  8. A novel whole-cell mechanism for long-term memory enhancement.

    Directory of Open Access Journals (Sweden)

    Iris Reuveni

    Full Text Available Olfactory-discrimination learning was shown to induce a profound long-lasting enhancement in the strength of excitatory and inhibitory synapses of pyramidal neurons in the piriform cortex. Notably, such enhancement was mostly pronounced in a sub-group of neurons, entailing about a quarter of the cell population. Here we first show that the prominent enhancement in the subset of cells is due to a process in which all excitatory synapses doubled their strength and that this increase was mediated by a single process in which the AMPA channel conductance was doubled. Moreover, using a neuronal-network model, we show how such a multiplicative whole-cell synaptic strengthening in a sub-group of cells that form a memory pattern, sub-serves a profound selective enhancement of this memory. Network modeling further predicts that synaptic inhibition should be modified by complex learning in a manner that much resembles synaptic excitation. Indeed, in a subset of neurons all GABAA-receptors mediated inhibitory synapses also doubled their strength after learning. Like synaptic excitation, Synaptic inhibition is also enhanced by two-fold increase of the single channel conductance. These findings suggest that crucial learning induces a multiplicative increase in strength of all excitatory and inhibitory synapses in a subset of cells, and that such an increase can serve as a long-term whole-cell mechanism to profoundly enhance an existing Hebbian-type memory. This mechanism does not act as synaptic plasticity mechanism that underlies memory formation but rather enhances the response of already existing memory. This mechanism is cell-specific rather than synapse-specific; it modifies the channel conductance rather than the number of channels and thus has the potential to be readily induced and un-induced by whole-cell transduction mechanisms.

  9. 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...

  10. Porous photonic crystal external cavity laser biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Qinglan [Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Peh, Jessie; Hergenrother, Paul J. [Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Cunningham, Brian T. [Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States)

    2016-08-15

    We report the design, fabrication, and testing of a photonic crystal (PC) biosensor structure that incorporates a porous high refractive index TiO{sub 2} dielectric film that enables immobilization of capture proteins within an enhanced surface-area volume that spatially overlaps with the regions of resonant electromagnetic fields where biomolecular binding can produce the greatest shifts in photonic crystal resonant wavelength. Despite the nanoscale porosity of the sensor structure, the PC slab exhibits narrowband and high efficiency resonant reflection, enabling the structure to serve as a wavelength-tunable element of an external cavity laser. In the context of sensing small molecule interactions with much larger immobilized proteins, we demonstrate that the porous structure provides 3.7× larger biosensor signals than an equivalent nonporous structure, while the external cavity laser (ECL) detection method provides capability for sensing picometer-scale shifts in the PC resonant wavelength caused by small molecule binding. The porous ECL achieves a record high figure of merit for label-free optical biosensors.

  11. 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.

  12. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

    2004-01-01

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  13. Personalized Whole-Cell Kinetic Models of Metabolism for Discovery in Genomics and Pharmacodynamics

    DEFF Research Database (Denmark)

    Bordbar, Aarash; McCloskey, Douglas; Zielinski, Daniel C

    2015-01-01

    Understanding individual variation is fundamental to personalized medicine. Yet interpreting complex phenotype data, such as multi-compartment metabolomic profiles, in the context of genotype data for an individual is complicated by interactions within and between cells and remains an unresolved...... challenge. Here, we constructed multi-omic, data-driven, personalized whole-cell kinetic models of erythrocyte metabolism for 24 healthy individuals based on fasting-state plasma and erythrocyte metabolomics and whole-genome genotyping. We show that personalized kinetic rate constants, rather than...

  14. Biosensor Regeneration: A Review of Common Techniques and Outcomes.

    Science.gov (United States)

    Goode, J A; Rushworth, J V H; Millner, P A

    2015-06-16

    Biosensors are ideally portable, low-cost tools for the rapid detection of pathogens, proteins, and other analytes. The global biosensor market is currently worth over 10 billion dollars annually and is a burgeoning field of interdisciplinary research that is hailed as a potential revolution in consumer, healthcare, and industrial testing. A key barrier to the widespread adoption of biosensors, however, is their cost. Although many systems have been validated in the laboratory setting and biosensors for a range of analytes are proven at the concept level, many have yet to make a strong commercial case for their acceptance. Though it is true with the development of cheaper electrodes, circuits, and components that there is a downward pressure on costs, there is also an emerging trend toward the development of multianalyte biosensors that is pushing in the other direction. One way to reduce the cost that is suitable for certain systems is to enable their reuse, thus reducing the cost per test. Regenerating biosensors is a technique that can often be used in conjunction with existing systems in order to reduce costs and accelerate the commercialization process. This article discusses the merits and drawbacks of regeneration schemes that have been proven in various biosensor systems and indicates parameters for successful regeneration based on a systematic review of the literature. It also outlines some of the difficulties encountered when considering the role of regeneration at the point of use. A brief meta-analysis has been included in this review to develop a working definition for biosensor regeneration, and using this analysis only ∼60% of the reported studies analyzed were deemed a success. This highlights the variation within the field and the need to normalize regeneration as a standard process across the field by establishing a consensus term.

  15. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

    Science.gov (United States)

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

    2015-10-01

    Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

  16. 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

  17. 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.

  18. Surface Patterning and Nanowire Biosensor Construction

    DEFF Research Database (Denmark)

    Iversen, Lars

    2008-01-01

    surface. A central limitation to this biosensor principle is the screening of analyte charge by mobile ions in electrolytes with physiological ionic strength. To overcome this problem, we propose to use as capture agents proteins which undergo large conformational changes. Using structure based protein...... charge prediction, we show how ligand induced changes in conformation of two model proteins, both being ligand binding domains from glutamate receptors, can lead to changes in electrostatic potential predicted to be sufficient for NW sensing. Finally we, demonstrate how InAs nanowires can....... In part I - “Surface Patterning” - glass and gold surfaces serve as spatially encoded immobilization supports for patterning of recombinant proteins and organic monolayers. First, we combine micro-contact printing with a reactive SNAP-tag protein to establish a general platform for templated protein...

  19. Enhanced productivity of gamma-amino butyric acid by cascade modifications of a whole-cell biocatalyst.

    Science.gov (United States)

    Yang, Xinwei; Ke, Chongrong; Zhu, Jiangming; Wang, Yan; Zeng, Wenchao; Huang, Jianzhong

    2018-04-01

    We previously developed a gamma-amino butyric acid (GABA)-producing strain of Escherichia coli, leading to production of 614.15 g/L GABA at 45 °C from L-glutamic acid (L-Glu) with a productivity of 40.94 g/L/h by three successive whole-cell conversion cycles. However, the increase in pH caused by the accumulation of GABA resulted in inactivation of the biocatalyst and consequently led to relatively lower productivity. In this study, by overcoming the major problem associated with the increase in pH during the production process, a more efficient biocatalyst was obtained through cascade modifications of the previously reported E. coli strain. First, we introduced four amino acid mutations to the codon-optimized GadB protein from Lactococcus lactis to shift its decarboxylation activity toward a neutral pH, resulting in 306.65 g/L of GABA with 99.14 mol% conversion yield and 69.8% increase in GABA productivity. Second, we promoted transportation of L-Glu and GABA by removing the genomic region encoding the C-plug of GadC (a glutamate/GABA antiporter) to allow its transport path to remain open at a neutral pH, which improved the GABA productivity by 16.8% with 99.3 mol% conversion of 3 M L-Glu. Third, we enhanced the expression of soluble GadB by introducing the GroESL molecular chaperones, leading to 20.2% improvement in GABA productivity, with 307.40 g/L of GABA and a 61.48 g/L/h productivity obtained in one cycle. Finally, we inhibited the degradation of GABA by inactivation of gadA and gadB from the E. coli genome, which resulted in almost no GABA degradation after 40 h. After the cascade system modifications, the engineered recombinant E. coli strain achieved a 44.04 g/L/h productivity with a 99.6 mol% conversion of 3 M L-Glu in a 5-L bioreactor, about twofold increase in productivity compared to the starting strain. This increase represents the highest GABA productivity by whole-cell bioconversion using L-Glu as a substrate in one cycle observed

  20. Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

    Science.gov (United States)

    Alderete, J F

    1984-06-01

    An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses during the development of lesions in animals inoculated subcutaneously and it reproducibly measured the individual classes immunoglobulins directed at T vaginalis. The colorimetric assay was also suitable for showing cross reactivity between trichomonal species as well as between different strains of T vaginalis. Conditions established for monitoring antibody to trichomanads in immunised rabbits or infected mice were equally effective for human materials, such as serum or vaginal washes. Serum from experimental animals or infected people showed high concentrations of IgG, IgA, and IgM antibody to trichomonads. Only antibodies of the IgG and IgA class were detected in vaginal washes from women with acute trichomoniasis. No IgE antibody to trichomonads was found under a variety of conditions in serum samples from patients or experimental animals.

  1. Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Takaya, Tomohiro; Koda, Risa; Adachi, Daisuke; Ogino, Chiaki; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering; Nakashima, Kazunori [Kobe Univ. (Japan). Organization of Advanced Science and Technology; Wada, Junpei; Bogaki, Takayuki [Ozeki Co., Nishinomiya-shi, Hyogo (Japan)

    2011-05-15

    In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5{sup '} untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL1) and double copies (pSENSU-FHL2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL1 and 2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time. (orig.)

  2. 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.

  3. Prediction of the limit of detection of an optical resonant reflection biosensor.

    Science.gov (United States)

    Hong, Jongcheol; Kim, Kyung-Hyun; Shin, Jae-Heon; Huh, Chul; Sung, Gun Yong

    2007-07-09

    A prediction of the limit of detection of an optical resonant reflection biosensor is presented. An optical resonant reflection biosensor using a guided-mode resonance filter is one of the most promising label-free optical immunosensors due to a sharp reflectance peak and a high sensitivity to the changes of optical path length. We have simulated this type of biosensor using rigorous coupled wave theory to calculate the limit of detection of the thickness of the target protein layer. Theoretically, our biosensor has an estimated ability to detect thickness change approximately the size of typical antigen proteins. We have also investigated the effects of the absorption and divergence of the incident light on the detection ability of the biosensor.

  4. Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis.

    Science.gov (United States)

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Liu, Long; Chen, Jian

    2014-01-01

    The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39μg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Aspergillus niger whole-cell catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids.

    Science.gov (United States)

    Rajapriya, Govindaraju; Morya, Vivek Kumar; Mai, Ngoc Lan; Koo, Yoon-Mo

    2018-04-01

    Synthesis of caffeic acid ester essentially requires an efficient esterification process to produce various kinds of medicinally important ester derivatives. In the present study, a comprehensive and comparative analysis of whole-cell catalyzed caffeic acid esters production in ionic liquids (ILs) media was performed. Olive oil induced mycelial mass of halotolerant Aspergillus niger (A.niger) EXF 4321 was freeze dried and used as a catalyst. To ensure maximum solubilization of caffeic acid for highest substrate loading several ILs were screened and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Emim][Tf 2 N]) was found to have the maximum solubility and favoured for enzymatic activity of freeze dried mycelia. The whole-cell catalyzed synthesis of caffeic acid phenethyl ester (CAPE) conditions were optimized and bioconversion up to 84% was achieved at a substrate molar ratio of 1:20 (caffeic acid:2-phenyl ethanol), 30°C for 12h. Results obtained during this study were encouraging and helpful to design a bioreactor system to produce caffeic acid derived esters. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Pathway-selective sensitization of Mycobacterium tuberculosis for target-based whole-cell screening

    Science.gov (United States)

    Abrahams, Garth L.; Kumar, Anuradha; Savvi, Suzana; Hung, Alvin W.; Wen, Shijun; Abell, Chris; Barry, Clifton E.; Sherman, David R.; Boshoff, Helena I.M.; Mizrahi, Valerie

    2012-01-01

    SUMMARY Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery but subsequent target elucidation often proves difficult. Conditional mutants that under-express essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA) and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered the mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery. PMID:22840772

  7. Production of human milk oligosaccharides by enzymatic and whole-cell microbial biotransformations.

    Science.gov (United States)

    Sprenger, Georg A; Baumgärtner, Florian; Albermann, Christoph

    2017-09-20

    Human milk oligosaccharides (HMO) are almost unique constituents of breast milk and are not found in appreciable amounts in cow milk. Due to several positive aspects of HMO for the development, health, and wellbeing of infants, production of HMO would be desirable. As a result, scientists from different disciplines have developed methods for the preparation of single HMO compounds. Here, we review approaches to HMO preparation by (chemo-)enzymatic syntheses or by whole-cell biotransformation with recombinant bacterial cells. With lactose as acceptor (in vitro or in vivo), fucosyltransferases can be used for the production of 2'-fucosyllactose, 3-fucosyllactose, or more complex fucosylated core structures. Sialylated HMO can be produced by sialyltransferases and trans-sialidases. Core structures as lacto-N-tetraose can be obtained by glycosyltransferases from chemical donor compounds or by multi-enzyme cascades; recent publications also show production of lacto-N-tetraose by recombinant Escherichia coli bacteria and approaches to obtain fucosylated core structures. In view of an industrial production of HMOs, the whole cell biotransformation is at this stage the most promising option to provide human milk oligosaccharides as food additive. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

    2016-07-28

    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

  9. Redox self-sufficient whole cell biotransformation for amination of alcohols.

    Science.gov (United States)

    Klatte, Stephanie; Wendisch, Volker F

    2014-10-15

    Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD(+) was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. 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...

  11. Recent Advances in Application of Biosensors in Tissue Engineering

    Science.gov (United States)

    Paul, Arghya; Lee, Yong-kyu; Jaffa, Ayad A.

    2014-01-01

    Biosensors research is a fast growing field in which tens of thousands of papers have been published over the years, and the industry is now worth billions of dollars. The biosensor products have found their applications in numerous industries including food and beverages, agricultural, environmental, medical diagnostics, and pharmaceutical industries and many more. Even though numerous biosensors have been developed for detection of proteins, peptides, enzymes, and numerous other biomolecules for diverse applications, their applications in tissue engineering have remained limited. In recent years, there has been a growing interest in application of novel biosensors in cell culture and tissue engineering, for example, real-time detection of small molecules such as glucose, lactose, and H2O2 as well as serum proteins of large molecular size, such as albumin and alpha-fetoprotein, and inflammatory cytokines, such as IFN-g and TNF-α. In this review, we provide an overview of the recent advancements in biosensors for tissue engineering applications. PMID:25165697

  12. Recent Advances in Application of Biosensors in Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Anwarul Hasan

    2014-01-01

    Full Text Available Biosensors research is a fast growing field in which tens of thousands of papers have been published over the years, and the industry is now worth billions of dollars. The biosensor products have found their applications in numerous industries including food and beverages, agricultural, environmental, medical diagnostics, and pharmaceutical industries and many more. Even though numerous biosensors have been developed for detection of proteins, peptides, enzymes, and numerous other biomolecules for diverse applications, their applications in tissue engineering have remained limited. In recent years, there has been a growing interest in application of novel biosensors in cell culture and tissue engineering, for example, real-time detection of small molecules such as glucose, lactose, and H2O2 as well as serum proteins of large molecular size, such as albumin and alpha-fetoprotein, and inflammatory cytokines, such as IFN-g and TNF-α. In this review, we provide an overview of the recent advancements in biosensors for tissue engineering applications.

  13. Analytical solution using computer algebra of a biosensor for detecting toxic substances in water

    Science.gov (United States)

    Rúa Taborda, María. Isabel

    2014-05-01

    In a relatively recent paper an electrochemical biosensor for water toxicity detection based on a bio-chip as a whole cell was proposed and numerically solved and analyzed. In such paper the kinetic processes in a miniaturized electrochemical biosensor system was described using the equations for specific enzymatic reaction and the diffusion equation. The numerical solution shown excellent agreement with the measured data but such numerical solution is not enough to design efficiently the corresponding bio-chip. For this reason an analytical solution is demanded. The object of the present work is to provide such analytical solution and then to give algebraic guides to design the bio-sensor. The analytical solution is obtained using computer algebra software, specifically Maple. The method of solution is the Laplace transform, with Bromwich integral and residue theorem. The final solution is given as a series of Bessel functions and the effective time for the bio-sensor is computed. It is claimed that the analytical solutions that were obtained will be very useful to predict further current variations in similar systems with different geometries, materials and biological components. Beside of this the analytical solution that we provide is very useful to investigate the relationship between different chamber parameters such as cell radius and height; and electrode radius.

  14. The benzoquinone-mediated electrochemical microbial biosensor for water biotoxicity assay

    International Nuclear Information System (INIS)

    Li, Jiuming; Yu, Yuan; Wang, Yuning; Qian, Jun; Zhi, Jinfang

    2013-01-01

    Graphical abstract: The mediator can participate in microorganism respiration, accept the electrons from respiratory chains, and therefore be reduced by microorganism. The re-oxidization currents of mediators on electrode can reflect the microbial activity, and when respiration is suppressed by toxicants, it can be detected by the resulting change of currents. Unlike other biotoxicity tests, which record the toxic effect after a fixed time for incubation of biocomponents and toxicants, this mediated whole cell biosensor can provide a real-time monitor of the microbial activity during the measurement. -- Abstract: A simple mediated microbial biosensor providing real-time monitoring of water quality and evaluation of biotoxicity was fabricated by entrapping Escherichia coli (E. coli) cells in gelatin on glassy carbon electrode with benzoquinone as the redox mediator. The biotoxicity assay was based on the respiratory activity of E. coli cells estimated by the oxidation current of microbially reduced benzoquinone. The neutrality and lipophilicity rendered benzoquinone better efficiency than ferricyanide in mediated microbial reactions. After the optimization of preparation conditions, the prepared microbial biosensors have measured several common toxicants with different concentrations. In addition, the biotoxicity of binary mixtures of heavy metals and wastewater were investigated. The fabricated biosensor exhibited good repeatability and stability in the biotoxicity measurements

  15. Advances in in-situ product recovery (ISPR) in whole cell biotechnology during the last decade.

    Science.gov (United States)

    Van Hecke, Wouter; Kaur, Guneet; De Wever, Heleen

    2014-11-15

    The review presents the state-of-the-art in the applications of in-situ product recovery (ISPR) in whole-cell biotechnology over the last 10years. It summarizes various ISPR-integrated fermentation processes for the production of a wide spectrum of bio-based products. A critical assessment of the performance of various ISPR concepts with respect to the degree of product enrichment, improved productivity, reduced process flows and increased yields is provided. Requirements to allow a successful industrial implementation of ISPR are also discussed. Finally, supporting technologies such as online monitoring, mathematical modeling and use of recombinant microorganisms with ISPR are presented. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Flow cytogenetic studies in chromosomes and whole cells for the detection of clastogenic effects

    International Nuclear Information System (INIS)

    Otto, F.J.; Oldiges, H.

    1980-01-01

    Flow cytometric measurements of the chromosomal DNA content have been used to develop a screening method for the detection of chemically- or physically-induced cytogenetic damage. The reproducibility of this flow cytogenetic assay was shown in a series of subcultures of a Chinese hamster cell clone. The accuracy and sensitivity was tested in cultures treated with chemical mutagens and x-rays. The clastogenic effectiveness was quantified and the dose-effect relationship was established by the increase of the coefficient of variation of the peak of the largest chromosome type in the flow histograms. Since structural chromosome aberrations cause an unequal division of the DNA at mitosis, it is expected that clastogenic effects can be detected also in whole cells of growing populations as an increased dispersion of the cellular DNA content. In order to test this feature, high resolution flow cytometric measurements were performed in x-irradiated hamster cells in vitro and mouse bone marrow cells in vivo

  17. Malaria chemoprophylaxis and the serologic response to measles and diphtheria-tetanus-whole-cell pertussis vaccines

    Directory of Open Access Journals (Sweden)

    Saliou Pierre

    2005-11-01

    Full Text Available Abstract Background Acute malaria has been associated with a decreased antibody response to tetanus and diphtheria toxoids, meningococcal, salmonella, and Hib vaccines. Interest in giving malaria drug therapy and prevention at the time of childhood immunizations has increased greatly following recent trials of intermittent preventive therapy during infancy (IPTi, stimulating this re-analysis of unpublished data. The effect of malaria chemoprophylaxis on vaccine response was studied following administration of measles vaccines and diphtheria-tetanus-whole cell pertussis (DTP vaccines. Methods In 1975, six villages divided into two groups of children ≤74 months of age from Burkina Faso, were assigned to receive amodiaquine hydrochloride chemoprophylaxis (CH+ every two weeks for seven months or no chemoprophylaxis (CH-. After five months, children in each group received either one dose of measles or two doses of DTP vaccines. Results For recipients of the measles vaccine, the seroconversion rates in CH+ and CH- children, respectively, were 93% and 96% (P > 0.05. The seroresponse rates in CH+ and CH- children respectively, were 73% and 86% for diphtheria (P > 0.05 and 77% and 91% for tetanus toxoid (P > 0.05. In a subset analysis, in which only children who strictly adhered to chemoprophylaxis criteria were included, there were, likewise, no significant differences in seroconversion or seroresponse for measles, diphtheria, or tetanus vaccines (P > 0.05. While analysis for pertussis showed a 43% (CH+ and 67% (CH- response (P Conclusion Malaria chemoprophylaxis prior to vaccination in malaria endemic settings did not improve or impair immunogenicity of DTP and measles vaccines. This is the first human study to look at the association between malaria chemoprophylaxis and the serologic response to whole-cell pertussis vaccine.

  18. Whole cell Deinococcus radiodurans ameliorates salt stress in Indian mustard through pyrroloquinoline quinone

    International Nuclear Information System (INIS)

    Srivastava, A.K.; Jadhav, P.; Suprasanna, P.; Rajpurohit, Y.S.; Misra, H.S.

    2015-01-01

    Salinity stress is considered as one of the major abiotic stresses limiting crop productivity. A variety of symbiotic and non-symbiotic bacteria are currently being used worldwide with the aim to boost built-in defense system in plants. Deinococcus radiodurans is a highly desiccation and radiation tolerant bacterium which synthesizes PQQ (pyrroloquinoline quinone) that has been shown to have a versatile role in crop productivity and as a general stress response regulator in bacteria and mammals. PQQ also acts as scavenger of reactive oxygen species and hence, can module redox signaling, one of the major regulator of stress tolerance in plants. In view of this, present research was conducted to evaluate the potential of whole cell D. radiodurans for ameliorating salt stress in plants. The soil colonization with wild-type cells led to partial amelioration of salt stress. The PQQ mutant showed an intermediate phenotype between wild-type seedlings and those grown on non-colonized soils which confirmed that the effects are largely associated with PQQ. The differential phenotype was also correlated with ROS level and ABA accumulation. The flame photometry data showed that there was no significant reduction in water soluble Na + level in control plant and those treated with either wild-type or PQQ mutant. Further, the elevated levels of antioxidant enzymes and reduced ascorbate in the plants treated with bacterial cells indicated its positive role in oxidative stress management. Although, the exact molecular basis to these effects is yet to be understood, present findings support the use of whole cell D. radiodurans for managing the growth and productivity of Indian mustard in salt affected fields. (author)

  19. A whole-cell bioreporter assay for quantitative genotoxicity evaluation of environmental samples.

    Science.gov (United States)

    Jiang, Bo; Li, Guanghe; Xing, Yi; Zhang, Dayi; Jia, Jianli; Cui, Zhisong; Luan, Xiao; Tang, Hui

    2017-10-01

    Whole-cell bioreporters have emerged as promising tools for genotoxicity evaluation, due to their rapidity, cost-effectiveness, sensitivity and selectivity. In this study, a method for detecting genotoxicity in environmental samples was developed using the bioluminescent whole-cell bioreporter Escherichia coli recA::luxCDABE. To further test its performance in a real world scenario, the E. coli bioreporter was applied in two cases: i) soil samples collected from chromium(VI) contaminated sites; ii) crude oil contaminated seawater collected after the Jiaozhou Bay oil spill which occurred in 2013. The chromium(VI) contaminated soils were pretreated by water extraction, and directly exposed to the bioreporter in two phases: aqueous soil extraction (water phase) and soil supernatant (solid phase). The results indicated that both extractable and soil particle fixed chromium(VI) were bioavailable to the bioreporter, and the solid-phase contact bioreporter assay provided a more precise evaluation of soil genotoxicity. For crude oil contaminated seawater, the response of the bioreporter clearly illustrated the spatial and time change in genotoxicity surrounding the spill site, suggesting that the crude oil degradation process decreased the genotoxic risk to ecosystem. In addition, the performance of the bioreporter was simulated by a modified cross-regulation gene expression model, which quantitatively described the DNA damage response of the E. coli bioreporter. Accordingly, the bioluminescent response of the bioreporter was calculated as the mitomycin C equivalent, enabling quantitative comparison of genotoxicities between different environmental samples. This bioreporter assay provides a rapid and sensitive screening tool for direct genotoxicity assessment of environmental samples. Copyright © 2017. Published by Elsevier Ltd.

  20. Oriented antibodies as versatile detection element in biosensors

    NARCIS (Netherlands)

    Trilling, A.K.

    2013-01-01

    The aim of this thesis is to explore orientation of detection elements on biosensor

    surfaces. To this end, different strategies were combined such as surface chemistry and protein functionalization, with the aim to generate a platform for oriented immobilization of antibodies

    in

  1. Orientation of llama antibodies strongly increases sensitivity of biosensors

    NARCIS (Netherlands)

    Trilling, A.K.; Hesselink, T.; Houwelingen, van A.; Cordewener, J.H.G.; Jongsma, M.A.; Schoffelen, S.; Hest, van J.C.M.; Zuilhof, J.T.; Beekwilder, J.

    2014-01-01

    Sensitivity of biosensors depends on theorientation of bio-receptors on the sensor surface.The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV)

  2. Synthesis and assessment of peptide-nanocellulosic biosensors

    Science.gov (United States)

    Nanocellulose is an ideal transducer surface for biosensors: it provides a high surface area, easily derivatized with bioactive molecules, and abrogates binding of proteins present in biological fluids where analytes and clinical biomarkers are of interest. Here an example of approaches to biosenso...

  3. 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.

  4. 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.

  5. More About Thin-Membrane Biosensor

    Science.gov (United States)

    Case, George D.; Worley, Jennings F., III

    1994-01-01

    Report presents additional information about device described in "Thin-Membrane Sensor With Biochemical Switch" (MFS-26121). Device is modular sensor that puts out electrical signal indicative of chemical or biological agent. Signal produced as membrane-crossing ion current triggered by chemical reaction between agent and recognition protein conjugated to channel blocker. Prototype of biosensor useful in numerous laboratory, industrial, or field applications; such as to detect bacterial toxins in food, to screen for disease-producing micro-organisms, or to warn of toxins or pollutants in air.

  6. 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.

  7. Wireless implantable electronic platform for chronic fluorescent-based biosensors.

    Science.gov (United States)

    Valdastri, Pietro; Susilo, Ekawahyu; Förster, Thilo; Strohhöfer, Christof; Menciassi, Arianna; Dario, Paolo

    2011-06-01

    The development of a long-term wireless implantable biosensor based on fluorescence intensity measurement poses a number of technical challenges, ranging from biocompatibility to sensor stability over time. One of these challenges is the design of a power efficient and miniaturized electronics, enabling the biosensor to move from bench testing to long term validation, up to its final application in human beings. In this spirit, we present a wireless programmable electronic platform for implantable chronic monitoring of fluorescent-based autonomous biosensors. This system is able to achieve extremely low power operation with bidirectional telemetry, based on the IEEE802.15.4-2003 protocol, thus enabling over three-year battery lifetime and wireless networking of multiple sensors. During the performance of single fluorescent-based sensor measurements, the circuit drives a laser diode, for sensor excitation, and acquires the amplified signals from four different photodetectors. In vitro functionality was preliminarily tested for both glucose and calcium monitoring, simply by changing the analyte-binding protein of the biosensor. Electronics performance was assessed in terms of timing, power consumption, tissue exposure to electromagnetic fields, and in vivo wireless connectivity. The final goal of the presented platform is to be integrated in a complete system for blood glucose level monitoring that may be implanted for at least one year under the skin of diabetic patients. Results reported in this paper may be applied to a wide variety of biosensors based on fluorescence intensity measurement.

  8. Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

    Science.gov (United States)

    Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

    2017-09-01

    Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

  9. Arsenic bioavailability in soils before and after soil washing: the use of Escherichia coli whole-cell bioreporters.

    Science.gov (United States)

    Yoon, Youngdae; Kang, Yerin; Chae, Yooeun; Kim, Sunghoon; Lee, Youngshim; Jeong, Seung-Woo; An, Youn-Joo

    2016-02-01

    We investigated the quantification of bioavailable arsenic in contaminated soils and evaluation of soil-washing processes in the aspect of bioavailability using a novel bacterial bioreporter developed in present study. The whole-cell bioreporter (WCB) was genetically engineered by fusing the promoter of nik operon from Escherichia coli and green fluorescent protein as a sensing domain and reporter domain. Among eight well-known hazardous heavy metals and metalloid, this system responded specifically to arsenic, thereby inferring association of As(III) with NikR inhibits the repression. Moreover, the response was proportional to the concentration of As(III), thereby it was capable to determine the amount of bioavailable arsenic quantitatively in contaminated soils. The bioavailable portion of arsenic was 5.9 (3.46-10.96) and 0.9 (0.27-1.74) % of total from amended and site soils, respectively, suggesting the bioavailability of arsenic in soils was related to the soil properties and duration of aging. On the other hand, only 1.37 (0.21-2.97) % of total arsenic was extracted into soil solutions and 19.88 (11.86-28.27) % of arsenic in soil solution was bioavailable. This result showed that the soluble arsenic is not all bioavailable and most of bioavailable arsenic in soils is water non-extractable. In addition, the bioavailable arsenic was increased after soil-washing while total amount was decreased, thereby suggesting the soil-washing processes release arsenic associated with soil materials to be bioavailable. Therefore, it would be valuable to have a tool to assess bioavailability and the bioavailability should be taken into consideration for soil remediation plans.

  10. Lignin and silicate based hydrogels for biosensor applications

    Science.gov (United States)

    Burrs, S. L.; Jairam, S.; Vanegas, D. C.; Tong, Z.; McLamore, E. S.

    2013-05-01

    Advances in biocompatible materials and electrocatalytic nanomaterials have extended and enhanced the field of biosensors. Immobilization of biorecognition elements on nanomaterial platforms is an efficient technique for developing high fidelity biosensors. Single layer (i.e., Langmuir-Blodgett) protein films are efficient, but disadvantages of this approach include high cost, mass transfer limitations, and Vromer competition for surface binding sites. There is a need for simple, user friendly protein-nanomaterial sensing membranes that can be developed in laboratories or classrooms (i.e., outside of the clean room). In this research, we develop high fidelity nanomaterial platforms for developing electrochemical biosensors using sustainable biomaterials and user-friendly deposition techniques. Catalytic nanomaterial platforms are developed using a combination of self assembled monolayer chemistry and electrodeposition. High performance biomaterials (e.g., nanolignin) are recovered from paper pulp waste and combined with proteins and nanomaterials to form active sensor membranes. These methods are being used to develop electrochemical biosensors for studying physiological transport in biomedical, agricultural, and environmental applications.

  11. 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...

  12. Surface complexation of neptunium (V) onto whole cells and cell componets of Shewanella alga

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Donald Timothy [Los Alamos National Laboratory; Deo, Randhir P [ASU; Rittmann, Bruce E [ASU; Songkasiri, Warinthorn [UNAFFILIATED

    2008-01-01

    We systematically quantified surface complexation of neptunium(V) onto whole cells of Shewanella alga strain BrY and onto cell wall and extracellular polymeric substances (EPS) of S. alga. We first performed acid and base titrations and used the mathematical model FITEQL with constant-capacitance surface-complexation to determine the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl site associated with amino acids (pK{sub a} {approx} 2.4), a carboxyl group not associated with amino acids (pK{sub a} {approx} 5), a phosphoryl site (pK{sub a} {approx} 7.2), and an amine site (pK{sub a} > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components at different pHs. Results show that solution pH influenced the speciation of Np(V) and each of the surface functional groups. We used the speciation sub-model of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, NpO{sub 2}{sup +} was the dominant form of Np(V), and its log K values for the low-pK{sub a} carboxyl, other carboxyl, and phosphoryl groups were 1.75, 1.75, and 2.5 to 3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH3+, which complexed with NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-}. The log K for NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-} complexed onto the amine groups was 3.1 to 3.6. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results point towards the important role of surface complexation in defining key actinide-microbiological interactions in the subsurface.

  13. 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...

  14. 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.

  15. 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.

  16. Co-Expression of ORFCma with PHB Depolymerase (PhaZCma ) in Escherichia coli Induces Efficient Whole-Cell Biodegradation of Polyesters.

    Science.gov (United States)

    Lee, Ming-Chieh; Liu, En-Jung; Yang, Cheng-Han; Hsiao, Li-Jung; Wu, Tzong-Ming; Li, Si-Yu

    2018-04-01

    Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZ Cma , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORF Cma (a putative periplasmic substrate binding protein that is within the same operon of phaZ Cma ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZ Cma has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORF Cma can potentially be a universal element for enhancing the secretion of recombinant protein. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Evaluation of Optical Detection Platforms for Multiplexed Detection of Proteins and the Need for Point-of-Care Biosensors for Clinical Use

    Directory of Open Access Journals (Sweden)

    Samantha Spindel

    2014-11-01

    Full Text Available This review investigates optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks encompassing multiple, rather than single, proteins. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots or with the use of unique identifiers/labels (such as spectral separation—different colored dyes, or unique beads—size or color. The strengths and weaknesses of conventional platforms such as immunoassays and new platforms involving protein arrays and lab-on-a-chip technology, including commercially-available devices, are discussed. Three major public health concerns are identified whereby detecting medically-relevant markers using Point-of-Care (POC multiplex assays could potentially allow for a more efficient diagnosis and treatment of diseases.

  18. 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.

  19. Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

    Science.gov (United States)

    Pijanka, Jacek K; Stone, Nicholas; Rutter, Abigail V; Forsyth, Nicholas; Sockalingum, Ganesh D; Yang, Ying; Sulé-Suso, Josep

    2013-09-07

    Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

  20. Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester.

    Science.gov (United States)

    Majewska, Paulina; Serafin, Monika; Klimek-Ochab, Magdalena; Brzezińska-Rodak, Małgorzata; Żymańczyk-Duda, Ewa

    2016-06-01

    A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68-93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50-55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids). Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Fast detection of extrasynaptic GABA with a whole-cell sniffer

    Directory of Open Access Journals (Sweden)

    Rasmus Kordt Christensen

    2014-05-01

    Full Text Available Gamma-amino-butyric acid (GABA is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a Human Embryonic Kidney (HEK cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a sniffer allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations.

  2. Fast detection of extrasynaptic GABA with a whole-cell sniffer.

    Science.gov (United States)

    Christensen, Rasmus K; Petersen, Anders V; Schmitt, Nicole; Perrier, Jean-François

    2014-01-01

    Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a "sniffer" allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations.

  3. Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM.

    Science.gov (United States)

    Zheng, Zhaojuan; Ma, Cuiqing; Gao, Chao; Li, Fengsong; Qin, Jiayang; Zhang, Haiwei; Wang, Kai; Xu, Ping

    2011-04-20

    Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal. A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l(-1)) and high productivity (2.3 g l(-1) h(-1)) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.

  4. Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM.

    Directory of Open Access Journals (Sweden)

    Zhaojuan Zheng

    2011-04-01

    Full Text Available Phenyllactic acid (PLA, a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA into PLA are equivocal.A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l(-1 and high productivity (2.3 g l(-1 h(-1 under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA.Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.

  5. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

    Science.gov (United States)

    Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

    2014-10-01

    Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

  6. Toxicity assessment and modelling of Moringa oleifera seeds in water purification by whole cell bioreporter.

    Science.gov (United States)

    Al-Anizi, Ali Adnan; Hellyer, Maria Theresa; Zhang, Dayi

    2014-06-01

    Moringa oleifera has been used as a coagulation reagent for drinking water purification, especially in developing countries such as Malawi. This research revealed the cytoxicity and genotoxicity of M. oleifera by Acinetobacter bioreporter. The results indicated that significant cytoxicity effects were observed when the powdered M. oleifera seeds concentration is from 1 to 50 mg/L. Through direct contact, ethanolic-water extraction and hexane extraction, the toxic effects of hydrophobic and hydrophilic components in M. oleifera seeds were distinguished. It suggested that the hydrophobic lipids contributed to the dominant cytoxicity, consequently resulting in the dominant genotoxicity in the water-soluble fraction due to limited dissolution when the M. oleifera seeds granule concentration was from 10 to 1000 mg/L. Based on cytoxicity and genotoxicity model, the LC50 and LC90 of M. oleifera seeds were 8.5 mg/L and 300 mg/L respectively and their genotoxicity was equivalent to 8.3 mg mitomycin C per 1.0 g dry M. oleifera seed. The toxicity of M. oleifera has also remarkable synergistic effects, suggesting whole cell bioreporter as an appropriate and complementary tool to chemical analysis for environmental toxicity assessment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Evaluating the impact of substrate and product concentration on a whole-cell biocatalyst during a Baeyer-Villiger reaction

    DEFF Research Database (Denmark)

    Shitu, J. O.; Chartrain, M.; Woodley, John

    2009-01-01

    The presence of high concentrations of substrate or product may impede the optimal functioning of a biocatalyst, more so in the case of whole cell biocatalysts where the metabolic status of the cells may be compromised. In this article we investigate these effects using as an example the Baeyer-V...

  8. Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.

    Science.gov (United States)

    Jose, Joachim; von Schwichow, Steffen

    2004-04-02

    Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

  9. Producing biodiesel from cotton seed oil using Rhizopus oryzae ATTC #34612 whole cell biocatalysts: Culture media and cultivation period optimization

    Science.gov (United States)

    The effect of culture medium composition and cultivation time on biodiesel production by Rhizopus oryzae ATCC #34612 whole cell catalysts, immobilized on novel rigid polyethylene biomass supports, was investigated. Supplementation of the medium with carbon sources led to higher lipase activity and i...

  10. Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

    DEFF Research Database (Denmark)

    Knudsen, Jenny Dahl; Hägglöf, Cecilia; Weber, Nora

    2016-01-01

    yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated. RESULTS: A series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol...

  11. A novel microfluidic chip electrophoresis strategy for simultaneous, label-free, multi-protein detection based on a graphene energy transfer biosensor.

    Science.gov (United States)

    Lin, Fengming; Zhao, Xiaochao; Wang, Jianshe; Yu, Shiyong; Deng, Yulin; Geng, Lina; Li, HuanJun

    2014-06-07

    A new type of high-throughput and parallel optical sensing platform with a single-color probe based on microfluidic chip electrophoresis combined with aptamer-carboxyfluorescein/graphene oxide energy transfer is reported here. Label-free protein multi-targets were detected, even in challenging complex samples without any pre-treatment.

  12. The remarkable similarity between the acid-base properties of ISFETs and proteins and the consequences for the design of ISFET biosensors

    NARCIS (Netherlands)

    Bergveld, Piet; van Hal, R.E.G.; van Hal, R.E.G.; Eijkel, Jan C.T.

    1995-01-01

    Studying the acid-base properties of protein molecules led us to reconsider the operational mechanism of ISFETs. Based on the site-dissociation model, applied to the amphoteric metal oxide gate materials used in ISFETs, the sensitivity of ISFETs is described in terms of the intrinsic buffer capacity

  13. Determination of hydrophobic coenzyme a esters and other lipids using a biosensor comprising a modified coenzyme a- and acyl-coa binding protein (acbp)

    DEFF Research Database (Denmark)

    2002-01-01

    , food and feed preparations, tissue extracts, acyl-CoA synthetase reaction media and various laboratory conditions using a modified Coenzyme A- and acyl-CoA binding protein (ACBP) is provided. Furthermore the invention relates to a construct comprising a peptide and a signal moiety for performing...

  14. 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.

  15. A novel nano-photonics biosensor concept for rapid molecular diagnostics

    Science.gov (United States)

    Klunder, Dion J. W.; van Herpen, Maarten M. J. W.; Kolesnychenko, Aleksey; Hornix, Eefje; Kahya, Nicoletta; de Boer, Ruth; Stapert, Henk

    2008-04-01

    We present a novel nano-photonics biosensor concept that offers an ultra-high surface specificity and excellent suppression of background signals due to the sample fluid on top of the biosensor. In our contribution, we will briefly discuss the operation principle and fabrication of the biosensor, followed by a more detailed discussion on the experimentally determined performance parameters. Recent results on detection of fluorescently labeled molecules in a highly fluorescent background will be shown, and we will give an outlook on real-time detection of bio-molecules such as proteins and nucleic acids.

  16. Theory and Applications of Surface Plasmon Resonance, Resonant Mirror, Resonant Waveguide Grating, and Dual Polarization Interferometry Biosensors

    Directory of Open Access Journals (Sweden)

    Billy W. Day

    2010-11-01

    Full Text Available Biosensors have been used extensively in the scientific community for several purposes, most notably to determine association and dissociation kinetics, protein-ligand, protein-protein, or nucleic acid hybridization interactions. A number of different types of biosensors are available in the field, each with real or perceived benefits over the others. This review discusses the basic theory and operational arrangements of four commercially available types of optical biosensors: surface plasmon resonance, resonant mirror, resonance waveguide grating, and dual polarization interferometry. The different applications these techniques offer are discussed from experiments and results reported in recently published literature. Additionally, recent advancements or modifications to the current techniques are also discussed.

  17. 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...

  18. 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, ...

  19. Engineering Pseudomonas stutzeri as a biogeochemical biosensor

    Science.gov (United States)

    Boynton, L.; Cheng, H. Y.; Del Valle, I.; Masiello, C. A.; Silberg, J. J.

    2016-12-01

    Biogeochemical cycles are being drastically altered as a result of anthropogenic activities, such as the burning of fossil fuels and the industrial production of ammonia. We know microbes play a major part in these cycles, but the extent of their biogeochemical roles remains largely uncharacterized due to inadequacies with culturing and measurement. While metagenomics and other -omics methods offer ways to reconstruct microbial communities, these approaches can only give an indication of the functional roles of microbes in a community. These -omics approaches are rapidly being expanded to the point of outpacing our knowledge of functional genes, which highlights an inherent need for analytical methods that non-invasively monitor Earth's processes in real time. Here we aim to exploit synthetic biology methods in order to engineer a ubiquitous denitrifying microbe, Pseudomonas stutzeri that can act as a biosensor in soil and marine environments. By using an easily cultivated microbe that is also common in many environments, we hope to develop a tool that allows us to zoom in on specific aspects of the nitrogen cycle. In order to monitor processes occurring at the genetic level in environments that cannot be resolved with fluorescence-based methods, such as soils, we have developed a system that instead relies on gas production by engineered microbial biosensors. P. stutzeri has been successfully engineered to release a gas, methyl bromide, which can continuously and non-invasively be measured by GC-MS. Similar to using Green Fluorescent Protein, GFP, in the biological sciences, the gene controlling gas production can be linked to those involved in denitrification, thereby creating a quantifiable gas signal that is correlated with microbial activity in the soil. Synthetically engineered microbial biosensors could reveal key aspects of metabolism in soil systems and offer a tool for characterizing the scope and degree of microbial impact on major biogeochemical cycles.

  20. The remarkable similarity between the acid-base properties of ISFETs and proteins and the consequences for the design of ISFET biosensors

    OpenAIRE

    Bergveld, Piet; van Hal, R.E.G.; van Hal, R.E.G.; Eijkel, Jan C.T.

    1995-01-01

    Studying the acid-base properties of protein molecules led us to reconsider the operational mechanism of ISFETs. Based on the site-dissociation model, applied to the amphoteric metal oxide gate materials used in ISFETs, the sensitivity of ISFETs is described in terms of the intrinsic buffer capacity of the oxide surface, ßs, and the electrical surface capacitance, Cs. The ISFET sensitivity towards changes in the bulk pH is fully described by the ratio ßs/Cs. Practical measurements support thi...

  1. 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.

  2. Calcium phosphate-gold nanoparticles nanocomposite for protein adsorption and mediator-free H{sub 2}O{sub 2} biosensor construction

    Energy Technology Data Exchange (ETDEWEB)

    Xu Qin [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China); Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou 225002 (China); Lu Guiju; Bian XiaoJun; Jin Gendi [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China); Wang Wei [School of Chemical and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051 (China); Hu Xiaoya, E-mail: xyhu@yzu.edu.cn [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China); Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou 225002 (China); Wang Yang; Yang Zhanjun [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China)

    2012-04-01

    This work reports a new method for the preparation and application of a kind of biocompatible calcium phosphate-gold nanoparticles (Ca{sub 3}(PO{sub 4}){sub 2}-AuNPs) nanocomposite. UV-vis spectroscopy and transmittance electron microscopy (TEM) have been used to monitor the formation process of the nanocomposite and to examine the interaction between calcium phosphate and gold nanoparticles (AuNPs). The nanocomposite has multiple sites and improved conductivity which make it suitable for the binding of proteins to construct electrochemical sensors. Myoglobin (Mb) adsorbed on the nanocomposite retained its native structure which was proved by Fourier transform infrared spectroscopy (FTIR). Direct electron transfer between the adsorbed Mb and the electrode was observed. Further results demonstrated that the adsorbed Mb has good electrocatalytic activity towards the reduction of H{sub 2}O{sub 2} in the absence of any mediator. Highlights: Black-Right-Pointing-Pointer Using gelatin modified gold nanoparticles to prepare needle-like calcium phosphate. Black-Right-Pointing-Pointer Calcium phosphate provides multiple sites for protein adsorption. Black-Right-Pointing-Pointer Gold nanoparticles act as electron tunneling. Black-Right-Pointing-Pointer Myoglobin adsorbed on the material showed direct electrochemistry and good catalysis.

  3. 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.

  4. Photonic crystals: emerging biosensors and their promise for point-of-care applications.

    Science.gov (United States)

    Inan, Hakan; Poyraz, Muhammet; Inci, Fatih; Lifson, Mark A; Baday, Murat; Cunningham, Brian T; Demirci, Utkan

    2017-01-23

    Biosensors are extensively employed for diagnosing a broad array of diseases and disorders in clinical settings worldwide. The implementation of biosensors at the point-of-care (POC), such as at primary clinics or the bedside, faces impediments because they may require highly trained personnel, have long assay times, large sizes, and high instrumental cost. Thus, there exists a need to develop inexpensive, reliable, user-friendly, and compact biosensing systems at the POC. Biosensors incorporated with photonic crystal (PC) structures hold promise to address many of the aforementioned challenges facing the development of new POC diagnostics. Currently, PC-based biosensors have been employed for detecting a variety of biotargets, such as cells, pathogens, proteins, antibodies, and nucleic acids, with high efficiency and selectivity. In this review, we provide a broad overview of PCs by explaining their structures, fabrication techniques, and sensing principles. Furthermore, we discuss recent applications of PC-based biosensors incorporated with emerging technologies, including telemedicine, flexible and wearable sensing, smart materials and metamaterials. Finally, we discuss current challenges associated with existing biosensors, and provide an outlook for PC-based biosensors and their promise at the POC.

  5. Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors.

    Science.gov (United States)

    Bereza-Malcolm, Lara; Aracic, Sanja; Kannan, Ruban; Mann, Gülay; Franks, Ashley E

    2017-08-15

    Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10µgml -1 , as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20-40min following exposure to 3µgml -1 cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The effect of cultivation media and washing whole-cell biocatalysts on monoamine oxidase catalyzed oxidative desymmetrization of 3-azabicyclo[3,3,0]octane

    DEFF Research Database (Denmark)

    Ramesh, Hemalata; Zajkoska, Petra; Rebros, Martin

    2016-01-01

    It is well known that washing whole-cells containing enzyme activities after fermentation, but prior to biocatalysis can improve their activity in the subsequent reaction. In this paper, we quantify the impact of both the fermentation media and cell washing on the performance of whole-cell biocat...

  7. Diabetes mellitus: biosensors for research and management.

    Science.gov (United States)

    Turner, A P; Pickup, J C

    1985-01-01

    The condition of diabetes mellitus is described with particular reference to the parameters that it would be desirable to monitor in order to improve management and understanding of the disease. Previous attention has largely focused on analysis of glucose, but many other intermediates of carbohydrate, fat and protein metabolism are deranged in diabetes and may be alternative measures of control. The need for laboratory analysers, self-monitoring, closed-loop devices and alarms are detailed and the problems associated with implantable sensors discussed. Progress in the development of biosensors is reviewed using glucose sensors as the main example. Electrochemical, optoelectronic and calorimetric approaches to sensing are considered and it is concluded that configurations based either on hydrogen peroxide detection or on mediated electron transfer are most likely to provide a raid route to in vivo monitoring. The extension of biosensor technology to tackle other important substrates is discussed, the principal hurdle to success being seen as the lack of long-term stability of the biological component.

  8. Development of a formaldehyde biosensor with application to synthetic methylotrophy.

    Science.gov (United States)

    Woolston, Benjamin M; Roth, Timothy; Kohale, Ishwar; Liu, David R; Stephanopoulos, Gregory

    2018-01-01

    Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is, therefore, of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD-dependent methanol dehydrogenase (Mdh) variants, the rate-limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross-talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion. Finally, we applied this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build-up while also reducing the burden of heterologous expression. This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy. © 2017 Wiley Periodicals, Inc.

  9. 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

  10. Whole-cell vaccine of Streptococcus agalactiae in Oreochromis sp. with immersion method

    Directory of Open Access Journals (Sweden)

    , Sukenda

    2015-05-01

    Full Text Available ABSTRACT The study was aimed to evaluate the efficacy of formalin-killed non-hemolytic Streptococcus agalactiae N14G and NK1 isolates whole-killed vaccine to prevent streptococcosis in tilapia. Ten fishes were reared in a tank 60x30x35 cm3 with an average body weight at 10.79±0.99 g. Fish was vaccinated through bath immersion at a concentration of 109 cfu/mL. Fish was subsequently challenged by intraperitonial injection of Streptococcus agalactiae 105 cfu/mL at 11 days post-vaccination. Parameters observed were survival, relative percent survival (RPS, total leukocyte, phagocytic activity, antibody titer, total erythrocyte, haemoglobin level, haematocrit level, dan water quality. Samplings were performed in day-0, 20, and 30 after vaccination. Both vaccines have shown higher survival (60% and RPS (40% when challenged with pathogenic Streptococcus N14G isolates than other treatments. Based on RPS percentage observed, those vaccine were still not sufficiently effective to combat S. agalactiae infection. Keywords: tilapia, bath immersion, Streptococcus agalactiae, whole-cell vaccine ABSTRAK Penelitian ini bertujuan untuk mengevaluasi efikasi vaksin formalin-killed cell Streptococcus agalactiae tipe isolat nonhemolitik N14G dan NK1 se utuh yang diberikan melalui perendaman dalam mencegah penyakit streptococcosis pada ikan nila. Ikan nila yang digunakan memiliki bobot 10,79±0,99 g, dipelihara sebanyak sepuluh ekor dalam akuarium ukuran 60x30x35 cm3. Ikan divaksinasi dengan metode perendaman dengan dosis 109 cfu/mL. Uji tantang dilakukan pada hari ke-11 pascavaksinasi dengan dosis 105 cfu/mL. Parameter yang diamati meliputi sintasan (SR, sintasan relatif/relative percent survival (RPS, total leukosit, aktivitas fagositik, titer antibodi, total eritrosit, kadar hemoglobin, kadar hematokrit, dan kualitas air. Pengamatan parameter dilakukan pada hari ke-0, ke-10, ke-20, dan ke-30. Hasil penelitian menunjukkan perlakuan kedua vaksin yang diinfeksi

  11. Regional analysis of whole cell currents from hair cells of the turtle posterior crista.

    Science.gov (United States)

    Brichta, Alan M; Aubert, Anne; Eatock, Ruth Anne; Goldberg, Jay M

    2002-12-01

    The turtle posterior crista is made up of two hemicristae, each consisting of a central zone containing type I and type II hair cells and a surrounding peripheral zone containing only type II hair cells and extending from the planum semilunatum to the nonsensory torus. Afferents from various regions of a hemicrista differ in their discharge properties. To see if afferent diversity is related to the basolateral currents of the hair cells innervated, we selectively harvested type I and II hair cells from the central zone and type II hair cells from two parts of the peripheral zone, one near the planum and the other near the torus. Voltage-dependent currents were studied with the whole cell, ruptured-patch method and characterized in voltage-clamp mode. We found regional differences in both outwardly and inwardly rectifying voltage-sensitive currents. As in birds and mammals, type I hair cells have a distinctive outwardly rectifying current (I(K,L)), which begins activating at more hyperpolarized voltages than do the outward currents of type II hair cells. Activation of I(K,L) is slow and sigmoidal. Maximal outward conductances are large. Outward currents in type II cells vary in their activation kinetics. Cells with fast kinetics are associated with small conductances and with partial inactivation during 200-ms depolarizing voltage steps. Almost all type II cells in the peripheral zone and many in the central zone have fast kinetics. Some type II cells in the central zone have large outward currents with slow kinetics and little inactivation. Although these currents resemble I(K,L), they can be distinguished from the latter both electrophysiologically and pharmacologically. There are two varieties of inwardly rectifying currents in type II hair cells: activation of I(K1) is rapid and monoexponential, whereas that of I(h) is slow and sigmoidal. Many type II cells either have both inward currents or only have I(K1); very few cells only have I(h). Inward currents are

  12. Biooxidation of 2-phenylethanol to phenylacetic acid by whole-cell Gluconobacter oxydans biocatalyst immobilized in polyelectrolyte complex capsules

    Czech Academy of Sciences Publication Activity Database

    Bertóková, A.; Vikartovská, A.; Bučko, M.; Gemeiner, P.; Tkáč, J.; Chorvát, D.; Štefuca, V.; Neděla, Vilém

    2015-01-01

    Roč. 33, č. 2 (2015), s. 111-120 ISSN 1024-2422 R&D Projects: GA ČR(CZ) GA14-22777S Institutional support: RVO:68081731 Keywords : Gluconobacter oxydans * natural flavors * phenylacetic acid * immobilized whole-cell biocatalyst * polyelectrolyte complex capsules * environmental scanning electron microscopy Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 0.892, year: 2015

  13. Biphasic whole-cell synthesis of R-2-octanol with recycling of the ionic liquid

    OpenAIRE

    Dennewald, Danielle

    2011-01-01

    Whole-cell biocatalysis in biphasic systems permits the synthesis of inhibiting chiral alcohols if appropriate non-water miscible ionic liquids are used. Taking the asymmetric reduction of 2-octanone to R-2-octanol by a recombinant Escherichia coli as a model reaction, a detailed characterisation of the biocatalytic reaction was performed with [HMPL][NTF] as ionic liquid. This made the asymmetric synthesis of R-2-octanol in a simple batch at a conversion > 99 % and at an enantiomeric excess >...

  14. Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil.

    Science.gov (United States)

    Andrade, Grazielle S S; Carvalho, Ana K F; Romero, Cintia M; Oliveira, Pedro C; de Castro, Heizir F

    2014-12-01

    The intracellular lipase production by Mucor circinelloides URM 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. Physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. Olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhance the intracellular lipase activity. Low inoculum level and poor aeration rate also provided suitable conditions to attain high lipase activity (64.8 ± 0.8 U g(-1)). The transesterification activity of the immobilized whole- cells was assayed and optimal reaction conditions for the ethanolysis of babassu oil were determined by experimental design. Statistical analysis showed that M. circinelloides whole-cells were able to produce ethyl esters at all tested conditions, with the highest yield attained (98.1 %) at 35 °C using an 1:6 oil-to-ethanol molar ratio. The biocatalyst operational stability was also assayed in a continuous packed bed reactor (PBR) charged with glutaraldehyde (GA) and Aliquat-treated cells revealing half-life of 43.0 ± 0.5 and 20.0 ± 0.8 days, respectively. These results indicate the potential of immobilized M. circinelloides URM 4182 whole-cells as a low-cost alternative to conventional biocatalysts in the production of ethyl esters from babassu oil.

  15. A novel biocatalytic approach to acetylation of 1-β-D-arabinofuranosylcytosine by Aspergillus oryzae whole cell in organic solvents.

    Science.gov (United States)

    Li, Xiao-Feng; Zhu, Zhen; Zhao, Guang-Lei; Yu, Yi-Gang; Lai, Fu-Rao; Wu, Hui

    2012-01-01

    Biocatalytic acylation of 1-β-D-arabinofuranosylcytosine (ara-C) was developed using whole cell of Aspergillus oryzae as a novel catalyst. (13)C nuclear magnetic resonance (NMR) analysis indicated that the whole-cell biocatalyst had more specific activity toward the 3'-hydroxyl group than 5'-hydroxyl group among the available hydroxyl groups in sugar moiety of ara-C. Except for glucose and maltose, 11 carbon sources supplemented to basal media, including Spans, Tweens, olive oil and oleic acid, exhibited notable enhancement effects on both the cell growth and the acylation reactions. It was suggested that the carbon sources containing controlled-release oleic acid were the important substrates for the production of fungal cell-bound lipase with specific activity, partially due to a gradual induction effect of their released oleic acid on the cell-bound lipase production. Despite the low initial reaction rate and substrate conversion, the addition of 2.0 g/l Span 80 resulted in a higher 3'-regioselectivity of the cells than 81%. By using Tween 85 at its optimum concentration of 5.0 g/l, however, the highest initial rates (3.2 mmol/l h) and substrate conversion (76%) of the whole-cell catalyzed acylation of ara-C can be achieved. It was also found that the 3'-regioselectivity of the cells showed observable increase by extending the culture time. And the activity of cell-bound lipase drastically increased in the early stage of cell growth and then declined in the late culture stage, whatever the culture media used. Our results thus indicated that A. oryzae whole cell was a promising green tool for biosynthesis of nucleoside esters with potential bioactivities.

  16. 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.

  17. Electro-biocatalytic production of formate from carbon dioxide using an oxygen-stable whole cell biocatalyst.

    Science.gov (United States)

    Hwang, Hyojin; Yeon, Young Joo; Lee, Sumi; Choe, Hyunjun; Jang, Min Gee; Cho, Dae Haeng; Park, Sehkyu; Kim, Yong Hwan

    2015-06-01

    The use of biocatalysts to convert CO2 into useful chemicals is a promising alternative to chemical conversion. In this study, the electro-biocatalytic conversion of CO2 to formate was attempted with a whole cell biocatalyst. Eight species of Methylobacteria were tested for CO2 reduction, and one of them, Methylobacterium extorquens AM1, exhibited an exceptionally higher capability to synthesize formate from CO2 by supplying electrons with electrodes, which produced formate concentrations of up to 60mM. The oxygen stability of the biocatalyst was investigated, and the results indicated that the whole cell catalyst still exhibited CO2 reduction activity even after being exposed to oxygen gas. From the results, we could demonstrate the electro-biocatalytic conversion of CO2 to formate using an obligate aerobe, M. extorquens AM1, as a whole cell biocatalyst without providing extra cofactors or hydrogen gas. This electro-biocatalytic process suggests a promising approach toward feasible way of CO2 conversion to formate. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. [Investigation of algae pollution in Xiliu Lake and identification of toxic cyanobacteria by whole-cell PCR].

    Science.gov (United States)

    Ban, Hai-qun; Zhuang, Dong-gang; Zhu, Jing-yuan; Ba, Yue

    2006-03-01

    To investigate the contaminative condition of the floating algae (especially toxic cyanobacteria) in Xiliu Lake, and establish a whole-cell PCR method for identifying the toxic cyanobacteria. The surface water of Xiliu Lake was sampled by plastic sampler from March, 2004, and the number of algae was counted by using blood cell counter. The phycocyanin intergenic spacer region (PC-IGS) and microcystin synthetase gene B (mcyB) were identified by whole-cell PCR in water samples, and the amplified product of mcyB was inserted into T vector and sequenced. Cyanobacteria, Chlorophyta, Bacillariophyta and Euglenophyta were main algae, and cyanobacteria was the dominant algae in summer and autumn. From July 7 to September 27,2 004, PC-IGS was detected positively in 11 samples, and from July 29 to September 27, mcyB was-detieted positively in 9 samples. Compared with the reported mcyB of Microcystis aeruginosa in Genbank, the homology of gene sequence was more than 97 t he homology of amino acid sequence was more than 94%. In summer and autumn toxic cyanobacteria could be detected in Xiliu Lake. Toxic cyanobacteria could be identified successfully by whole-cell PCR.

  19. Liquid crystal interfaces: Experiments, simulations and biosensors

    Science.gov (United States)

    Popov, Piotr

    Interfacial phenomena are ubiquitous and extremely important in various aspects of biological and industrial processes. For example, many liquid crystal applications start by alignment with a surface. The underlying mechanisms of the molecular organization of liquid crystals at an interface are still under intensive study and continue to be important to the display industry in order to develop better and/or new display technology. My dissertation research has been devoted to studying how complex liquid crystals can be guided to organize at an interface, and to using my findings to develop practical applications. Specifically, I have been working on developing biosensors using liquid-crystal/surfactant/lipid/protein interactions as well as the alignment of low-symmetry liquid crystals for potential new display and optomechanical applications. The biotechnology industry needs better ways of sensing biomaterials and identifying various nanoscale events at biological interfaces and in aqueous solutions. Sensors in which the recognition material is a liquid crystal naturally connects the existing knowledge and experience of the display and biotechnology industries together with surface and soft matter sciences. This dissertation thus mainly focuses on the delicate phenomena that happen at liquid interfaces. In the introduction, I start by defining the interface and discuss its structure and the relevant interfacial forces. I then introduce the general characteristics of biosensors and, in particular, describe the design of biosensors that employ liquid crystal/aqueous solution interfaces. I further describe the basic properties of liquid crystal materials that are relevant for liquid crystal-based biosensing applications. In CHAPTER 2, I describe the simulation methods and experimental techniques used in this dissertation. In CHAPTER 3 and CHAPTER 4, I present my computer simulation work. CHAPTER 3 presents insight of how liquid crystal molecules are aligned by

  20. Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli

    DEFF Research Database (Denmark)

    Siedler, Solvej; Stahlhut, Steen Gustav; Malla, Sailesh

    2014-01-01

    This study describes the construction of two flavonoid biosensors, which can be applied for metabolic engineering of Escherichia coli strains. The biosensors are based on transcriptional regulators combined with autofluorescent proteins. The transcriptional activator FdeR from Herbaspirillum...... and externally added flavonoid concentration. The QdoR-biosensor was successfully applied for detection of kaempferol production in vivo at the single cell level by fluorescence-activated cell sorting. Furthermore, the amount of kaempferol produced highly correlated with the specific fluorescence of E. coli...... cells containing a flavonol synthase from Arabidopsis thaliana (fls1). We expect the designed biosensors to be applied for isolation of genes involved in flavonoid biosynthetic pathways. © 2013 The Authors....

  1. Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection.

    Science.gov (United States)

    Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin

    2016-04-01

    Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

  2. 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.

  3. Porous silicon biosensor for the detection of autoimmune diseases

    Science.gov (United States)

    Jane, Andrew O.; Szili, Endre J.; Reed, Joanne H.; Gordon, Tom P.; Voelcker, Nicolas H.

    2007-12-01

    Advances in porous silicon (pSi) technology have led to the development of new sensitive biosensors. The unique optical properties of pSi renders the material a perfect candidate for optical transducers exploiting photoluminescence or white light interference effects. The ability of biosensors exploiting these transduction mechanisms to quickly and accurately detect biological target molecules affords an alternative to current bioassays such as enzyme-linked immunosorbent assays (ELISAs). Here, we present a pSi biosensor that was developed to detect antibodies against the autoimmune protein La. This protein is associated with autoimmune diseases including rheumatic disorders, systematic lupus erythematosus (SLE) and Sjogren's syndrome (SS). A fast and sensitive detection platform such as the one described here can be applied to the rapid diagnosis of these debilitating autoimmune diseases. The immobilisation of the La protein onto pSi films gave a protein receptor-decorated sensor matrix. A cascade of immunological reactions was then initiated to detect anti-La antibody on the functionalised pSi surface. In the presence of o-phenylenediamine (OPD), horseradish peroxidase (HRP)/H IIO II catalysed the formation of an oxidised radical species that accelerated pSi corrosion. pSi corrosion was detected as a blue-shift in the generated interference pattern, corresponding to a decrease in the effective optical thickness (EOT) of the pSi film. Compared to an ELISA, the pSi biosensor could detect the anti-La antibody at a similar concentration (500 - 125 ng/ml). Furthermore, we found that the experimental process can be significantly shortened resulting in detection of the anti-La antibody in 80 minutes compared to a minimum of 5 hours required for ELISA.

  4. Biocatalytic anti-Prelog reduction of prochiral ketones with whole cells of Acetobacter pasteurianus GIM1.158.

    Science.gov (United States)

    Du, Peng-Xuan; Wei, Ping; Lou, Wen-Yong; Zong, Min-Hua

    2014-06-10

    Enantiomerically pure alcohols are important building blocks for production of chiral pharmaceuticals, flavors, agrochemicals and functional materials and appropriate whole-cell biocatalysts offer a highly enantioselective, minimally polluting route to these valuable compounds. At present, most of these biocatalysts follow Prelog's rule, and thus the (S)-alcohols are usually obtained when the smaller substituent of the ketone has the lower CIP priority. Only a few anti-Prelog (R)-specific whole cell biocatalysts have been reported. In this paper, the biocatalytic anti-Prelog reduction of 2-octanone to (R)-2-octanol was successfully conducted with high enantioselectivity using whole cells of Acetobacter pasteurianus GIM1.158. Compared with other microorganisms investigated, Acetobacter pasteurianus GIM1.158 was shown to be more effective for the reduction reaction, affording much higher yield, product enantiomeric excess (e.e.) and initial reaction rate. The optimal temperature, buffer pH, co-substrate and its concentration, substrate concentration, cell concentration and shaking rate were 35°C, 5.0, 500 mmol/L isopropanol, 40 mmol/L, 25 mg/mL and 120 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 89.5% and >99.9%, respectively, in 70 minutes. Compared with the best available data in aqueous system (yield of 55%), the yield of (R)-2-octanol was greatly increased. Additionally, the efficient whole-cell biocatalytic process was feasible on a 200-mL preparative scale and the chemical yield increased to 95.0% with the product e.e. being >99.9%. Moreover, Acetobacter pasteurianus GIM1.158 cells were proved to be capable of catalyzing the anti-Prelog bioreduction of other prochiral carbonyl compounds with high efficiency. Via an effective increase in the maximum yield and the product e.e. with Acetobacter pasteurianus GIM1.158 cells, these results open the way to use of whole cells of this microorganism for

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. Silica suspended waveguide splitter-based biosensor

    Science.gov (United States)

    Harrison, M. C.; Hawk, R. M.; Armani, A. M.

    2012-03-01

    Recently, a novel integrated optical waveguide 50/50 splitter was developed. It is fabricated using standard lithographic methods, a pair of etching steps and a laser reflow step. However, unlike other integrated waveguide splitters, the waveguide is elevated off of the silicon substrate, improving its interaction with biomolecules in solution and in a flow field. Additionally, because it is fabricated from silica, it has very low optical loss, resulting in a high signal-to-noise ratio, making it ideal for biosensing. By functionalizing the device using an epoxy-silane method using small samples and confining the protein solutions to the device, we enable highly efficient detection of CREB with only 1 μL of solution. Therefore, the waveguide coupler sensor is representative of the next generation of ultra-sensitive optical biosensors, and, when combined with microfluidic capabilities, it will be an ideal candidate for a more fully-realized lab-on-a-chip device.

  11. Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

    Science.gov (United States)

    Akopiants, Konstantin; Zhou, Rui-Zhe; Mohapatra, Susovan; Valerie, Kristoffer; Lees-Miller, Susan P; Lee, Kyung-Jong; Chen, David J; Revy, Patrick; de Villartay, Jean-Pierre; Povirk, Lawrence F

    2009-07-01

    XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

  12. 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)

  13. Assessing the bioavailability of organic contaminants using a novel bioluminescent biosensor

    International Nuclear Information System (INIS)

    Keane, A.; Phoenix, P.; Lau, P.C.K.; Ghoshal, S.

    2002-01-01

    The limited rate and extent of biodegradation in contaminated soils is often attributed to a lack of bioavailability of hydrophobic organic compounds. To date, the majority of studies aimed at assessing bioavailability and modes of bacterial uptake have relied upon quantification of microbial degradation rates in comparison to rates of dissolution or desorption in corresponding abiotic systems. Several studies have indicated the possibility of a direct uptake mechanism for sorbed or separate phase compounds. However, there is a lack of direct evidence to support these claims. To address the need for a direct measurement technique for microbial bioavailability, we have constructed a whole-cell bioluminescent biosensor, Pseudomonas putida F1G4 (PpF1G4), by fusing lux genes that encode for bioluminescence to the solvent efflux pump (sep) promoter element in PpF1G4, which is induced by the presence of target organic compounds. When the biosensor microorganism is exposed to an inducing compound, the bioluminescence system is activated and the cell produces an intensity of visible light (λ = 495 nm) that is directly related to the level of exposure to the contaminant. Batch experiments were carried out to assess whether the biosensor is able to sense the presence of toluene, a representative target compound, contained in a NAPL. Preliminary results show that while PpF1G4 responds to toluene in the aqueous phase, the biosensor does not appear to emit a significant bioluminescence signal in response to the toluene present in the NAPL. Ongoing research is focusing on optimizing the experimental procedure to fully explore this issue. (author)

  14. Fabrication of Si-based planar type patch clamp biosensor using silicon on insulator substrate

    International Nuclear Information System (INIS)

    Zhang, Z.L.; Asano, T.; Uno, H.; Tero, R.; Suzui, M.; Nakao, S.; Kaito, T.; Shibasaki, K.; Tominaga, M.; Utsumi, Y.; Gao, Y.L.; Urisu, T.

    2008-01-01

    The aim of this paper is to fabricate the planar type patch clamp ion-channel biosensor, which is suitable for the high throughput screening, using silicon-on-insulator (SOI) substrate. The micropore with 1.2 μm diameter is formed through the top Si layer and the SiO 2 box layer of the SOI substrate by focused ion beam (FIB). Then the substrate is assembled into the microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with transient receptor potential vanilloid type 1 (TRPV1) is positioned on the micropore and the whole-cell configuration is formed by the suction. Capsaicin is added to the extracellular solution as a ligand molecule, and the channel current showing the desensitization unique to TRPV1 is measured successfully

  15. Fabrication of Si-based planar type patch clamp biosensor using silicon on insulator substrate

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Z.L.; Asano, T. [Graduate University for Advanced Studies, Myodaiji, Okazaki, 444-8585 (Japan); Uno, H. [Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan); Tero, R. [Graduate University for Advanced Studies, Myodaiji, Okazaki, 444-8585 (Japan); Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan); Suzui, M.; Nakao, S. [Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan); Kaito, T. [SII NanoTechnology Inc., 36-1, Takenoshita, Oyama-cho, Sunto-gun, Shizuoka, 410-1393 (Japan); Shibasaki, K.; Tominaga, M. [Okazaki Institute for Integrative Bioscience, 5-1, Higashiyama, Myodaiji, Okazaki, 444-8787 (Japan); Utsumi, Y. [Laboratory of Advanced Science and Technology for Industry, University of Hyogo, 3-1-2, Koto, Kamigori, Ako-gun, Hyogo, 678-1205 (Japan); Gao, Y.L. [Department of Physics and Astronomy, Rochester University, Rochester, New York 14627 (United States); Urisu, T. [Graduate University for Advanced Studies, Myodaiji, Okazaki, 444-8585 (Japan); Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan)], E-mail: urisu@ims.ac.jp

    2008-03-03

    The aim of this paper is to fabricate the planar type patch clamp ion-channel biosensor, which is suitable for the high throughput screening, using silicon-on-insulator (SOI) substrate. The micropore with 1.2 {mu}m diameter is formed through the top Si layer and the SiO{sub 2} box layer of the SOI substrate by focused ion beam (FIB). Then the substrate is assembled into the microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with transient receptor potential vanilloid type 1 (TRPV1) is positioned on the micropore and the whole-cell configuration is formed by the suction. Capsaicin is added to the extracellular solution as a ligand molecule, and the channel current showing the desensitization unique to TRPV1 is measured successfully.

  16. Oriented Immobilization of His-Tagged Protein on a Redox Active Thiol Derivative of DPTA-Cu(II Layer Deposited on a Gold Electrode—The Base of Electrochemical Biosensors

    Directory of Open Access Journals (Sweden)

    Hanna Radecka

    2013-09-01

    Full Text Available This paper concerns the development of an electrochemical biosensor for the determination of Aβ16–23' and Aβ1–40 peptides. The His-tagged V and VC1 domains of Receptor for Advanced Glycation end Products (RAGE immobilized on a gold electrode surface were used as analytically active molecules. The immobilization of His6–RAGE domains consists of: (i formation of a mixed layer of N-acetylcysteamine (NAC and the thiol derivative of pentetic acid (DPTA; (ii complexation of Cu(II by DPTA; (iii oriented immobilization of His6–RAGE domains via coordination bonds between Cu(II sites from DPTA–Cu(II complex and imidazole nitrogen atoms of a histidine tag. Each modification step was controlled by cyclic voltammetry (CV, Osteryoung square-wave voltammetry (OSWV, and atomic force microscopy (AFM. The applicability of the proposed biosensor was tested in the presence of human plasma, which had no influence on its performance. The detection limits for Aβ1–40 determination were 1.06 nM and 0.80 nM, in the presence of buffer and human plasma, respectively. These values reach the concentration level of Aβ1–40 which is relevant for determination of its soluble form in human plasma, as well as in brain. This indicates the promising future application of biosensor presented for early diagnosis of neurodegenerative diseases.

  17. 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.

  18. 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

  19. Biosensors Used for Quantification of Nitrates in Plants

    Directory of Open Access Journals (Sweden)

    Romero-Galindo Raul

    2016-01-01

    Full Text Available Nitrogen is essential for the plant because it is used for the production of chlorophyll, proteins, nucleic acids, amino acids, and other cellular compounds; nitrogen is available in two forms: ammonium and nitrate. Several tools have been used to quantify nitrates in plants such as the Kjeldahl method and Dumas combustion digestion; however, they are destructive and long time-consuming methods. To solve these disadvantages, methods such as selective electrodes, optical sensors, reflectometers, and images based sensors have been developed; nonetheless, all these techniques show interference when carrying out measurements. Currently, biosensors based on genetic constructions, based on the response of promoter gene fused to Gene Fluorescent Protein (GFP, are gaining popularity, because they improve the accuracy of measurements of nitrate by avoiding the interference of carriers ion, high salt conditions, and other factors. The present review shows the different methods to quantify the nitrogen in plants; later, a biosensors perspective is presented, mainly focused on biosensors based on organism genetically modified. The review presents a list of promoter and reporter genes that could be used to develop different kind of sensors, and a perspective of sensors to measure quantitatively the nitrogen is presented.

  20. The Production of Biodiesel from Cottonseed Oil Using Rhizopus oryzae Whole Cell Biocatalysts

    Science.gov (United States)

    Athalye, Sneha Kishor

    Biodiesel is an environmentally friendly alternative to fossil fuels which have become increasingly expensive in recent times. An alternate approach to alkaline biodiesel production is needed as catalyst miscibility with the glycerol by-product, generation of large amounts of waste water, and saponification of the feedstock are major disadvantages associated with the process. Lipases are water soluble enzymes which act as catalysts in many lipid based reactions. Reuse of lipases can significantly reduce cost of enzymatic biodiesel production; however retention of lipolytic activity still remains a challenge. Use of microbial cells immobilized on various surfaces like sponge, foam and plastics as biocatalysts instead of extracted enzyme could help overcome this problem. A novel, rigid biomass support with high surface area made from recyclable polyethylene (Bioblok(TM)) was used in this study. Several fungal and bacterial species have been reported to possess appreciable levels of lipase activity. The biomass production and immobilization as well as lipase activity of three different species; Candida rugosa (ATCC #38772), Aspergillus oryzae (ATCC #58299), and Rhizopus oryzae (ATTC #34612) were tested. C. rugosa did not attach well to the support particles while A.oryzae had lower biomass accumulation of 6.1 g (dry cell wt)/L compared to 11.8 g (dry cell wt)/L for R.oryzae. Hence Rhizopus oryzae, fungal specie with cell surface bound lipase was selected for the current study. The study investigated the influence of media composition and growth time of the R.oryzae whole cell biocatalysts, immobilized on the BSPs, for FAME production from cottonseed oil. R.oryzae BSPs grown in basal media supplemented with 1% (w/v) of glucose or oil or both for 48 h, 72 h or 90 h were used in a 36 h transesterification reaction with cottonseed oil and methanol. BSPs grown in both glucose and oil supplemented medium for 72 h had the highest conversion of 22.4% (wt/wt) and a biomass

  1. Artificial neural networks for classification in metabolomic studies of whole cells using 1H nuclear magnetic resonance.

    LENUS (Irish Health Repository)

    Brougham, D F

    2011-01-01

    We report the successful classification, by artificial neural networks (ANNs), of (1)H NMR spectroscopic data recorded on whole-cell culture samples of four different lung carcinoma cell lines, which display different drug resistance patterns. The robustness of the approach was demonstrated by its ability to classify the cell line correctly in 100% of cases, despite the demonstrated presence of operator-induced sources of variation, and irrespective of which spectra are used for training and for validation. The study demonstrates the potential of ANN for lung carcinoma classification in realistic situations.

  2. 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.

  3. Correlation between the physicochemical properties of organic solvents and their biocompatibility toward epoxide hydrolase activity in whole-cells of a yeast, Rhodotorulasp

    CSIR Research Space (South Africa)

    Lotter, J

    2004-08-01

    Full Text Available in whole-cells of the yeast Rhodotorula sp. UOFS Y-0448 was investigated. No formal correlation between solvent biocompatibility and physicochemical properties was deductible, although the introduction of hydroxyl groups increased biocompatibility. 1...

  4. Direct evidence of advantage of using nanosized zeolite Beta for ISFET-based biosensor construction

    International Nuclear Information System (INIS)

    Soy, Esin; Galioglu, Sezin; Soldatkin, Oleksandr O.; Dzyadevych, Sergei V.; Warzywoda, Juliusz; Sacco, Albert; Akata, Burcu

    2013-01-01

    Analytical characteristics of urease- and butyrylcholinesterase (BuChE)- based ion sensitive field-effect transistor (ISFET) biosensors were investigated by the incorporation of zeolite Beta nanoparticles with varying Si/Al ratios. The results obtained by the zeolite-modified ISFET transducers suggested that the Si/Al ratio strongly influenced the biosensor performances due to the electrostatic interactions among enzyme, substrate, and zeolite surface as well as the nature of the enzymatic reaction. Using relatively small nanoparticles (62.7 ± 10, 76.2 ± 10, and 77.1 ± 10 nm) rather than larger particles, that are widely used in the literature, allow us to produce more homogenous products which will give more control over the quantity of materials used on the electrode surface and ability to change solely Si/Al ratio without changing other parameters such as particle size, pore volume, and surface area. This should enable the investigation of the individual effect of changing acidic and electronic nature of this material on the biosensor characteristics. According to our results, high biosensor sensitivity is evident on nanosize and submicron size particles, with the former resulting in higher performance. The sensitivity of biosensors modified by zeolite particles is higher than that to the protein for both types of biosensors. Most significantly, our results show that the performance of constructed ISFET-type biosensors strongly depends on Si/Al ratio of employed zeolite Beta nanoparticles as well as the type of enzymatic reaction employed. All fabricated biosensors demonstrated high signal reproducibility and stability for both BuChE and urease.

  5. Facile fabrication of gold nanoparticle on zein ultrafine fibers and their application for catechol biosensor

    International Nuclear Information System (INIS)

    Chen, Xiaodong; Li, Dawei; Li, Guohui; Luo, Lei; Ullah, Naseeb; Wei, Qufu; Huang, Fenglin

    2015-01-01

    Graphical abstract: (A) Formation mechanism of A-CZNF and (B) reaction principle and formation mechanism of A-CZUF biosensor. - Highlights: • We utilized the hydrophobic protein nanofibers to fabricate a laccase-based biosensor for the first time. • The composite containing gold nanoparticles was prepared by combining electrospinning and one-step reduction method, which is a novel nanomaterial. • It is noticeable that the laccase biosensor showed a high electrochemical response and electrochemical activity toward catechol. • The novel biosensor will offer a simple, convenient and high efficient method for detecting polyphenolic compounds in environment. - Abstract: A novel laccase biosensor based on a new composite of laccase–gold nanoparticles (Au NPs)-crosslinked zein ultrafine fibers (CZUF) has been fabricated for catechol determination in real solution samples. Firstly, crosslinked zein ultrafine fibers containing gold nanoparticles (A-CZUF) were prepared by combining electrospinning and one-step reduction method using poly(ethyleneimine) (PEI) as reducing and crosslinking agent. A smooth morphology and relative average distribution of A-CZUF were depicted by scanning electron microscope (SEM) and transmission electron microscopy (TEM). The Fourier transform infrared spectroscopy (FT-IR) analysis indicated that PEI molecules attached to the surface of the zein ultrafine fibers via the reaction of functional groups between PEI and glyoxal. The results obtained from ultraviolet visible spectroscopy (UV–vis spectroscopy), X-ray diffraction (XRD) and thermal gravimetric analysis (TGA) for A-CZUF confirmed the existence of Au NPS coated on the surface of CZUF. Square wave voltammetry (SWV) and cyclic voltammetry (CV) were used to detect the electrochemical performance of the proposed biosensor. The results demonstrated that this biosensor possessed a high sensitive detection to catechol, which was attributed to the direct electron transfer (DET

  6. A surface plasmon resonance biosensor for direct detection of the rabies virus

    Directory of Open Access Journals (Sweden)

    Jing Xu

    2012-01-01

    Full Text Available A surface plasmon resonance biosensor chip was constructed for detection of rabies virus. For the construction of the biosensor chip, N protein specific antibody and N protein specific antibody combined with G protein specific antibody of rabies virus were linked on two different flow cells on one CM5 chip, respectively. The chip was tested for the detection of rabies virus antigens using the crude extract of rabies virus from infected BHK cell strain culture. Tenfold serial dilutions of SRV9 strain virus-infected cell cultures were tested by the biosensor chip to establish the detection limit. The limit detection was approximately 70 pg/ml of nucleoprotein and glycoprotein. The biosensor chip developed in this study was employed for the detection of rabies virus in five suspect infectious specimens of brain tissue from guinea pigs; the results were compared by fluorescent antibody test. Surface plasmon resonance biosensor chip could be a useful automatic tool for prompt detection of rabies virus infection.

  7. 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.

  8. 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

  9. Pseudomonas fluorescens HK44: Lessons Lerned from a Model Whole-Cell Bioreporter with a Broad Application History

    Czech Academy of Sciences Publication Activity Database

    Trögl, J.; Chauhan, A.; Ripp, S.; Layton, A.C.; Kuncová, Gabriela; Sayler, G.S.

    2012-01-01

    Roč. 12, č. 2 (2012), s. 1544-1571 ISSN 1424-8220 R&D Projects: GA MŠk ME 892; GA MŠk ME 893 Grant - others:USDA(US) 2009-39210-20230 Institutional research plan: CEZ:AV0Z40720504 Keywords : bioluminiscence * bioreporter * biosensors Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.953, year: 2012

  10. Killed Whole-Cell Oral Cholera Vaccine Induces CCL20 Secretion by Human Intestinal Epithelial Cells in the Presence of the Short-Chain Fatty Acid, Butyrate

    Directory of Open Access Journals (Sweden)

    Ju-Ri Sim

    2018-01-01

    Full Text Available Short-chain fatty acids (SCFAs, such as acetate, butyrate, and propionate, modulate immune responses in the gut. However, the effect of SCFAs on mucosal vaccine-induced immune cell migration is poorly understood. Here, we investigated whether SCFAs modulate chemokine expression induced by the killed whole-cell oral cholera vaccine, Shanchol™, in human intestinal epithelial cells. Shanchol™ induced expression of CCL2, CCL5, CCL20, and CXCL10 at the mRNA level, but not at the protein level. Interestingly, CCL20 secretion was substantially increased by co-stimulation with Shanchol™ and butyrate, while neither acetate nor propionate showed such effect. Enhanced CCL20 secretion was associated with GPR109A activation, and histone deacetylase (HDAC inhibition. In addition, co-treatment with Shanchol™ and butyrate synergistically increased the secretion of adenosine triphosphate (ATP. Moreover, CCL20 secretion was decreased by inhibiting the extracellular ATP receptor P2X7. However, neither inflammasomes nor caspases were involved in CCL20 production. The culture supernatant of cells treated with Shanchol™ and butyrate augmented human immature dendritic cell migration. Collectively, these results suggest that butyrate enhances Shanchol™-induced CCL20 production in human intestinal epithelial cells via HDAC inhibition and ATP-P2X7 signaling by activating GPR109A. These effects potentially enhance the mucosal immune responses in the gut induced by this oral cholera vaccine.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Carbohydrate-based electrochemical biosensor for detection of a cancer biomarker in human plasma.

    Science.gov (United States)

    Devillers, Marion; Ahmad, Lama; Korri-Youssoufi, Hafsa; Salmon, Laurent

    2017-10-15

    Autocrine motility factor (AMF) is a tumor-secreted cytokine that stimulates tumor cell motility in vitro and metastasis in vivo. AMF could be detected in serum or urine of cancer patients with worse prognosis. Reported as a cancer biomarker, AMF secretion into body fluids might be closely related to metastases formation. In this study, a sensitive and specific carbohydrate-based electrochemical biosensor was designed for the detection and quantification of a protein model of AMF, namely phosphoglucose isomerase from rabbit muscle (RmPGI). Indeed, RmPGI displays high homology with AMF and has been shown to have AMF activity. The biosensor was constructed by covalent binding of the enzyme substrate d-fructose 6-phosphate (F6P). Immobilization was achieved on a gold surface electrode following a bottom-up approach through an aminated surface obtained by electrochemical patterning of ethylene diamine and terminal amine polyethylene glycol chain to prevent non-specific interactions. Carbohydrate-protein interactions were quantified in a range of 10 fM to 100nM. Complex formation was analyzed through monitoring of the redox couple Fe 2+ /Fe 3+ by electrochemical impedance spectroscopy and square wave voltammetry. The F6P-biosensor demonstrates a detection limit of 6.6 fM and high selectivity when compared to other non-specific glycolytic proteins such as d-glucose-6-phosphate dehydrogenase. Detection of protein in spiked plasma was demonstrated and accuracy of 95% is obtained compared to result obtained in PBS (phosphate buffered saline). F6P-biosensor is a very promising proof of concept required for the design of a carbohydrate-based electrochemical biosensor using the enzyme substrate as bioreceptor. Such biosensor could be generalized to detect other protein biomarkers of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

    Science.gov (United States)

    Romaniuk, Joseph A. H.; Cegelski, Lynette

    2015-01-01

    The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936

  14. Fluorescent Biosensors Based on Single-Molecule Counting.

    Science.gov (United States)

    Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

    2016-09-20

    fluorescence signals by specific in vitro/in vivo fluorescent labeling, and consequently, the fluorescent molecules indicate the presence of target molecules. The resultant fluorescence signals may be simply counted by either microfluidic device-integrated confocal microscopy or total internal reflection fluorescence-based single-molecule imaging. We have developed a series of single-molecule counting-based biosensors which can be classified as separation-free and separation-assisted assays. As a proof-of-concept, we demonstrate the applications of single-molecule counting-based biosensors for sensitive detection of various target biomolecules such as DNAs, miRNAs, proteins, enzymes, and intact cells, which may function as the disease-related biomarkers. Moreover, we give a summary of future directions to expand the usability of single-molecule counting-based biosensors including (1) the development of more user-friendly and automated instruments, (2) the discovery of new fluorescent labels and labeling strategies, and (3) the introduction of new concepts for the design of novel biosensors. Due to their high sensitivity, good selectivity, rapidity, and simplicity, we believe that the single-molecule counting-based fluorescent biosensors will indubitably find wide applications in biological research, clinical diagnostics, and drug discovery.

  15. Nonlinear Impedance of Whole Cells Near an Electrode as a Probe of Mitochondrial Activity

    Directory of Open Access Journals (Sweden)

    John H. Miller Jr.

    2011-04-01

    Full Text Available By simultaneously measuring the bulk media and electrode interface voltages of a yeast (Saccharomyces cerevisiae suspension subjected to an AC voltage, a yeast-dependent nonlinear response was found only near the current injection electrodes. Computer simulation of yeast near a current injection electrode found an enhanced voltage drop across the yeast near the electrode due to slowed charging of the electrode interfacial capacitance. This voltage drop is sufficient to induce conformation change in membrane proteins. Disruption of the mitochondrial electron transport chain is found to significantly change the measured nonlinear current response, suggesting nonlinear impedance can be used as a non-invasive probe of cellular metabolic activity.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. Tolerance of a knotted near infrared fluorescent protein to random circular permutation

    Science.gov (United States)

    Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

    2016-01-01

    Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

  2. Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

    Science.gov (United States)

    Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

    2016-07-12

    Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

  3. 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.

  4. Orientation of llama antibodies strongly increases sensitivity of biosensors.

    Science.gov (United States)

    Trilling, Anke K; Hesselink, Thamara; van Houwelingen, Adèle; Cordewener, Jan H G; Jongsma, Maarten A; Schoffelen, Sanne; van Hest, Jan C M; Zuilhof, Han; Beekwilder, Jules

    2014-10-15

    Sensitivity of biosensors depends on the orientation of bio-receptors on the sensor surface. The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV) were used for making biosensors, and azides were introduced in the VHH to function as bioorthogonal reactive groups. The importance of the orientation of bio-receptors was addressed by comparing sensors with randomly oriented VHH (with multiple exposed azide groups) to sensors with uniformly oriented VHH (with only a single azide group). A surface plasmon resonance (SPR) chip exposing cyclooctyne was reacted to azide functionalized VHH domains, using click chemistry. Comparison between randomly and uniformly oriented bio-receptors showed up to 800-fold increase in biosensor sensitivity. This technique may increase the containment of infectious diseases such as FMDV as its strongly enhanced sensitivity may facilitate early diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Micropatterning of 3D Microenvironments for Living Biosensor Applications

    Directory of Open Access Journals (Sweden)

    William F. Hynes

    2014-02-01

    Full Text Available Micro-scale printing and patterning of living cells has multiple applications including tissue engineering, cell signaling assays, and the fabrication of cell-based biosensors. In this work, a molecular printing instrument, the Bioforce Nano eNabler, was modified to enable micron-scale “quill-pen” based printing of mammalian cells in a 3D hyaluronan/gelatin based hydrogel. Specifically, photo-initiated “thiol-ene” click chemistry was used to couple the thiol groups of thiolated hyaluronan/thiolated gelatin to the alkene groups of 4-arm polyethylene glycol (PEG-norbornene molecules. Rapid photopolymerization enabled direct printing and controlled curing of living cells within the hydrogel matrix. The resulting hydrogels were biocompatible with human adipose-derived stem cells, NIH-3T3 cells, and mouse embryonic stem cells. The utility of this printing approach was also explored for cell-based biosensors. Micro-printed cells expressing a redox sensitive variant of the green fluorescent protein (roGFP-R12 showed a measurable fluorescent response to addition of oxidizing and then reducing agents. This work represents a novel approach to micron-scale cell patterning, and its potential for living, cell-based biosensors.

  6. 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.

  7. GMR biosensor arrays: a system perspective.

    Science.gov (United States)

    Hall, D A; Gaster, R S; Lin, T; Osterfeld, S J; Han, S; Murmann, B; Wang, S X

    2010-05-15

    Giant magnetoresistive biosensors are becoming more prevalent for sensitive, quantifiable biomolecular detection. However, in order for magnetic biosensing to become competitive with current optical protein microarray technology, there is a need to increase the number of sensors while maintaining the high sensitivity and fast readout time characteristic of smaller arrays (1-8 sensors). In this paper, we present a circuit architecture scalable for larger sensor arrays (64 individually addressable sensors) while maintaining a high readout rate (scanning the entire array in less than 4s). The system utilizes both time domain multiplexing and frequency domain multiplexing in order to achieve this scan rate. For the implementation, we propose a new circuit architecture that does not use a classical Wheatstone bridge to measure the small change in resistance of the sensor. Instead, an architecture designed around a transimpedance amplifier is employed. A detailed analysis of this architecture including the noise, distortion, and potential sources of errors is presented, followed by a global optimization strategy for the entire system comprising the magnetic tags, sensors, and interface electronics. To demonstrate the sensitivity, quantifiable detection of two blindly spiked samples of unknown concentrations has been performed at concentrations below the limit of detection for the enzyme-linked immunosorbent assay. Lastly, the multiplexing capability and reproducibility of the system was demonstrated by simultaneously monitoring sensors functionalized with three unique proteins at different concentrations in real-time. 2010 Elsevier B.V. All rights reserved.

  8. A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol.

    Science.gov (United States)

    Brixius-Anderko, Simone; Schiffer, Lina; Hannemann, Frank; Janocha, Bernd; Bernhardt, Rita

    2015-09-15

    Synthetic glucocorticoids like methylprednisolone (medrol) are of high pharmaceutical interest and represent powerful drugs due to their anti-inflammatory and immunosuppressive effects. Since the chemical hydroxylation of carbon atom 21, a crucial step in the synthesis of the medrol precursor premedrol, exhibits a low overall yield because of a poor stereo- and regioselectivity, there is high interest in a more sustainable and efficient biocatalytic process. One promising candidate is the mammalian cytochrome P450 CYP21A2 which is involved in steroid hormone biosynthesis and performs a selective oxyfunctionalization of C21 to provide the precursors of aldosterone, the main mineralocorticoid, and cortisol, the most important glucocorticoid. In this work, we demonstrate the high potential of CYP21A2 for a biotechnological production of premedrol, an important precursor of medrol. We successfully developed a CYP21A2-based whole-cell system in Escherichia coli by coexpressing the cDNAs of bovine CYP21A2 and its redox partner, the NADPH-dependent cytochrome P450 reductase (CPR), via a bicistronic vector. The synthetic substrate medrane was selectively 21-hydroxylated to premedrol with a max. yield of 90 mg L(-1) d(-1). To further improve the biocatalytic activity of the system by a more effective electron supply, we exchanged the CPR with constructs containing five alternative redox systems. A comparison of the constructs revealed that the redox system with the highest endpoint yield converted 70 % of the substrate within the first 2 h showing a doubled initial reaction rate compared with the other constructs. Using the best system we could increase the overall yield of premedrol to a maximum of 320 mg L(-1) d(-1) in shaking flasks. Optimization of the biotransformation in a bioreactor could further improve the premedrol gain to a maximum of 0.65 g L(-1) d(-1). We successfully established a CYP21-based whole-cell system for the biotechnological production of premedrol

  9. Methods of reducing non-specific adsorption in microfluidic biosensors

    International Nuclear Information System (INIS)

    Choi, Seokheun; Chae, Junseok

    2010-01-01

    Non-specific adsorption (NSA) of biomolecules is a persistent challenge in microfluidic biosensors. Microfluidic biosensors often have immobilized bioreceptors such as antibodies, enzymes, DNAs, etc, via linker molecules such as SAMs (self-assembled monolayers) to enhance immobilization. However, the linker molecules are very susceptible to NSA, causing false responses and decreasing sensitivity. In this paper, we present design methods to reduce the NSA of alkanethiol SAMs, which are popular linker molecules on microfluidic biosensors. Three design parameters were studied for two different chain-length SAMs (n = 2 and 10): (i) SAM incubation time, (ii) surface roughness [0.8 nm and 4.4 nm RMS (root mean square)] and (iii) gold crystal re-growth along (1 1 1) the target orientation. NSA was monitored by surface plasmon resonance (SPR). The results suggest that increased SAM incubation time reduces NSA, and that short-chain SAMs respond more favorably than the long-chain SAMs. Both SAMs were shown to be sensitive to surface roughness, and long-chain SAMs reduced NSA by 75%. Gold crystal re-growth along (1 1 1) the target orientation profoundly reduced NSA on the short-chain SAM. On a gold surface where surface roughness was 0.8 nm and there was strong directional alignment along the (1 1 1) gold crystal, final concentrations of nonspecifically bound proteins were 0.05 ng mm −2 (fibrinogen) and 0.075 ng mm −2 (lysozyme)—significantly lower than other known methods. The results show that optimizing three parameters (SAM incubation time, gold surface roughness and gold crystal orientation) improved SAM sensitivity for fibrinogen–anti-fibrinogen conjugates by a factor of 5 in 2.94 pM, suggesting that the methods are effective for reducing NSA in microfluidic biosensors.

  10. Whole cell probing with fluorescently labelled probes for in situ analysis of microbial populations

    International Nuclear Information System (INIS)

    Blackall, L.L.

    2005-01-01

    Until 1965, microbiologists struggled with simplicity of bacterial morphology and phenotypic characters in an attempt to construct a phylogenetic division for the prokaryotes. Then, it was found that molecular sequences were the source of much evolutionary information. Consequently, the way from phenotypic to genotypic characteristics for evolutionary inference was clear. Ribosomes within biological cells are the sites of protein synthesis. They are composed of a mixture of nucleic acids [ribosomal RiboNucleic Acids (rRNA)] and proteins and have an average size of 70s in bacteria. Because of their role in cell survival, maintenance and reproduction, rRNAs and their genes are described as being evolutionally conserved. Other genes can also be used to infer evolutionary relationships, and phylogenies inferred from all these molecules tend to concur. Comparative analyses of small subunit rRNA gene sequences were used in the 1980s to create a phylogeny or natural division for life on earth. It is composed of three domains - Bacteria, Archaea and Eucarya. The database of small subunit rRNA sequences is very large and allowed this broad comparative analysis to be done. In addition, the databases of these gene sequences are cumulative and constitute a growing resource available by modern communication channels to all researchers. The phylogenetic information has been used to clarify classification and taxonomic anomalies in the Bacteria and Archaea. Within the Bacteria, the small subunit rRNA is the 16S rRNA and the genes that code for this molecule are 16S rDNAs. In most cases, the 16S rDNA is exactly transcribed to form the 16S rRNA - i.e. the primary nucleic acid sequences of these two molecules are the same. Additionally, ribosomes of Bacteria contain the larger 23S rRNA (genes = 23R rDNAs), and sequence information from 23S rDNAs is also used to address evolutionary relationships between different Bacteria

  11. The Need and Potential of Biosensors to Detect Dioxins and Dioxin-Like Polychlorinated Biphenyls along the Milk, Eggs and Meat Food Chain

    Directory of Open Access Journals (Sweden)

    Aize Kijlstra

    2011-12-01

    Full Text Available Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs are hazardous toxic, ubiquitous and persistent chemical compounds, which can enter the food chain and accumulate up to higher trophic levels. Their determination requires sophisticated methods, expensive facilities and instruments, well-trained personnel and expensive chemical reagents. Ideally, real-time monitoring using rapid detection methods should be applied to detect possible contamination along the food chain in order to prevent human exposure. Sensor technology may be promising in this respect. This review gives the state of the art for detecting possible contamination with dioxins and DL-PCBs along the food chain of animal-source foods. The main detection methods applied (i.e., high resolution gas-chromatography combined with high resolution mass-spectrometry (HRGC/HRMS and the chemical activated luciferase gene expression method (CALUX bioassay, each have their limitations. Biosensors for detecting dioxins and related compounds, although still under development, show potential to overcome these limitations. Immunosensors and biomimetic-based biosensors potentially offer increased selectivity and sensitivity for dioxin and DL-PCB detection, while whole cell-based biosensors present interpretable biological results. The main shortcoming of current biosensors, however, is their detection level: this may be insufficient as limits for dioxins and DL-PCBs for food and feedstuffs are in pg per gram level. In addition, these contaminants are normally present in fat, a difficult matrix for biosensor detection. Therefore, simple and efficient extraction and clean-up procedures are required which may enable biosensors to detect dioxins and DL-PCBs contamination along the food chain.

  12. The Need and Potential of Biosensors to Detect Dioxins and Dioxin-Like Polychlorinated Biphenyls along the Milk, Eggs and Meat Food Chain

    Science.gov (United States)

    Chobtang, Jeerasak; de Boer, Imke J. M.; Hoogenboom, Ron L. A. P.; Haasnoot, Willem; Kijlstra, Aize; Meerburg, Bastiaan G.

    2011-01-01

    Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs) are hazardous toxic, ubiquitous and persistent chemical compounds, which can enter the food chain and accumulate up to higher trophic levels. Their determination requires sophisticated methods, expensive facilities and instruments, well-trained personnel and expensive chemical reagents. Ideally, real-time monitoring using rapid detection methods should be applied to detect possible contamination along the food chain in order to prevent human exposure. Sensor technology may be promising in this respect. This review gives the state of the art for detecting possible contamination with dioxins and DL-PCBs along the food chain of animal-source foods. The main detection methods applied (i.e., high resolution gas-chromatography combined with high resolution mass-spectrometry (HRGC/HRMS) and the chemical activated luciferase gene expression method (CALUX bioassay)), each have their limitations. Biosensors for detecting dioxins and related compounds, although still under development, show potential to overcome these limitations. Immunosensors and biomimetic-based biosensors potentially offer increased selectivity and sensitivity for dioxin and DL-PCB detection, while whole cell-based biosensors present interpretable biological results. The main shortcoming of current biosensors, however, is their detection level: this may be insufficient as limits for dioxins and DL-PCBs for food and feedstuffs are in pg per gram level. In addition, these contaminants are normally present in fat, a difficult matrix for biosensor detection. Therefore, simple and efficient extraction and clean-up procedures are required which may enable biosensors to detect dioxins and DL-PCBs contamination along the food chain. PMID:22247688

  13. The need and potential of biosensors to detect dioxins and dioxin-like polychlorinated biphenyls along the milk, eggs and meat food chain.

    Science.gov (United States)

    Chobtang, Jeerasak; de Boer, Imke J M; Hoogenboom, Ron L A P; Haasnoot, Willem; Kijlstra, Aize; Meerburg, Bastiaan G

    2011-01-01

    Dioxins and dioxin-like polychlorinated biphenyls (DL-PCBs) are hazardous toxic, ubiquitous and persistent chemical compounds, which can enter the food chain and accumulate up to higher trophic levels. Their determination requires sophisticated methods, expensive facilities and instruments, well-trained personnel and expensive chemical reagents. Ideally, real-time monitoring using rapid detection methods should be applied to detect possible contamination along the food chain in order to prevent human exposure. Sensor technology may be promising in this respect. This review gives the state of the art for detecting possible contamination with dioxins and DL-PCBs along the food chain of animal-source foods. The main detection methods applied (i.e., high resolution gas-chromatography combined with high resolution mass-spectrometry (HRGC/HRMS) and the chemical activated luciferase gene expression method (CALUX bioassay)), each have their limitations. Biosensors for detecting dioxins and related compounds, although still under development, show potential to overcome these limitations. Immunosensors and biomimetic-based biosensors potentially offer increased selectivity and sensitivity for dioxin and DL-PCB detection, while whole cell-based biosensors present interpretable biological results. The main shortcoming of current biosensors, however, is their detection level: this may be insufficient as limits for dioxins and DL-PCBs for food and feedstuffs are in pg per gram level. In addition, these contaminants are normally present in fat, a difficult matrix for biosensor detection. Therefore, simple and efficient extraction and clean-up procedures are required which may enable biosensors to detect dioxins and DL-PCBs contamination along the food chain.

  14. 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.

  15. Comparative analysis of different whole cell immobilized Aspergillus niger catalysts for gluconic acid fermentation using pretreated cane molasses

    Energy Technology Data Exchange (ETDEWEB)

    Subba Rao, D. (Div. of Biochemical Engineering, Dept. of Chemical Engineering, Indian Inst. of Tech., Madras (India)); Panda, T. (Div. of Biochemical Engineering, Dept. of Chemical Engineering, Indian Inst. of Tech., Madras (India))

    1994-10-01

    To compare the efficiency of various whole cell immobilization techniques for the production of gluconic acid by Aspergillus niger were investigated using potassium ferrocyanide-treated cane molasses as the substrate. The techniques followed were: (1) Calcium alginate entrapment, (2) cross-linking with glutaraldehyde after cell permeabilization with (a) acetone, (b) toluene and (c) isopropanol and (3) development of granular catalyst. A comparative analysis of yield has revealed that calcium alginate entrapment was the most suitable technique as it had given the maximum product yield (0.40 g gluconic acid/g total reducing sugar supplied). The properties of immobilized A. niger in sodium alginate gel have been thoroughly investigated and compared with those of free cells under most suitable conditions of fermentation. (orig.)

  16. Pseudomonas fluorescens HK44: Lessons Learned from a Model Whole-Cell Bioreporter with a Broad Application History

    Directory of Open Access Journals (Sweden)

    Gary S. Sayler

    2012-02-01

    Full Text Available Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE phenotype directly linked to a catabolic (naphthalene degradative pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands, liquid (water, wastewater, and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution.

  17. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...... in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test, The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high...

  18. Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

    Science.gov (United States)

    Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

    2017-03-15

    Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h.

  19. A fluorescent probe distinguishes between inhibition of early and late steps of lipopolysaccharide biogenesis in whole cells

    Science.gov (United States)

    Moison, Eileen; Xie, Ran; Zhang, Ge; Lebar, Matthew D.; Meredith, Timothy C.; Kahne, Daniel

    2017-01-01

    Lipopolysaccharide (LPS) biogenesis in Gram-negative organisms involves its biosynthesis in the cytoplasm and subsequent transport across three cellular compartments to the cell surface. We developed a fluorescent probe that allows us to determine the spatial distribution of LPS in whole cells. We show that polymyxin B nonapeptide (PMBN) containing a dansyl fluorophore specifically binds to LPS in membranes. We show that this probe detects decreases in LPS levels on the cell surface when LPS biosynthesis is inhibited at an early step. We also can detect accumulation of LPS in particular subcellular locations when LPS assembly is blocked during transport, allowing us to differentiate inhibitors targeting early and late stages of LPS biogenesis. PMID:28248483

  20. Comparison of immune responses to a killed bivalent whole cell oral cholera vaccine between endemic and less endemic settings.

    Science.gov (United States)

    Desai, Sachin N; Akalu, Zenebe; Teferi, Mekonnen; Manna, Byomkesh; Teshome, Samuel; Park, Ju Yeon; Yang, Jae Seung; Kim, Deok Ryun; Kanungo, Suman; Digilio, Laura

    2016-02-01

    Studies on safety, immunogenicity and efficacy of the killed, bivalent whole cell oral cholera vaccine (Shanchol) have been conducted in historically endemic settings of Asia. Recent cholera vaccination campaigns in Haiti and Guinea have also demonstrated favourable immunogenicity and effectiveness in nonendemic outbreak settings. We performed a secondary analysis, comparing immune responses of Shanchol from two randomised controlled trials performed in an endemic and a less endemic area (Addis Ababa) during a nonoutbreak setting. While Shanchol may offer some degree of immediate protection in primed populations living in cholera endemic areas, as well as being highly immunogenic in less endemic settings, understanding the characteristics of immune responses in each of these areas is vital in determining ideal dosing strategies that offer the greatest public health impact to populations from areas with varying degrees of cholera endemicity. © 2015 John Wiley & Sons Ltd.

  1. Immunogenicity of a killed bivalent (O1 and O139 whole cell oral cholera vaccine, Shanchol, in Haiti.

    Directory of Open Access Journals (Sweden)

    Richelle C Charles

    2014-05-01

    Full Text Available Studies of the immunogenicity of the killed bivalent whole cell oral cholera vaccine, Shanchol, have been performed in historically cholera-endemic areas of Asia. There is a need to assess the immunogenicity of the vaccine in Haiti and other populations without historical exposure to Vibrio cholerae.We measured immune responses after administration of Shanchol, in 25 adults, 51 older children (6-17 years, and 47 younger children (1-5 years in Haiti, where cholera was introduced in 2010. A≥4-fold increase in vibriocidal antibody titer against V. cholerae O1 Ogawa was observed in 91% of adults, 74% of older children, and 73% of younger children after two doses of Shanchol; similar responses were observed against the Inaba serotype. A≥2-fold increase in serum O-antigen specific polysaccharide IgA antibody levels against V. cholerae O1 Ogawa was observed in 59% of adults, 45% of older children, and 61% of younger children; similar responses were observed against the Inaba serotype. We compared immune responses in Haitian individuals with age- and blood group-matched individuals from Bangladesh, a historically cholera-endemic area. The geometric mean vibriocidal titers after the first dose of vaccine were lower in Haitian than in Bangladeshi vaccinees. However, the mean vibriocidal titers did not differ between the two groups after the second dose of the vaccine.A killed bivalent whole cell oral cholera vaccine, Shanchol, is highly immunogenic in Haitian adults and children. A two-dose regimen may be important in Haiti, and other populations lacking previous repeated exposures to V. cholerae.

  2. Stability enhancement of an atomic force microscope for long-term force measurement including cantilever modification for whole cell deformation

    Science.gov (United States)

    Weafer, P. P.; McGarry, J. P.; van Es, M. H.; Kilpatrick, J. I.; Ronan, W.; Nolan, D. R.; Jarvis, S. P.

    2012-09-01

    Atomic force microscopy (AFM) is widely used in the study of both morphology and mechanical properties of living cells under physiologically relevant conditions. However, quantitative experiments on timescales of minutes to hours are generally limited by thermal drift in the instrument, particularly in the vertical (z) direction. In addition, we demonstrate the necessity to remove all air-liquid interfaces within the system for measurements in liquid environments, which may otherwise result in perturbations in the measured deflection. These effects severely limit the use of AFM as a practical tool for the study of long-term cell behavior, where precise knowledge of the tip-sample distance is a crucial requirement. Here we present a readily implementable, cost effective method of minimizing z-drift and liquid instabilities by utilizing active temperature control combined with a customized fluid cell system. Long-term whole cell mechanical measurements were performed using this stabilized AFM by attaching a large sphere to a cantilever in order to approximate a parallel plate system. An extensive examination of the effects of sphere attachment on AFM data is presented. Profiling of cantilever bending during substrate indentation revealed that the optical lever assumption of free ended cantilevering is inappropriate when sphere constraining occurs, which applies an additional torque to the cantilevers "free" end. Here we present the steps required to accurately determine force-indentation measurements for such a scenario. Combining these readily implementable modifications, we demonstrate the ability to investigate long-term whole cell mechanics by performing strain controlled cyclic deformation of single osteoblasts.

  3. Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.

    Science.gov (United States)

    Krawczyk, Z; Wu, C

    1987-08-15

    We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.

  4. Whole-cell response to nitrogen deprivation in the diatom Phaeodactylum tricornutum.

    Science.gov (United States)

    Alipanah, Leila; Rohloff, Jens; Winge, Per; Bones, Atle M; Brembu, Tore

    2015-10-01

    Algal growth is strongly affected by nitrogen (N) availability. Diatoms, an ecologically important group of unicellular algae, have evolved several acclimation mechanisms to cope with N deprivation. In this study, we integrated physiological data with transcriptional and metabolite data to reveal molecular and metabolic modifications in N-deprived conditions in the marine diatom Phaeodactylum tricornutum. Physiological and metabolite measurements indicated that the photosynthetic capacity and chlorophyll content of the cells decreased, while neutral lipids increased in N-deprived cultures. Global gene expression analysis showed that P. tricornutum responded to N deprivation through an increase in N transport, assimilation, and utilization of organic N resources. Following N deprivation, reduced biosynthesis and increased recycling of N compounds like amino acids, proteins, and nucleic acids was observed at the transcript level. The majority of the genes associated with photosynthesis and chlorophyll biosynthesis were also repressed. Carbon metabolism was restructured through downregulation of the Calvin cycle and chrysolaminarin biosynthesis, and co-ordinated upregulation of glycolysis, the tricarboxylic acid cycle, and pyruvate metabolism, leading to funnelling of carbon sources to lipid metabolism. Finally, reallocation of membrane lipids and induction of de novo triacylglycerol biosynthesis directed cells to accumulation of neutral lipids. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  5. Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Berg, van den W.A.M.; Wiel, van de D.F.M.; Schaaper, W.M.M.; Korf, J.; Berkel, van W.J.H.

    2007-01-01

    The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50 ± 10%), reconstitutable dimers (20 ± 10%)

  6. The application of neoglycopeptides in the development of sensitive surface plasmon resonance-based biosensors

    NARCIS (Netherlands)

    Maljaars, C.E.P.; de Souza, A.C.; Halkes, K.M.; Upton, P.J.; Reeman, S.M.; André, S.; Gabius, H.-J.; McDonnell, M.B.; Kamerling, J.P.

    2008-01-01

    The development of a biosensor based on surface plasmon resonance is described for the detection of carbohydrate-binding proteins in solution on a Biacore 2000 instrument, using immobilized glycopeptides as ligands. Their selection was based on previous screenings of solid-phase glycopeptide

  7. Immobilization of HRP in Mesoporous Silica and Its Application for the Construction of Polyaniline Modified Hydrogen Peroxide Biosensor

    Directory of Open Access Journals (Sweden)

    Chien-Chung Chen

    2009-06-01

    Full Text Available Polyaniline (PANI, an attractive conductive polymer, has been successfully applied in fabricating various types of enzyme-based biosensors. In this study, we have employed mesoporous silica SBA-15 to stably entrap horseradish peroxidase (HRP, and then deposited the loaded SBA-15 on the PANI modified platinum electrode to construct a GA/SBA-15(HRP/PANI/Pt biosensor. The mesoporous structures and morphologies of SBA-15 with or without HRP were characterized. Enzymatic protein assays were employed to evaluate HRP immobilization efficiency. Our results demonstrated that the constructed biosensor displayed a fine linear correlation between cathodic response and H2O2 concentration in the range of 0.02 to 18.5 mM, with enhanced sensitivity. In particular, the current approach provided the PANI modified biosensor with improved stability for multiple measurements.

  8. Nanostructured silicon-based biosensors for the selective identification of analytes of social interest

    International Nuclear Information System (INIS)

    D'Auria, Sabato; Champdore, Marcella de; Aurilia, Vincenzo; Parracino, Antonietta; Staiano, Maria; Vitale, Annalisa; Rossi, Mose; Rea, Ilaria; Rotiroti, Lucia; Rossi, Andrea M; Borini, Stefano; Rendina, Ivo; Stefano, Luca De

    2006-01-01

    Small analytes such as glucose, L-glutamine (Gln), and ammonium nitrate are detected by means of optical biosensors based on a very common nanostructured material, porous silicon (PSi). Specific recognition elements, such as protein receptors and enzymes, were immobilized on hydrogenated PSi wafers and used as probes in optical sensing systems. The binding events were optically transduced as wavelength shifts of the porous silicon reflectivity spectrum or were monitored via changes of the fluorescence emission. The biosensors described in this article suggest a general approach for the development of new sensing systems for a wide range of analytes of high social interest

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Identification of Salmonella Typhimurium-specific DNA aptamers developed using whole-cell SELEX and FACS analysis.

    Science.gov (United States)

    Moon, Jihea; Kim, Giyoung; Lee, Sangdae; Park, Saetbyeol

    2013-11-01

    Conventional methods for detection of infective organisms, such as Salmonella, are complicated and require multiple steps, and the need for rapid detection has increased. Biosensors show great potential for rapid detection of pathogens. In turn, aptamers have great potential for biosensor assay development, given their small size, ease of synthesis and labeling, lack of immunogenicity, a lower cost of production than antibodies, and high target specificity. In this study, ssDNA aptamers specific to Salmonella Typhimurium were obtained by a whole bacterium-based systematic evolution of ligands by exponential enrichment (SELEX) procedure and applied to probing S. Typhimurium. After 10 rounds of selection with S. Typhimurium as the target and Salmonella Enteritidis, Escherichia coli and Staphylococcus aureus as counter targets, the highly enriched oligonucleic acid pool was sorted using flow cytometry. In total, 12 aptamer candidates from different families were sequenced and grouped. Fluorescent analysis demonstrated that aptamer C4 had particularly high binding affinity and selectivity; this aptamer was then further characterized. © 2013 Elsevier B.V. All rights reserved.

  14. Photonic Crystal Biosensor Based on Optical Surface Waves

    Directory of Open Access Journals (Sweden)

    Giovanni Dietler

    2013-02-01

    Full Text Available A label-free biosensor device based on registration of photonic crystal surface waves is described. Angular interrogation of the optical surface wave resonance is used to detect changes in the thickness of an adsorbed layer, while an additional simultaneous detection of the critical angle of total internal reflection provides independent data of the liquid refractive index. The abilities of the device are demonstrated by measuring of biotin molecule binding to a streptavidin monolayer, and by measuring association and dissociation kinetics of immunoglobulin G proteins. Additionally, deposition of PSS / PAH polyelectrolytes is recorded in situ resulting calculation of PSS and PAH monolayer thicknesses separately.

  15. Integration of fractal biosensor in a digital microfluidic platform

    KAUST Repository

    Mashraei, Yousof

    2015-11-01

    Fractal capacitive electrodes have been successfully integrated into a digital microfluidic open-platform. These electrodes perform actuation and withstand voltages up to 300V without insulation-layer breakdown. They were used to quantify the concentration levels of C-reactive protein (CRP) to determine the risk of cardiovascular disease. The capacitance increased sevenfold and stabilized in less than 5 minutes. The sensor shows a decreasing trend of capacitance readouts with the increase of concentrations. The same immunoassay was tested with untreated electrodes and showed no significant response, which suggests that immobilization was necessary. This configuration allows the electrodes to be used as biosensors.

  16. 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

  17. Prussian Blue acts as a mediator in a reagentless cytokinin biosensor

    International Nuclear Information System (INIS)

    Kowalska, Marta; Tian Faming; Smehilova, Maria; Galuszka, Petr; Frebort, Ivo; Napier, Richard; Dale, Nicholas

    2011-01-01

    Highlights: · An electrochemical biosensor for detection of the plant hormone cytokinin. · Constitutive expression system for large-scale protein production. · CKX enzyme entrapment in sol-gel film on the surface of a PrB-modified electrode. · Prussian Blue as an electron mediator between the enzyme and the electrode. · The biosensor was sensitive to micromolar concentrations of several cytokinins. - Abstract: An electrochemical biosensor for detection of the plant hormone cytokinin is introduced. Cytokinin homeostasis in tissues of many lower and higher plants is controlled largely by the activity of cytokinin dehydrogenase (CKX, EC 1.5.99.12) that catalyzes an irreversible cleavage of N 6 -side chain of cytokinins. Expression of Arabidopsis thaliana CKX2 from Pichia pastoris was used to prepare purified AtCKX2 as the basis of the cytokinin biosensor. Prussian Blue (PrB) was electrodeposited on Pt microelectrodes prior to deposition of the enzyme in a sol-gel matrix. The biosensor gave amperometric responses to several cytokinins. These responses depended on the presence of both the enzyme and the Prussian Blue. Thus Prussian Blue must act as an electron mediator between the FAD centre in CKX2 and the Pt surface.

  18. 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.

  19. 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

  20. Synthesis and utilization of carbon nanotubes for fabrication of electrochemical biosensors

    International Nuclear Information System (INIS)

    Lawal, Abdulazeez T.

    2016-01-01

    Graphical abstract: Carbon nanotubes. - Highlights: • This review discusses synthesis and applications of carbon nanotubes sensors. • The review summarizes contributions of carbon nanotube to electrochemical biosensor. • Good electrical conductivity makes carbon nanotubes a good material for biosensors. • Carbon nanotubes promotes electron transfer that aids biosensing of biomolecules. - Abstract: This review summarizes the most recent contributions in the fabrication of carbon nanotubes-based electrochemical biosensors in recent years. It discusses the synthesis and application of carbon nanotubes to the assembly of carbon nanotube-based electrochemical sensors, its analytical performance and future expectations. An increasing number of reviews and publications involving carbon nanotubes sensors have been reported ever since the first design of carbon nanotube electrochemical biosensors. The large surface area and good electrical conductivity of carbon nanotubes allow them to act as “electron wire” between the redox center of an enzyme or protein and an electrode's surface, which make them very excellent material for the design of electrochemical biosensors. Carbon nanotubes promote the different rapid electron transfers that facilitate accurate and selective detection of cytochrome-c, β-nicotinamide adenine dinucleotide, hemoglobin and biomolecules, such as glucose, cholesterol, ascorbic acid, uric acid, dopamine pesticides, metals ions and hydrogen peroxide.

  1. Synthesis and utilization of carbon nanotubes for fabrication of electrochemical biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Lawal, Abdulazeez T., E-mail: abdul.lawal@yahoo.com

    2016-01-15

    Graphical abstract: Carbon nanotubes. - Highlights: • This review discusses synthesis and applications of carbon nanotubes sensors. • The review summarizes contributions of carbon nanotube to electrochemical biosensor. • Good electrical conductivity makes carbon nanotubes a good material for biosensors. • Carbon nanotubes promotes electron transfer that aids biosensing of biomolecules. - Abstract: This review summarizes the most recent contributions in the fabrication of carbon nanotubes-based electrochemical biosensors in recent years. It discusses the synthesis and application of carbon nanotubes to the assembly of carbon nanotube-based electrochemical sensors, its analytical performance and future expectations. An increasing number of reviews and publications involving carbon nanotubes sensors have been reported ever since the first design of carbon nanotube electrochemical biosensors. The large surface area and good electrical conductivity of carbon nanotubes allow them to act as “electron wire” between the redox center of an enzyme or protein and an electrode's surface, which make them very excellent material for the design of electrochemical biosensors. Carbon nanotubes promote the different rapid electron transfers that facilitate accurate and selective detection of cytochrome-c, β-nicotinamide adenine dinucleotide, hemoglobin and biomolecules, such as glucose, cholesterol, ascorbic acid, uric acid, dopamine pesticides, metals ions and hydrogen peroxide.

  2. Alpha-fetoprotein detection by using a localized surface plasmon coupled fluorescence fiber-optic biosensor

    Science.gov (United States)

    Chang, Ying-Feng; Chen, Ran-Chou; Li, Ying-Chang; Yu, Chih-Jen; Hsieh, Bao-Yu; Chou, Chien

    2007-11-01

    Alpha-fetoprotein (AFP) detection by using a localized surface plasmon coupled fluorescence (LSPCF) fiber-optic biosensor is setup and experimentally demonstrated. It is based on gold nanoparticle (GNP) and coupled with localized surface plasmon wave on the surface of GNP. In this experiment, the fluorophores are labeled on anti-AFP which are bound to protein A conjugated GNP. Thus, LSPCF is excited with high efficiency in the near field of localized surface plasmon wave. Therefore, not only the sensitivity of LSPCF biosensor is enhanced but also the specific selectivity of AFP is improved. Experimentally, the ability of real time measurement in the range of AFP concentration from 0.1ng/ml to 100ng/ml was detected. To compare with conventional methods such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), the LSPCF fiber-optic biosensor performs higher or comparable detection sensitivity, respectively.

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

    Directory of Open Access Journals (Sweden)

    Kenzo Maehashi

    2009-07-01

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

  4. IGZO thin film transistor biosensors functionalized with ZnO nanorods and antibodies.

    Science.gov (United States)

    Shen, Yi-Chun; Yang, Chun-Hsu; Chen, Shu-Wen; Wu, Shou-Hao; Yang, Tsung-Lin; Huang, Jian-Jang

    2014-04-15

    We demonstrate a biosensor structure consisting of an IGZO (Indium-Gallium-Zinc-Oxide) TFT (thin film transistor) and an extended sensing pad. The TFT acts as the sensing and readout device, while the sensing pad ensures the isolation of biological solution from the transistor channel layer, and meanwhile increases the sensing area. The biosensor is functionalized by first applying ZnO nanorods to increase the surface area for attracting electrical charges of EGFR (epidermal growth factor receptor) antibodies. The device is able to selectively detect 36.2 fM of EGFR in the total protein solution of 0.1 ng/ml extracted from squamous cell carcinoma (SCC). Furthermore, the conjugation duration of the functionalized device with EGFR can be limited to 3 min, implying that the biosensor has the advantage for real-time detection. © 2013 Elsevier B.V. All rights reserved.

  5. Aptamer-functionalized nano-biosensors.

    Science.gov (United States)

    Chiu, Tai-Chia; Huang, Chih-Ching

    2009-01-01

    Nanomaterials have become one of the most interesting sensing materials because of their unique size- and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs), metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs). We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused.

  6. Aptamer-Functionalized Nano-Biosensors

    Directory of Open Access Journals (Sweden)

    Tai-Chia Chiu

    2009-12-01

    Full Text Available Nanomaterials have become one of the most interesting sensing materials because of their unique size- and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs, metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs. We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused.

  7. Autonomous electrochemical biosensors: A new vision to direct methanol fuel cells.

    Science.gov (United States)

    Sales, M Goreti F; Brandão, Lúcia

    2017-12-15

    A new approach to biosensing devices is demonstrated aiming an easier and simpler application in routine health care systems. Our methodology considered a new concept for the biosensor transducing event that allows to obtain, simultaneously, an equipment-free, user-friendly, cheap electrical biosensor. The use of the anode triple-phase boundary (TPB) layer of a passive direct methanol fuel cell (DMFC) as biosensor transducer is herein proposed. For that, the ionomer present in the anode catalytic layer of the DMFC is partially replaced by an ionomer with molecular recognition capability working as the biorecognition element of the biosensor. In this approach, fuel cell anode catalysts are modified with a molecularly imprinted polymer (plastic antibody) capable of protein recognition (ferritin is used as model protein), inserted in a suitable membrane electrode assembly (MEA) and tested, as initial proof-of-concept, in a non-passive fuel cell operation environment. The anchoring of the ionomer-based plastic antibody on the catalyst surface follows a simple one-step grafting from approach through radical polymerization. Such modification increases fuel cell performance due to the proton conductivity and macroporosity characteristics of the polymer on the TPB. Finally, the response and selectivity of the bioreceptor inside the fuel cell showed a clear and selective signal from the biosensor. Moreover, such pioneering transducing approach allowed amplification of the electrochemical response and increased biosensor sensitivity by 2 orders of magnitude when compared to a 3-electrodes configuration system. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

    Science.gov (United States)

    Nguyen, Hoang-Minh; Mathiesen, Geir; Stelzer, Elena Maria; Pham, Mai Lan; Kuczkowska, Katarzyna; Mackenzie, Alasdair; Agger, Jane W; Eijsink, Vincent G H; Yamabhai, Montarop; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

    2016-10-04

    Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach

  9. 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...

  10. 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...

  11. 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...

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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 ...

  17. Cytochrome c biosensor for determination of trace levels of cyanide and arsenic compounds

    International Nuclear Information System (INIS)

    Fuku, Xolile; Iftikar, Faiza; Hess, Euodia; Iwuoha, Emmanuel; Baker, Priscilla

    2012-01-01

    Highlights: ► Cytochrome c biosensor for detection of KCN, As 2 O 3 and Fe 2 K (CN) was constructed. ► Detection limits in the range of 4.3–9.1 μM for the analytes were obtained using CV, SWV and EIS. ► The detection limits for the biosensor were significantly lower than current EPA and WHO guidelines. - Abstract: An electrochemical method based on a cytochrome c biosensor was developed, for the detection of selected arsenic and cyanide compounds. Boron doped diamond (BDD) electrode was used as a transducer, onto which cytochrome c was immobilised and used for direct determination of Prussian blue, potassium cyanide and arsenic trioxide. The sensitivity as calculated from cyclic voltammetry (CV) and square wave voltammetry (SWV), for each analyte in phosphate buffer (pH = 7) was found to be in the range of (1.1–4.5) × 10 −8 A μM −1 and the detection limits ranged from 4.3 to 9.1 μM. The biosensor is therefore able to measure significantly lower than current Environmental Protection Agency (EPA) and World Health Organisation (WHO) guidelines, for these types of analytes. The protein binding was monitored as a decrease in biosensor peak currents by SWV and as an increase in biosensor charge transfer resistance by electrochemical impedance spectroscopy (EIS). EIS provided evidence that the electrocatalytic advantage of BDD electrode was not lost upon immobilisation of cytochrome c. The interfacial kinetics of the biosensor was modelled as equivalent electrical circuit based on electrochemical impedance spectroscopy data. UV–vis spectroscopy was used to confirm the binding of the protein in solution by monitoring the intensity of the soret bands and the Q bands. FTIR was used to characterise the protein in the immobilised state and to confirm that the protein was not denatured upon binding to the pre-treated bare BDD electrode. SNFTIR of cyt c immobilised at platinum electrode, was used to study the effect of oxidation state on the surface bond

  18. 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.

  19. 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)

  20. Lipid Microarray Biosensor for Biotoxin Detection.

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.; Edel, Joshua B.; Meyer, Grant D.; Craighead, Harold G.

    2006-05-01

    We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates by TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4

  1. 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.

  2. 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.

  3. Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

    Science.gov (United States)

    Annecchino, Luca A; Morris, Alexander R; Copeland, Caroline S; Agabi, Oshiorenoya E; Chadderton, Paul; Schultz, Simon R

    2017-08-30

    Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethybenzene, and xylene (BTEX) sensing

    International Nuclear Information System (INIS)

    Applegate, B.M.; Kehrmeyer, S.R.; Sayler, G.S.

    1998-01-01

    A tod-luxCDABE fusion was constructed and introduced into the chromosome of Pseudomonas putida F1, yielding the strain TVA8. This strain was used to examine the induction of the tod operon when exposed to benzene, toluene, ethylbenzene, and xylene (BTEX) compounds and aqueous solutions of JP-4 jet fuel constituents. Since this system contained the complete lux cassette (luxCDABE), bacterial bioluminescence in response to putative chemical inducers of the tod operon was measured on-line in whole cells without added aldehyde substrate. There was an increasing response to toluene concentrations from 30 microg/liter to 50 mg/liter, which began to saturate at higher concentrations. The detection limit was 30 microg/liter. There was a significant light response to benzene, m- and p-xylenes, phenol, and water-soluble JP-4 jet fuel components, but there was no bioluminescence response upon exposure to o-xylene. The transposon insertion was stable and had no negative effect on cell growth

  5. Effect of external mass transfer on activation energy of butyl oleate ester synthesis using a whole cell bio catalyst

    International Nuclear Information System (INIS)

    Shahhoseini, Sh.; Nasernejad, B.; Vahabzadeh, F.

    2016-01-01

    In the present research, synthesis of butyl oleate ester from oleic acid and butanol using loofa-immobilized Rhizopus oryzae as a whole cell biocatalyst (LIC) was studied in which hexane was used as the hydrophobic solvent. Decrease of mass transfer limitations as result of the interface formation between the two immiscible substrates, positively affected on the reaction progress (87% as the ester product yielded within 10 h). By applying Arrhenius equation, the activation energy of the ester synthesis was determined as Ea=18.2 kJ/mol within temperature range of 15-45°C. It was notable to test appearance of the nonlinearity in Arrhenius plot which was indicative of presence of two sections. The reaction limited region was 15-35°C; Ea=27 kJ/mol and diffusion limited region was >35°C; Ea=6.8 kJ/mol. Eventually, in this research, influence of external mass transfer on activation energy with reference to the catalytic role of the LIC in the ester synthesis was discussed.

  6. Respon Imun Spesifik Larva Ikan Mas (Cyprinus carpio melalui Imunitas Maternal yang Diberi Vaksin Inaktif Whole Cell Aeromonas salmonicida

    Directory of Open Access Journals (Sweden)

    Syohibahttul Islamiyah BAHAR

    2017-02-01

    Full Text Available Aeromonas salmonicida are specific bacteria that can cause infections and death to the cultivation of carp (Cyprinus carpio during larval stage. Death in carp can be prevented by a vaccine, but the vaccine can only be given on the seed over the age of 3 weeks. Maternal vaccination needs to be done to improve the immune system of the larvae by means of inactivated whole cell vaccine A. salmonicida on broodstock ready to spawn. Aims to determine the effectiveness of vaccines on breeders carp to the parent antibody titer test and larvae, as well Survival Rate (SR and the Relative Percent Survival (RPS larvae. This research was conducted with a completely randomized design, 4 treatments A (control; B (0.3 ml/kg; C (0.4 ml/kg; D (0.5 ml/kg and 3 repetitions. The results show that the antibody titer of 0.3 ml dose capable of providing agglutination reaction to pitting 7th (64x dilution in broodstock, and vaccine doses 0,4ml on broodstock able to give agglutination reaction to the larvae until all 6 wells (32x dilution. A dose of 0.4 ml/kg resulted the highest SR and RPS with 96.11% and 81.25% respectively. Clinical symptoms of redness in control larvae was spread throughout the body whereas on the vaccine treatment was only in certain body parts. Keywords: A. salmonicida, vaccines, maternal immunity, larva, specific immune respon

  7. Biodiesel production from waste cooking oil in a magnetically fluidized bed reactor using whole-cell biocatalysts

    International Nuclear Information System (INIS)

    Chen, Guanyi; Liu, Jing; Yao, Jingang; Qi, Yun; Yan, Beibei

    2017-01-01

    Highlights: • A MFBR system was used for biodiesel production from waste cooking oil. • Reaction parameters were optimized by response surface methodology. • Transesterification using MWCBs in MFBR obtained a max yield of 91.8% after 48 h. • The MWCBs can be reused in MFBR for 10 cycles with maintaining 87.5% yield. • The MFBR using MWCBs was an efficient system for large-scale biodiesel industry. - Abstract: Biodiesel production from catalytic transesterification of waste cooking oil (WCO) was investigated in a magnetically fluidized bed reactor (MFBR) over Pseudomonas mendocina cells immobilized in magnetic microspheres. The effects of methanol to oil molar ratio (MOMR), magnetic field intensity, biocatalysts concentration and reactant flow rate on biodiesel production were investigated. Optimization of the selected parameters was carried out for maximum biodiesel production using response surface methodology with support of Design-Expert software. The parameters optimized with response surface methodology were MOMR of 3.74:1, magnetic field intensity of 136.63 Oe, biocatalysts concentration of 10.21 wt.% and reactant flow rate of 16.97 mL/min. An experimental biodiesel yield of 91.8% was obtained at 35 °C after 48 h with these optimized parameters. Moreover, the magnetic whole-cell biocatalysts (MWCBs) exhibited good reusability in MFBR that 87.5% biodiesel yield could still be achieved after 10 cycles. The results suggested that MWCBs catalyzed transesterification in the MFBR system would have broad application prospects in biodiesel production.

  8. Neurons of the rat suprachiasmatic nucleus show a circadian rhythm in membrane properties that is lost during prolonged whole-cell recording

    NARCIS (Netherlands)

    Schaap, J.; Bos, N. P.; de Jeu, M. T.; Geurtsen, A. M.; Meijer, J. H.; Pennartz, C. M.

    1999-01-01

    The suprachiasmatic nucleus is commonly considered to contain the main pacemaker of behavioral and hormonal circadian rhythms. Using whole-cell patch-clamp recordings, the membrane properties of suprachiasmatic nucleus neurons were investigated in order to get more insight in membrane physiological

  9. Different IgG-subclass distributions after whole-cell and acellular pertussis infant primary vaccinations in healthy and pertussis infected children

    NARCIS (Netherlands)

    Hendrikx, Lotte H.; Schure, Rose-Minke; Ozturk, Kemal; de Rond, Lia G. H.; de Greeff, S. C.; Sanders, Elisabeth A. M.; Berbers, Guy A. M.; Buisman, Anne-Marie

    2011-01-01

    The distribution of IgG-subclasses provides insight in the immunological mechanisms of protection against whooping cough. We investigated the effect of Dutch whole-cell pertussis and acellular pertussis vaccines administered in infancy on the IgG-subclass distributions in healthy children aged 12

  10. Acute necrotizing encephalopathy secondary to diphtheria, tetanus toxoid and whole-cell pertussis vaccination: diffusion-weighted imaging and proton MR spectroscopy findings

    International Nuclear Information System (INIS)

    Aydin, Hale; Ozgul, Esra; Agildere, Ahmet Muhtesem

    2010-01-01

    We present a previously healthy 6-month-old boy who was admitted to our hospital with lethargy, hypotonia and focal clonic seizures 6 days following diptheria, tetanus toxoid and whole-cell pertussis vaccination. A diagnosis of acute necrotising encephalopathy was made with the aid of MRI, including diffusion-weighted imaging and proton MR spectroscopy. (orig.)

  11. Acute necrotizing encephalopathy secondary to diphtheria, tetanus toxoid and whole-cell pertussis vaccination: diffusion-weighted imaging and proton MR spectroscopy findings

    Energy Technology Data Exchange (ETDEWEB)

    Aydin, Hale; Ozgul, Esra; Agildere, Ahmet Muhtesem [Baskent University Hospital, Department of Radiology, Ankara (Turkey)

    2010-07-15

    We present a previously healthy 6-month-old boy who was admitted to our hospital with lethargy, hypotonia and focal clonic seizures 6 days following diptheria, tetanus toxoid and whole-cell pertussis vaccination. A diagnosis of acute necrotising encephalopathy was made with the aid of MRI, including diffusion-weighted imaging and proton MR spectroscopy. (orig.)

  12. Whole-cell or acellular pertussis vaccination in infancy determines IgG subclass profiles to DTaP booster vaccination

    NARCIS (Netherlands)

    van der Lee, Saskia; Sanders, Elisabeth A.M.; Berbers, Guy A M; Buisman, Anne-Marie

    2018-01-01

    Introduction Duration of protection against pertussis is shorter in adolescents who have been immunized with acellular pertussis (aP) in infancy compared with adolescents who received whole-cell pertussis (wP) vaccines in infancy, which is related to immune responses elicited by these priming

  13. Whole-cell or acellular pertussis vaccination in infancy determines IgG subclass profiles to DTaP booster vaccination.

    NARCIS (Netherlands)

    van der Lee, Saskia; Sanders, Elisabeth A M; Berbers, Guy A M; Buisman, Anne-Marie

    2018-01-01

    Duration of protection against pertussis is shorter in adolescents who have been immunized with acellular pertussis (aP) in infancy compared with adolescents who received whole-cell pertussis (wP) vaccines in infancy, which is related to immune responses elicited by these priming vaccines. To better

  14. A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

    Science.gov (United States)

    Adachi, Daisuke; Koh, FookHee; Hama, Shinji; Ogino, Chiaki; Kondo, Akihiko

    2013-05-10

    To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. 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.

  16. Highly sensitive nano-porous lattice biosensor based on localized surface plasmon resonance and interference.

    Science.gov (United States)

    Yeom, Se-Hyuk; Kim, Ok-Geun; Kang, Byoung-Ho; Kim, Kyu-Jin; Yuan, Heng; Kwon, Dae-Hyuk; Kim, Hak-Rin; Kang, Shin-Won

    2011-11-07

    We propose a design for a highly sensitive biosensor based on nanostructured anodized aluminum oxide (AAO) substrates. A gold-deposited AAO substrate exhibits both optical interference and localized surface plasmon resonance (LSPR). In our sensor, application of these disparate optical properties overcomes problems of limited sensitivity, selectivity, and dynamic range seen in similar biosensors. We fabricated uniform periodic nanopore lattice AAO templates by two-step anodizing and assessed their suitability for application in biosensors by characterizing the change in optical response on addition of biomolecules to the AAO template. To determine the suitability of such structures for biosensing applications, we immobilized a layer of C-reactive protein (CRP) antibody on a gold coating atop an AAO template. We then applied a CRP antigen (Ag) atop the immobilized antibody (Ab) layer. The shift in reflectance is interpreted as being caused by the change in refractive index with membrane thickness. Our results confirm that our proposed AAO-based biosensor is highly selective toward detection of CRP antigen, and can measure a change in CRP antigen concentration of 1 fg/ml. This method can provide a simple, fast, and sensitive analysis for protein detection in real-time.

  17. A New, Sensitive Marine Microalgal Recombinant Biosensor Using Luminescence Monitoring for Toxicity Testing of Antifouling Biocides

    Science.gov (United States)

    Sanchez-Ferandin, Sophie; Leroy, Fanny; Bouget, François-Yves

    2013-01-01

    In this study, we propose the use of the marine green alga Ostreococcus tauri, the smallest free-living eukaryotic cell known to date, as a new luminescent biosensor for toxicity testing in the environment. Diuron and Irgarol 1051, two antifouling biocides commonly encountered in coastal waters, were chosen to test this new biosensor along with two degradation products of diuron. The effects of various concentrations of the antifoulants on four genetic constructs of O. tauri (based on genes involved in photosynthesis, cell cycle, and circadian clock) were compared using 96-well culture microplates and a luminometer to automatically measure luminescence over 3 days. This was compared to growth inhibition of O. tauri wild type under the same conditions. Luminescence appeared to be more sensitive than growth inhibition as an indicator of toxicity. Cyclin-dependent kinase (CDKA), a protein involved in the cell cycle, fused to luciferase (CDKA-Luc) was found to be the most sensitive of the biosensors, allowing an accurate determination of the 50% effective concentration (EC50) after only 2 days (diuron, 5.65 ± 0.44 μg/liter; Irgarol 1015, 0.76 ± 0.10 μg/liter). The effects of the antifoulants on the CDKA-Luc biosensor were then compared to growth inhibition in natural marine phytoplankton. The effective concentrations of diuron and Irgarol 1051 were found to be similar, indicating that this biosensor would be suitable as a reliable ecotoxicological test. The advantage of this biosensor over cell growth inhibition testing is that the process can be easily automated and could provide a high-throughput laboratory approach to perform short-term toxicity tests. The ability to genetically transform and culture recombinant O. tauri gives it huge potential for screening many other toxic compounds. PMID:23144143

  18. 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.

  19. Integration of Fractal Biosensor in a Digital Microfluidic Platform

    KAUST Repository

    Mashraei, Yousof

    2016-06-08

    The digital microfluidic (DMF) platform introduces many applications in biomedical assays. If it is to be commercially available to the public, it needs to have the essential features of smart sensing and a compact size. In this work, we report on a fractal electrode biosensor that is used for both droplet actuation and sensing C-reactive protein (CRP) concentration levels to assess cardiac disease risk. Our proposed electrode is the first two-terminal electrode design to be integrated into DMF platforms. A simulation of the electrical field distribution shows reduced peak intensities and uniform distribution of the field. When compared to a V-notch square electrode, the fractal electrode shows a superior performance in both aspects, i.e. field uniformity and intensity. These improvements are translated into a successful and responsive actuation of a water droplet with 100V. Likewise, the effective dielectric strength is improved by a 33% increase in the fractal electrode breakdown voltage. Additionally, the capability of the fractal electrode to work as a capacitive biosensor is evaluated with CRP quantification test. Selected fractal electrodes undergo a surface treatment to immobilize anti-CRP antibodies on their surface. The measurement shows a response to the added CRP in capacitance within three minutes. When the untreated electrodes were used for quantification, there was no significant change in capacitance, and this suggested that immobilization was necessary. The electrodes configuration in the fabricated DMF platform allows the fractal electrodes to be selectively used as biosensors, which means the device could be integrated into point-of-care applications.

  20. 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.

  1. Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

    Directory of Open Access Journals (Sweden)

    Iris Bosschem

    Full Text Available Helicobacter suis (H. suis is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin, administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC/lysate and intranasal immunization with Cholera toxin (CT/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

  2. Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry.

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    Julie Gagnaire

    Full Text Available The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF mass spectrometry (MS, correlate delta-toxin expression with accessory gene regulator (agr status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively. In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

  3. Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G.

    Science.gov (United States)

    Lin, Baixue; Fan, Keqiang; Zhao, Jian; Ji, Junjie; Wu, Linjun; Yang, Keqian; Tao, Yong

    2015-08-11

    Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate-dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host β-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).

  4. Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection.

    Directory of Open Access Journals (Sweden)

    Meritxell García-Quintanilla

    Full Text Available The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010, one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (<1.0 endotoxin unit/106 cells compared to wild type cells. Immunization with formalin inactivated IB010 produced a robust antibody response consisting of both IgG1 and IgG2c subtypes. Mice immunized with IB010 had significantly lower post-infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.

  5. New naphthalene whole-cell bioreporter for measuring and assessing naphthalene in polycyclic aromatic hydrocarbons contaminated site.

    Science.gov (United States)

    Sun, Yujiao; Zhao, Xiaohui; Zhang, Dayi; Ding, Aizhong; Chen, Cheng; Huang, Wei E; Zhang, Huichun

    2017-11-01

    A new naphthalene bioreporter was designed and constructed in this work. A new vector, pWH1274_Nah, was constructed by the Gibson isothermal assembly fused with a 9 kb naphthalene-degrading gene nahAD (nahAa nahAb nahAc nahAd nahB nahF nahC nahQ nahE nahD) and cloned into Acinetobacter ADPWH_lux as the host, capable of responding to salicylate (the central metabolite of naphthalene). The ADPWH_Nah bioreporter could effectively metabolize naphthalene and evaluate the naphthalene in natural water and soil samples. This whole-cell bioreporter did not respond to other polycyclic aromatic hydrocarbons (PAHs; pyrene, anthracene, and phenanthrene) and demonstrated a positive response in the presence of 0.01 μM naphthalene, showing high specificity and sensitivity. The bioluminescent response was quantitatively measured after a 4 h exposure to naphthalene, and the model simulation further proved the naphthalene metabolism dynamics and the salicylate-activation mechanisms. The ADPWH_Nah bioreporter also achieved a rapid evaluation of the naphthalene in the PAH-contaminated site after chemical spill accidents, showing high consistency with chemical analysis. The engineered Acinetobacter variant had significant advantages in rapid naphthalene detection in the laboratory and potential in situ detection. The state-of-the-art concept of cloning PAHs-degrading pathway in salicylate bioreporter hosts led to the construction and assembly of high-throughput PAH bioreporter array, capable of crude oil contamination assessment and risk management. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Influences of Mg Doping on the Electrochemical Performance of TiO2 Nanodots Based Biosensor Electrodes

    Directory of Open Access Journals (Sweden)

    M. S. H. Al-Furjan

    2014-01-01

    Full Text Available Electrochemical biosensors are essential for health monitors to help in diagnosis and detection of diseases. Enzyme adsorptions on biosensor electrodes and direct electron transfer between them have been recognized as key factors to affect biosensor performance. TiO2 has a good protein adsorption ability and facilitates having more enzyme adsorption and better electron transfer. In this work, Mg ions are introduced into TiO2 nanodots in order to further improve electrode performance because Mg ions are considered to have good affinity with proteins or enzymes. Mg doped TiO2 nanodots on Ti substrates were prepared by spin-coating and calcining. The effects of Mg doping on the nanodots morphology and performance of the electrodes were investigated. The density and size of TiO2 nanodots were obviously changed with Mg doping. The sensitivity of 2% Mg doped TiO2 nanodots based biosensor electrode increased to 1377.64 from 897.8 µA mM−1 cm−2 and its KMapp decreases to 0.83 from 1.27 mM, implying that the enzyme achieves higher catalytic efficiency due to better affinity of the enzyme with the Mg doped TiO2. The present work could provide an alternative to improve biosensor performances.

  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. Positioning of the sensor cell on the sensing area using cell trapping pattern in incubation type planar patch clamp biosensor.

    Science.gov (United States)

    Wang, Zhi-Hong; Takada, Noriko; Uno, Hidetaka; Ishizuka, Toru; Yawo, Hiromu; Urisu, Tsuneo

    2012-08-01

    Positioning the sensor cell on the micropore of the sensor chip and keeping it there during incubation are problematic tasks for incubation type planar patch clamp biosensors. To solve these problems, we formed on the Si sensor chip's surface a cell trapping pattern consisting of a lattice pattern with a round area 5 μm deep and with the micropore at the center of the round area. The surface of the sensor chip was coated with extra cellular matrix collagen IV, and HEK293 cells on which a chimera molecule of channel-rhodopsin-wide-receiver (ChR-WR) was expressed, were then seeded. We examined the effects of this cell trapping pattern on the biosensor's operation. In the case of a flat sensor chip without a cell trapping pattern, it took several days before the sensor cell covered the micropore and formed an almost confluent state. As a result, multi-cell layers easily formed and made channel current measurements impossible. On the other hand, the sensor chip with cell trapping pattern easily trapped cells in the round area, and formed the colony consisted of the cell monolayer covering the micropore. A laser (473 nm wavelength) induced channel current was observed from the whole cell arrangement formed using the nystatin perforation technique. The observed channel current characteristics matched measurements made by using a pipette patch clamp. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Dynamic monitoring of transmembrane potential changes: a study of ion channels using an electrical double layer-gated FET biosensor.

    Science.gov (United States)

    Pulikkathodi, Anil Kumar; Sarangadharan, Indu; Chen, Yi-Hong; Lee, Geng-Yen; Chyi, Jen-Inn; Lee, Gwo-Bin; Wang, Yu-Lin

    2018-03-27

    In this research, we have designed, fabricated and characterized an electrical double layer (EDL)-gated AlGaN/GaN high electron mobility transistor (HEMT) biosensor array to study the transmembrane potential changes of cells. The sensor array platform is designed to detect and count circulating tumor cells (CTCs) of colorectal cancer (CRC) and investigate cellular bioelectric signals. Using the EDL FET biosensor platform, cellular responses can be studied in physiological salt concentrations, thereby eliminating complex automation. Upon investigation, we discovered that our sensor response follows the transmembrane potential changes of captured cells. Our whole cell sensor platform can be used to monitor the dynamic changes in the membrane potential of cells. The effects of continuously changing electrolyte ion concentrations and ion channel blocking using cadmium are investigated. This methodology has the potential to be used as an electrophysiological probe for studying ion channel gating and the interaction of biomolecules in cells. The sensor can also be a point-of-care diagnostic tool for rapid screening of diseases.

  10. In situ targeting TEM8 via immune response and polypeptide recognition by wavelength-modulated surface plasmon resonance biosensor

    Science.gov (United States)

    Wang, Yimin; Luo, Zewei; Liu, Kunping; Wang, Jie; Duan, Yixiang

    2016-01-01

    There is an increasing interest in real-time and in situ monitoring of living cell activities in life science and medicine. This paper reports a whole cell sensing protocol over the interface of Au film coupled in a wavelength-modulated surface plasmon resonance (WMSPR) biosensor. With dual parabolic mirrors integrated in the sensor, the compact and miniaturized instrument shows satisfactory refractive index sensitivity (2220 nm/RIU) and a high resolution of resonance wavelength shift of 0.3 nm to liquid samples. The affinity interactions between the biomarker of human tumor endothelial marker 8 (TEM8) and antibody (Ab) or specific polypeptide (PEP) were firstly introduced to WMSPR biosensor analysis. Both the interaction events of Ab-cell and PEP-cell over the Au film interface can be recognized by the sensor and the balance time of interactions is about 20 min. The concentration range of Ab for quantitative monitoring of the TEM8 expression on human colon carcinoma SW620 cells was investigated. The present low-cost and time-saving method provides a time resolution of binding specificity between Ab/PEP and TEM8 for real-time analysis of antigen on living tumor cell surface. PMID:26822761

  11. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Bo [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China); Zhang, Shu [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Lang, Qiaolin [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Song, Jianxia; Han, Lihui [Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Liu, Aihua, E-mail: liuah@qibebt.ac.cn [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China)

    2015-07-16

    Highlights: • E. coli surface-dispalyed Gldh exhibiting excellent enzyme activity and stability. • Sensitive amperometric biosensor for glutamate using Gldh-bacteria and MWNTs. • The glutamate biosensor exhibited high specificity and stability. - Abstract: A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP{sup +}-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP{sup +} involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.

  12. Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

    Science.gov (United States)

    Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S.

    2017-01-01

    ABSTRACT Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28112405

  13. Trends in Biosensors for HPV: Identification and Diagnosis

    Directory of Open Access Journals (Sweden)

    Isaac A. M. Frías

    2015-01-01

    Full Text Available The conventional methodologies used for the detection of human papillomavirus (HPV present actually robust and reproducible advantages. However, at the same time, they involve complex protocols that sometimes are difficult to popularize. Over the first half of XX century, the adequate treatment of complex and delicate processes from a simple instrumental base seemed a fundamental and intrinsic contradiction. However, interdisciplinary trends have allowed the manipulation of tissues, proteins, and nucleic acids through innovative increasingly smaller devices. The proper diagnosis of HPV has seen great advances since biosensor researchers are employing its virus strains as models to study the interactions between the biorecognition element and the transducer. Additionally, all recent improvements and trends that material sciences, biotechnology, and data processing scientists excel for biosensors can be applied for the HPV detection platforms. In this review, we highlight the recent trends on materials, nanomaterials, and transducers for the specific detection and differentiation of HPV strains. The most influential methods for the detection and identification of these papillomaviruses include optical, electrochemical, and piezoelectric transducers; we will visit their sensibility and advantages. Additionally, we highlight the factors that contributed to the increasing importance of these biodevices as potential substitutes to conventional diagnostic methods.

  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. 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.

  16. Electrochemical Affinity Biosensors Based on Disposable Screen-Printed Electrodes for Detection of Food Allergens

    Science.gov (United States)

    Vasilescu, Alina; Nunes, Gilvanda; Hayat, Akhtar; Latif, Usman; Marty, Jean-Louis

    2016-01-01

    Food allergens are proteins from nuts and tree nuts, fish, shellfish, wheat, soy, eggs or milk which trigger severe adverse reactions in the human body, involving IgE-type antibodies. Sensitive detection of allergens in a large variety of food matrices has become increasingly important considering the emergence of functional foods and new food manufacturing technologies. For example, proteins such as casein from milk or lysozyme and ovalbumin from eggs are sometimes used as fining agents in the wine industry. Nonetheless, allergen detection in processed foods is a challenging endeavor, as allergen proteins are degraded during food processing steps involving heating or fermentation. Detection of food allergens was primarily achieved via Enzyme-Linked Immuno Assay (ELISA) or by chromatographic methods. With the advent of biosensors, electrochemical affinity-based biosensors such as those incorporating antibodies and aptamers as biorecognition elements were also reported in the literature. In this review paper, we highlight the success achieved in the design of electrochemical affinity biosensors based on disposable screen-printed electrodes towards detection of protein allergens. We will discuss the analytical figures of merit for various disposable screen-printed affinity sensors in relation to methodologies employed for immobilization of bioreceptors on transducer surface. PMID:27827963

  17. A novel and robust recombinant Pichia pastoris yeast whole cell biocatalyst with intracellular overexpression of a Thermomyces lanuginosus lipase: preparation, characterization and application in biodiesel production.

    Science.gov (United States)

    Yan, Jinyong; Zheng, Xianliang; Li, Shengying

    2014-01-01

    A novel and robust recombinant Pichia pastoris yeast whole cell catalyst (WCC) with functional intracellular expression of Thermomyces lanuginosus lipase (Tll) was constructed and characterized for biodiesel production from waste cooking oils. This permeabilized WCC was able to convert waste cooking oils to biodiesel with 82% yield within 84 h at 6% dosage whole cells. The WCC showed two fold catalytic activity of 0.73 U/mg DCW compared to its commercial counterpart Lipozyme TLIM (immobilized Tll). Short chain alcohol tolerance of this WCC was significantly improved compared to Lipozyme TLIM. This beneficial property enabled it to catalyze biodiesel production efficiently with one step addition of methanol. The reusability of this biocatalyst retained 78% activity after three batch cycles. This easily prepared and cost-effective WCC showed better catalytic performance than Lipozyme TLIM with respect to biodiesel yield and productivity, thus suggesting a promising cost-effective biocatalyst for biodiesel production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst

    DEFF Research Database (Denmark)

    Kiss, Flora M.; Lundemo, Marie Therese; Zapp, Josef

    2015-01-01

    and drug precursors. Results: In this work, we demonstrate the conversion of a synthetic testosterone derivative, cyproterone acetate, by CYP106A2 under in vitro and in vivo conditions. Using a Bacillus megaterium whole-cell system overexpressing CYP106A2, sufficient amounts of product for structure...... describe the successful scale-up of cyproterone acetate conversion from shake flasks to bioreactors, using the CYP106A2 enzyme in a whole-cell system. The substrate was converted to its main human metabolite, 15 β-hydroxycyproterone acetate, a highly interesting drug candidate, due to its retained...... antiandrogen activity but significantly lower progestogen properties than the mother compound. Optimization of the process led to an improvement from 55% to 98% overall conversion, with a product formation of 0.43 g/L, approaching industrial process requirements and a future large-scale application....

  19. 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.

  20. 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.

  1. A hydrogel biosensor for high selective and sensitive detection of amyloid-beta oligomers.

    Science.gov (United States)

    Sun, Liping; Zhong, Yong; Gui, Jie; Wang, Xianwu; Zhuang, Xiaorong; Weng, Jian

    2018-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive and memory impairment. It is the most common neurological disease that causes dementia. Soluble amyloid-beta oligomers (AβO) in blood or cerebrospinal fluid (CSF) are the pathogenic biomarker correlated with AD. A simple electrochemical biosensor using graphene oxide/gold nanoparticles (GNPs) hydrogel electrode was developed in this study. Thiolated cellular prion protein (PrP C ) peptide probe was immobilized on GNPs of the hydrogel electrode to construct an AβO biosensor. Electrochemical impedance spectroscopy was utilized for AβO analysis. The specific binding between AβO and PrP C probes on the hydrogel electrode resulted in an increase in the electron-transfer resistance. The biosensor showed high specificity and sensitivity for AβO detection. It could selectively differentiate AβO from amyloid-beta (Aβ) monomers or fibrils. Meanwhile, it was highly sensitive to detect as low as 0.1 pM AβO in artificial CSF or blood plasma. The linear range for AβO detection is from 0.1 pM to 10 nM. This biosensor could be used as a cost-effective tool for early diagnosis of AD due to its high electrochemical performance and bionic structure.

  2. 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....

  3. 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

  4. Protection against cholera from killed whole-cell oral cholera vaccines: a systematic review and meta-analysis.

    Science.gov (United States)

    Bi, Qifang; Ferreras, Eva; Pezzoli, Lorenzo; Legros, Dominique; Ivers, Louise C; Date, Kashmira; Qadri, Firdausi; Digilio, Laura; Sack, David A; Ali, Mohammad; Lessler, Justin; Luquero, Francisco J; Azman, Andrew S

    2017-10-01

    Killed whole-cell oral cholera vaccines (kOCVs) are becoming a standard cholera control and prevention tool. However, vaccine efficacy and direct effectiveness estimates have varied, with differences in study design, location, follow-up duration, and vaccine composition posing challenges for public health decision making. We did a systematic review and meta-analysis to generate average estimates of kOCV efficacy and direct effectiveness from the available literature. For this systematic review and meta-analysis, we searched PubMed, Embase, Scopus, and the Cochrane Review Library on July 9, 2016, and ISI Web of Science on July 11, 2016, for randomised controlled trials and observational studies that reported estimates of direct protection against medically attended confirmed cholera conferred by kOCVs. We included studies published on any date in English, Spanish, French, or Chinese. We extracted from the published reports the primary efficacy and effectiveness estimates from each study and also estimates according to number of vaccine doses, duration, and age group. The main study outcome was average efficacy and direct effectiveness of two kOCV doses, which we estimated with random-effect models. This study is registered with PROSPERO, number CRD42016048232. Seven trials (with 695 patients with cholera) and six observational studies (217 patients with cholera) met the inclusion criteria, with an average two-dose efficacy of 58% (95% CI 42-69, I 2 =58%) and effectiveness of 76% (62-85, I 2 =0). Average two-dose efficacy in children younger than 5 years (30% [95% CI 15-42], I 2 =0%) was lower than in those 5 years or older (64% [58-70], I 2 =0%; pcholera for at least 3 years. One kOCV dose provides at least short-term protection, which has important implications for outbreak management. kOCVs are effective tools for cholera control. The Bill & Melinda Gates Foundation. Copyright This is an Open Access article published under the CC BY 3.0 IGO license which permits

  5. Vaccination in Nile tilapia broodstock with whole cell vaccine and disease resistance in its fry against Aeromonas hydrophila

    Directory of Open Access Journals (Sweden)

    Sukenda Sukenda

    2017-07-01

    Full Text Available ABSTRACT The aim of this study was to analyze the effectivity of vaccination in Nile tilapia broodstock with whole cell vaccine and disease resistance in fry tilapia against Aeromonas hydrophila. Tilapia Nirwana strain that used for this had average body weight of 185±13.23 g and were maintained in ponds sizing of (2.5×2.5×1 m3. Vaccinations that has been done through intraperitoneal injection using dose of 0.1 mL/fish, meanwhile the fish for control was injected by phosphate buffered saline (PBS. This study used complete randomized design with two treatments and three replications. Antibody level was measured by using indirect enzyme-linked immunosorbent assay (ELISA method in the broodstock, egg, and fry.  Challenge test in fry tilapia performed at the age of 5, 10, and 15 days. The results showed that vaccination in tilapia broodstock delivered a significant antibody level in broodstock, eggs, and fry (P<0.05 compared to the control. Relative percent survival of offspring at 5, 10, and 15 days were 78.26%, 70.59%, and 65.52%, respectively.  As a conclusion, vaccination in tilapia broodstock was effective to improve specific and non-specific immunity, and protect fry tilapia from A. hydrophila infection through maternal immunity. Keywords: vaccination, antibody, maternal immunity, tilapia, Aeromonas hydrophila  ABSTRAK Penelitian ini bertujuan untuk menganalisis efikasi vaksinasi pada induk nila dengan vaksin sel utuh dan ketahanan benih yang dihasilkan terhadap Aeromonas hydrophila. Ikan nila stain Nirwana yang digunakan dalam penelitian memiliki bobot rata-rata 185±13,23 g dan ikan dipelihara dalam kolam (2,5×2,5×1 m3. vaksinasi dilakukan melalui penyuntikan intraperitoneal dengan dosis 0,1 mL/ikan, sementara itu ikan kontrol disuntik dengan phosphate buffered saline (PBS. Penelitian ini menggunakan rancangan acak lengkap dengan dua perlakuan dan tiga ulangan. Tingkat antibodi diukur dengan menggunakan metode indirect enzyme

  6. The Touch and Zap Method for In Vivo Whole-Cell Patch Recording of Intrinsic and Visual Responses of Cortical Neurons and Glial Cells

    Science.gov (United States)

    Schramm, Adrien E.; Marinazzo, Daniele; Gener, Thomas; Graham, Lyle J.

    2014-01-01

    Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe “Touch and Zap”, an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the “Touch”. By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or “Zap”, as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi

  7. Permeability changes and incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of Chinese hamsters affected by MEA and low temperature

    International Nuclear Information System (INIS)

    Ermekova, V.M.; Kondakova, N.V.; Levitman, M.Kh.; Saugabaeva, K.M.; Ehjdus, L.Kh.

    1976-01-01

    Action of MEA and low temperature (20degC) on the incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of chinese hamsters has been studied. It has been found that each of the above-mentioned factors equally decreases the label uptake into the cell and DNA. It is concluded that MEA and low temperature do not substantially influence the rate of DNA synthesis

  8. The Touch and Zap method for in vivo whole-cell patch recording of intrinsic and visual responses of cortical neurons and glial cells.

    Science.gov (United States)

    Schramm, Adrien E; Marinazzo, Daniele; Gener, Thomas; Graham, Lyle J

    2014-01-01

    Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe "Touch and Zap", an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the "Touch". By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or "Zap", as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi

  9. 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.

  10. Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Huy, Tran Quang, E-mail: huytq@nihe.org.vn [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam); Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi [National Institute of Hygiene and Epidemiology (NIHE), No1 Yersin St., Hanoi (Viet Nam); Tuan, Mai Anh, E-mail: tuanma-itims@mail.hut.edu.vn [International Training Institute for Materials Science (ITIMS), Hanoi University of Science and Technology (HUST), No1 Dai Co Viet, Hanoi (Viet Nam)

    2011-06-01

    In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

  11. Highly sensitive voltammetric biosensor for nitric oxide based on its high affinity with hemoglobin

    International Nuclear Information System (INIS)

    Fan Chunhai; Liu Xinjian; Pang Jiantao; Li Genxi; Scheer, Hugo

    2004-01-01

    Although heme protein-based, amperometric nitric oxide (NO) biosensors have been well documented in previous studies, most have been conducted in anaerobic conditions. Herein we report a novel hemoglobin-based NO biosensor that is not only very sensitive but also usable in air. The heme protein was entrapped in a sodium montmorillonite film, which was immobilized at a pyrolytic graphite electrode surface. Film-entrapped hemoglobin can directly exchange electrons with the electrode, and this process has proven to favor the catalytic reduction of oxygen. In addition, NO induced a cathodic potential shift of the catalytic reduction peak of oxygen. This potential shift was proportional to the logarithm of NO concentration ranging from 4.0 x 10 -11 to 5.0 x 10 -6 mol/L. The detection limit has been estimated to be 20 pM, approximately four orders lower than previously reported amperometric detectors

  12. Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.

    Science.gov (United States)

    Zirpel, Bastian; Stehle, Felix; Kayser, Oliver

    2015-09-01

    The Δ9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of Δ9-tetrahydrocannabinolic acid (THCA) using whole cells. Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml(-1) (intracellular) and 44 pkat ml(-1) (extracellular) after 192 h of cultivation at 15 °C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l(-1)) THCA could be produced by 10.5 gCDW l(-1) before enzyme activity was lost. Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production.

  13. Label-free detection of glycoproteins by the lectin biosensor down to attomolar level using gold nanoparticles

    Science.gov (United States)

    Bertok, Tomas; Sediva, Alena; Katrlik, Jaroslav; Gemeiner, Pavol; Mikula, Milan; Nosko, Martin; Tkac, Jan

    2016-01-01

    We present here an ultrasensitive electrochemical biosensor based on a lectin biorecognition capable to detect concentrations of glycoproteins down to attomolar (aM) level by investigation of changes in the charge transfer resistance (Rct) using electrochemical impedance spectroscopy (EIS). On polycrystalline gold modified by an aminoalkanethiol linker layer, gold nanoparticles were attached. A Sambucus nigra agglutinin was covalently immobilised on a mixed self-assembled monolayer formed on gold nanoparticles and finally, the biosensor surface was blocked by poly(vinylalcohol). The lectin biosensor was applied for detection of sialic acid containing glycoproteins fetuin and asialofetuin. Building of a biosensing interface was carefully characterised by a broad range of techniques such as electrochemistry, EIS, atomic force microscopy, scanning electron microscopy and surface plasmon resonance with the best performance of the biosensor achieved by application of HS-(CH2)11-NH2 linker and gold nanoparticles with a diameter of 20 nm. The lectin biosensor responded to an addition of fetuin (8.7% of sialic acid) with sensitivity of (338 ± 11) Ω decade-1 and to asialofetuin (≤ 0.5% of sialic acid) with sensitivity of (109 ± 10) Ω decade-1 with a blank experiment with oxidised asialofetuin (without recognisable sialic acid) revealing sensitivity of detection of (79 ± 13) Ω decade-1. These results suggest the lectin biosensor responded to changes in the glycan amount in a quantitative way with a successful validation by a lectin microarray. Such a biosensor device has a great potential to be employed in early biomedical diagnostics of diseases such as arthritis or cancer, which are connected to aberrant glycosylation of protein biomarkers in biological fluids. PMID:23601864

  14. A novel nitrite biosensor based on the direct electron transfer hemoglobin immobilized in the WO3 nanowires with high length–diameter ratio

    International Nuclear Information System (INIS)

    Liu, Hui; Duan, Congyue; Yang, Chenhui; Chen, Xianjin; Shen, Wanqiu; Zhu, Zhenfeng

    2015-01-01

    WO 3 nanowires (WO 3 NWs) with high length–diameter ratio have been synthesized through a simple synthetic route without any additive and then used to immobilize hemoglobin (Hb) to fabricate a mediator-free biosensor. The morphology and structure of WO 3 NWs were characterized by scanning electron microscopy, transmission electronic microscopy and X-ray diffraction. Spectroscopic and electrochemical results revealed that WO 3 NWs are an excellent immobilization matrix with biocompatibility for redox protein, affording good protein bioactivity and stability. Meanwhile, due to unique morphology and property of the WO 3 nanowires, the direct electron transfer of Hb is facilitated and the prepared biosensors displayed good performance for the detection of nitrite with a wide linear range of 1 to 4200 μM, as well as an extremely low detection limit of 0.28 μM. The WO 3 nanowires with high length–diameter ratio could be a promising matrix for the fabrication of mediator-free biosensors, and may find wide potential applications in environmental analysis and biomedical detection. - Highlights: • The WO 3 NWs with high length–diameter ratio have been synthesized. • The WO 3 NWs were used to immobilize Hb to fabricate a mediator-free biosensor. • The biosensor displays a wide linear range of 1–4200 μM for nitrite. • The biosensor exhibits an extremely low detection limit of 0.28 μM for nitrite

  15. 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

  16. 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

  17. 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...

  18. Bioluminescent bacteria: lux genes as environmental biosensors

    OpenAIRE

    Nunes-Halldorson,Vânia da Silva; Duran,Norma Letícia

    2003-01-01

    Bioluminescent bacteria are widespread in natural environments. Over the years, many researchers have been studying the physiology, biochemistry and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology through the use of luminescent genes as biosensors for environmental studies. This paper will review the chronology of scientific discoveries on bacterial bioluminescence and the current applications of bioluminescence in env...

  19. Microbial Biosensors for Selective Detection of Disaccharides

    Science.gov (United States)

    Seven microbial strains were screened for their ability to detect disaccharides as components of Clark-type oxygen biosensors. Sensors responded to varying degrees to maltose, cellobiose, sucrose, and melibiose, but none responded strongly to lactose. Although microbial sensors are relatively nons...

  20. Methods for using redox liposome biosensors

    Science.gov (United States)

    Cheng, Quan; Stevens, Raymond C.

    2002-01-01

    The present invention provides methods and compositions for detecting the presence of biologically-important analytes by using redox liposome biosensors. In particular, the present invention provides liposome/sol-gel electrodes suitable for the detection of a wide variety of organic molecules, including but not limited to bacterial toxins.

  1. Boar taint detection using parasitoid biosensors

    Science.gov (United States)

    To evaluate the potential for a non-stinging wasp to be used as a biosensor in the pig industry, we trained wasps to 3 individual chemicals associated with boar taint. Training consisted of presenting the odors to hungry wasps while they were feeding on sugar. This associates the chemical with a fo...

  2. Clinical Assessment Applications of Ambulatory Biosensors

    Science.gov (United States)

    Haynes, Stephen N.; Yoshioka, Dawn T.

    2007-01-01

    Ambulatory biosensor assessment includes a diverse set of rapidly developing and increasingly technologically sophisticated strategies to acquire minimally disruptive measures of physiological and motor variables of persons in their natural environments. Numerous studies have measured cardiovascular variables, physical activity, and biochemicals…

  3. Development and Applications of Portable Biosensors.

    Science.gov (United States)

    Srinivasan, Balaji; Tung, Steve

    2015-08-01

    The significance of microfluidics-based and microelectromechanical systems-based biosensors has been widely acknowledged, and many reviews have explored their potential applications in clinical diagnostics, personalized medicine, global health, drug discovery, food safety, and forensics. Because health care costs are increasing, there is an increasing need to remotely monitor the health condition of patients by point-of-care-testing. The demand for biosensors for detection of biological warfare agents has increased, and research is focused on ways of producing small portable devices that would allow fast, accurate, and on-site detection. In the past decade, the demand for rapid and accurate on-site detection of plant disease diagnosis has increased due to emerging pathogens with resistance to pesticides, increased human mobility, and regulations limiting the application of toxic chemicals to prevent spread of diseases. The portability of biosensors for on-site diagnosis is limited due to various issues, including sample preparation techniques, fluid-handling techniques, the limited lifetime of biological reagents, device packaging, integrating electronics for data collection/analysis, and the requirement of external accessories and power. Many microfluidic, electronic, and biological design strategies, such as handling liquids in biosensors without pumps/valves, the application of droplet-based microfluidics, paper-based microfluidic devices, and wireless networking capabilities for data transmission, are being explored. © 2015 Society for Laboratory Automation and Screening.

  4. Biosensor discovery of thyroxine transport disrupting chemicals

    NARCIS (Netherlands)

    Marchesini, G.R.; Meimaridou, A.; Haasnoot, W.; Meulenberg, E.; Albertus, F.; Mizuguchi, M.; Takeuchi, M.; Irth, H.; Murk, A.J.

    2008-01-01

    Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two

  5. Fiber optic-based regenerable biosensor

    Science.gov (United States)

    Sepaniak, Michael J.; Vo-Dinh, Tuan

    1993-01-01

    A fiber optic-based regenerable biosensor. The biosensor is particularly suitable for use in microscale work in situ. In one embodiment, the biosensor comprises a reaction chamber disposed adjacent the distal end of a waveguide and adapted to receive therein a quantity of a sample containing an analyte. Leading into the chamber is a plurality of capillary conduits suitable for introducing into the chamber antibodies or other reagents suitable for selective interaction with a predetermined analyte. Following such interaction, the contents of the chamber may be subjected to an incident energy signal for developing fluorescence within the chamber that is detectable via the optical fiber and which is representative of the presence, i.e. concentration, of the selected analyte. Regeneration of the biosensor is accomplished by replacement of the reagents and/or the analyte, or a combination of these, at least in part via one or more of the capillary conduits. The capillary conduits extend from their respective terminal ends that are in fluid communication with the chamber, away from the chamber to respective location(s) remote from the chamber thereby permitting in situ location of the chamber and remote manipulation and/or analysis of the activity with the chamber.

  6. 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.

  7. Facile screening of potential xenoestrogens by an estrogen receptor-based reusable optical biosensor.

    Science.gov (United States)

    Liu, Lanhua; Zhou, Xiaohong; Lu, Yun; Shan, Didi; Xu, Bi; He, Miao; Shi, Hanchang; Qian, Yi

    2017-11-15

    The apparent increase in hormone-induced cancers and disorders of the reproductive tract has led to a growing demand for new technologies capable of screening xenoestrogens. We reported an estrogen receptor (ER)-based reusable fiber biosensor for facile screening estrogenic compounds in environment. The bioassay is based on the competition of xenoestrogens with 17β-estradiol (E 2 ) for binding to the recombinant receptor of human estrogen receptor α (hERα) protein, leaving E 2 free to bind to fluorophore-labeled anti-E 2 monoclonal antibody. Unbound anti-E 2 antibody then binds to the immobilized E 2 -protein conjugate on the fiber surface, and is detected by fluorescence emission induced by evanescent field. As expected, the stronger estrogenic activity of xenoestrogen would result in the weaker fluorescent signal. Three estrogen-agonist compounds, diethylstilbestrol (DES), 4-n-nonylphenol (NP) and 4-n-octylphenol (OP), were chosen as a paradigm for validation of this assay. The rank order of estrogenic potency determined by this biosensor was DES>OP>NP, which were consistent with the published results in numerous studies. Moreover, the E 2 -protein conjugate modified optical fiber was robust enough for over 300 sensing cycles with the signal recoveries ranging from 90% to 100%. In conclusion, the biosensor is reusable, reliable, portable and amenable to on-line operation, providing a facile, efficient and economical alternative to screen potential xenoestrogens in environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. An enzymatic glucose biosensor based on a glassy carbon electrode modified with cylinder-shaped titanium dioxide nanorods

    International Nuclear Information System (INIS)

    Yang, Zhanjun; Xu, Youbao; Li, Juan; Jian, Zhiqin; Yu, Suhua; Zhang, Yongcai; Hu, Xiaoya; Dionysiou, Dionysios D.

    2015-01-01

    We describe a highly sensitive electrochemical enzymatic glucose biosensor. A glassy carbon electrode was modified with cylinder-shaped titanium dioxide nanorods (TiO 2 -NRs) for the immobilization of glucose oxidase. The modified nanorods and the enzyme biosensor were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The glucose oxidase on the TiO 2 -NRs displays a high activity and undergoes fast surface-controlled electron transfer. A pair of well-defined quasi-reversible redox peaks was observed at −0.394 and −0.450 V. The TiO 2 -NRs provide a good microenvironment to facilitate the direct electron transfer between enzyme and electrode surface. The biosensor has two linear response ranges, viz. from 2.0 to 52 μM, and 0.052 to 2.3 mM. The lower detection limit is 0.5 μM, and the sensitivity is 68.58 mA M −1 cm −2 . The glucose biosensor is selective, well reproducible, and stable. In our perception, the cylindrically shaped TiO 2 -NRs provide a promising support for the immobilization of proteins and pave the way to the development of high-performance biosensors. (author)

  9. 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

  10. A novel glucose biosensor based on phosphonic acid-functionalized silica nanoparticles for sensitive detection of glucose in real samples

    International Nuclear Information System (INIS)

    Zhao, Wenbo; Fang, Yi; Zhu, Qinshu; Wang, Kuai; Liu, Min; Huang, Xiaohua; Shen, Jian

    2013-01-01

    An effective strategy for preparation amperometric biosensor by using the phosphonic acid-functionalized silica nanoparticles (PFSi NPs) as special modified materials is proposed. In such a strategy, glucose oxidase (GOD) was selected as model protein to fabricate glucose biosensor in the presence of phosphonic acid-functionalized silica nanoparticles (PFSi NPs). The PFSi NPs were first modified on the surface of glassy carbon (GC) electrode, then, GOD was adsorbed onto the PFSi NPs film by drop-coating. The PFSi NPs were characterized by transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR) spectra. The interaction of PFSi NPs with GOD was investigated by the circular dicroism spectroscopy (CD). The results showed PFSi NPs could essentially maintain the native conformation of GOD. The direct electron transfer of GOD on (PFSi NPs)/GCE electrode exhibited excellent electrocatalytic activity for the oxidation of glucose. The proposed biosensor modified with PFSi NPs displayed a fast amperometric response (5 s) to glucose, a good linear current–time relation over a wide range of glucose concentrations from 5.00 × 10 −4 to 1.87 × 10 −1 M, and a low detection limit of 2.44 × 10 −5 M (S/N = 3). Moreover, the biosensor can be used for assessment of the concentration of glucose in many real samples (relative error < 3%). The GOD biosensor modified with PFSi NPs will have essential meaning and practical application in future that attributed to the simple method of fabrication and good performance

  11. Construction and application of a zinc-specific biosensor for assessing the immobilization and bioavailability of zinc in different soils

    International Nuclear Information System (INIS)

    Liu Pulin; Huang Qiaoyun; Chen Wenli

    2012-01-01

    The inducibility and specificity of different czcRS operons in Pseudomonas putida X4 were studied by lacZ gene fusions. The data of β-glycosidase activity confirmed that the czcR3 promoter responded quantitatively to zinc. A zinc-specific biosensor, P. putida X4 (pczcR3GFP), was constructed by fusing a promoterless enhanced green fluorescent protein (egfp) gene with the czcR3 promoter in the chromosome of P. putida X4. In water extracts of four different soils amended with zinc, the reporter strain detected about 90% of the zinc content of the samples. Both the bioavailability assessment and the sequential extraction analysis demonstrated that the immobilization of zinc was highly dependent on the physico-chemical properties of soils. The results also showed that the lability of zinc decreased over time. It is concluded that the biosensor constitutes an alternative system for the convenient evaluation of zinc toxicity in the environment. - Highlights: ► A zinc-specific bacterial biosensor was developed. ► Four spiked soils were used to test the application of this biosensor. ► The bioavailable zinc in soil-water extracts decreased due to aging. ► The immobilization and speciation of zinc were highly dependent on the soil type. - The immobilization and bioavailability of zinc in soil were investigated as a function of soil type and aging by a newly constructed zinc-specific biosensor coupled with chemical analysis.

  12. Current Status of HbA1c Biosensors

    Science.gov (United States)

    Lin, Hua; Yi, Jun

    2017-01-01

    Glycated hemoglobin (HbA1c) is formed via non-enzymatic glycosylation reactions at the α–amino group of βVal1 residues in the tetrameric Hb, and it can reflect the ambient glycemic level over the past two to three months. A variety of HbA1c detection methods, including chromatography, immunoassay, enzymatic measurement, electrochemical sensor and capillary electrophoresis have been developed and used in research laboratories and in clinics as well. In this review, we summarize the current status of HbA1c biosensors based on the recognition of the sugar moiety on the protein and also their applications in the whole blood sample measurements. PMID:28777351

  13. Smart electrochemical biosensors: From advanced materials to ultrasensitive devices

    Energy Technology Data Exchange (ETDEWEB)

    Sadik, Omowunmi A., E-mail: osadik@binghamton.ed [Department of Chemistry, Center for Advanced Sensors and Environmental Monitoring (CASE), State University of New York-Binghamton, P.O. Box 6000, Binghamton, NY 13902 (United States); Mwilu, Samuel K.; Aluoch, Austin [Department of Chemistry, Center for Advanced Sensors and Environmental Monitoring (CASE), State University of New York-Binghamton, P.O. Box 6000, Binghamton, NY 13902 (United States)

    2010-05-30

    The specificity, simplicity, and inherent miniaturization afforded by advances in modern electronics have allowed electrochemical sensors to rival the most advanced optical protocols. One major obstacle in implementing electrochemistry for studying biomolecular reaction is its inadequate sensitivity. Recent reports however showed unprecedented sensitivities for biomolecular recognition using enhanced electronic amplification provided by new classes of electrode materials (e.g. carbon nanotubes, metal nanoparticles, and quantum dots). Biosensor technology is one area where recent advances in nanomaterials are pushing the technological limits of electrochemical sensitivities, thus allowing for the development of new sensor chemistries and devices. This work focuses on our recent work, based on metal-enhanced electrochemical detection, and those of others in combining advanced nanomaterials with electrochemistry for the development of smart sensors for proteins, nucleic acids, drugs and cancer cells.

  14. Smart electrochemical biosensors: From advanced materials to ultrasensitive devices

    International Nuclear Information System (INIS)

    Sadik, Omowunmi A.; Mwilu, Samuel K.; Aluoch, Austin

    2010-01-01

    The specificity, simplicity, and inherent miniaturization afforded by advances in modern electronics have allowed electrochemical sensors to rival the most advanced optical protocols. One major obstacle in implementing electrochemistry for studying biomolecular reaction is its inadequate sensitivity. Recent reports however showed unprecedented sensitivities for biomolecular recognition using enhanced electronic amplification provided by new classes of electrode materials (e.g. carbon nanotubes, metal nanoparticles, and quantum dots). Biosensor technology is one area where recent advances in nanomaterials are pushing the technological limits of electrochemical sensitivities, thus allowing for the development of new sensor chemistries and devices. This work focuses on our recent work, based on metal-enhanced electrochemical detection, and those of others in combining advanced nanomaterials with electrochemistry for the development of smart sensors for proteins, nucleic acids, drugs and cancer cells.

  15. A hydrogel biosensor for high selective and sensitive detection of amyloid-beta oligomers

    Directory of Open Access Journals (Sweden)

    Sun LP

    2018-02-01

    Full Text Available Liping Sun,1 Yong Zhong,1 Jie Gui,1 Xianwu Wang,1 Xiaorong Zhuang,2 Jian Weng1 1Key Laboratory of Biomedical Engineering of Fujian Province, Research Center of Biomedical Engineering of Xiamen, Department of Biomaterials, College of Materials, Xiamen University, 2Department of Neurology, The Affiliated Zhongshan Hospital of Xiamen University, Xiamen, People’s Republic of China Background: Alzheimer’s disease (AD is a neurodegenerative disorder characterized by progressive cognitive and memory impairment. It is the most common neurological disease that causes dementia. Soluble amyloid-beta oligomers (AβO in blood or cerebrospinal fluid (CSF are the pathogenic biomarker correlated with AD. Methods: A simple electrochemical biosensor using graphene oxide/gold nanoparticles (GNPs hydrogel electrode was developed in this study. Thiolated cellular prion protein (PrPC peptide probe was immobilized on GNPs of the hydrogel electrode to construct an AβO biosensor. Electrochemical impedance spectroscopy was utilized for AβO analysis. Results: The specific binding between AβO and PrPC probes on the hydrogel electrode resulted in an increase in the electron-transfer resistance. The biosensor showed high specificity and sensitivity for AβO detection. It could selectively differentiate AβO from amyloid-beta (Aβ monomers or fibrils. Meanwhile, it was highly sensitive to detect as low as 0.1 pM AβO in artificial CSF or blood plasma. The linear range for AβO detection is from 0.1 pM to 10 nM. Conclusion: This biosensor could be used as a cost-effective tool for early diagnosis of AD due to its high electrochemical performance and bionic structure. Keywords: Alzheimer’s disease, amyloid-beta oligomer, graphene, gold nanoparticles, biosensor

  16. Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact ganglia

    Science.gov (United States)

    Rugiero, François; Gola, Maurice; Kunze, Wolf A A; Reynaud, Jean-Claude; Furness, John B; Clerc, Nadine

    2002-01-01

    Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of −47 ± 6 mV and input resistances (Rin) of 713 ± 49 MΩ at voltages ranging from −90 to −40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inward-rectifier current (IKir) decreased Rin to 103 ± 10 MΩ. AH neurones had resting potentials of −57 ± 4 mV and Rin was 502 ± 27 MΩ. Rin fell to 194 ± 16 MΩ upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, Ih, and to IKir. Resting potential and Rin exhibited a low sensitivity to changes in [K+]o in both AH and S neurones. This indicates that both cells have a low background K+ permeability. The cationic current, Ih, contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of −72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e-fold change. Ih has relatively fast, voltage-dependent kinetics, with on and off time constants in the range of 50–350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium-dependent current that was poorly sensitive to TTX. In AH neurones, the post-action-potential slow hyperpolarizing current, IAHP, displayed large variation from cell to cell. IAHP appeared to be highly Ca2+ sensitive, since its activation with either membrane depolarization or caffeine (1 mm) was not prevented by perfusing the cell with 10 mm BAPTA. We determined the identity of the Ca2+ channels linked to IAHP. Action potentials of AH neurones that were elongated by TEA (10 mm) were similarly shortened and IAHP was suppressed with each of the three Ω-conotoxins GVIA, MVIIA and MVIIC (0.3–0.5 μm), but not with Ω-agatoxin IVA (0.2 μm). There was no

  17. Immunodetection of salivary biomarkers by an optical microfluidic biosensor with polyethylenimine-modified polythiophene-C70 organic photodetectors.

    Science.gov (United States)

    Dong, Tao; Pires, Nuno Miguel Matos

    2017-08-15

    This work reports a novel optical microfluidic biosensor with highly sensitive organic photodetectors (OPDs) for absorbance-based detection of salivary protein biomarkers at the point of care. The compact and miniaturized biosensor has comprised OPDs made of polythiophene-C 70 bulk heterojunction for the photoactive layer; whilst a calcium-free cathode interfacial layer, made of linear polyethylenimine, was incorporated to the photodetectors to enhance the low cost. The OPDs realized onto a glass chip were aligned to antibody-functionalized chambers of a poly(methyl methacrylate) microfluidic chip, in where immunogold-silver assays were conducted. The biosensor has detected IL-8, IL-1β and MMP-8 protein in spiked saliva with high detection specificity and short analysis time exhibiting detection limits between 80pgmL -1 and 120pgmL -1 . The result for IL-8 was below the clinical established cut-off of 600pgmL -1 , which revealed the potential of the biosensor to early detection of oral cancer. The detection limit was also comparable to other previously reported immunosensors performed with bulky instrumentation or using inorganic photodetectors. The optical detection sensitivity of the polythiophene-C 70 OPD was enhanced by optimizing the thickness of the photoactive layer and anode interfacial layer prior to the saliva immunoassays. Further, the biosensor was tested with unspiked human saliva samples, and the results of measuring IL-8 and IL-1β were in statistical agreement with those provided by two commercial assays of ELISA. The optical microfluidic biosensor reported hereby offers an attractive and cost-effective tool to diagnostics or screening purposes at the point of care. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Biosensors in the small scale: methods and technology trends.

    Science.gov (United States)

    Senveli, Sukru U; Tigli, Onur

    2013-03-01

    This study presents a review on biosensors with an emphasis on recent developments in the field. A brief history accompanied by a detailed description of the biosensor concepts is followed by rising trends observed in contemporary micro- and nanoscale biosensors. Performance metrics to quantify and compare different detection mechanisms are presented. A comprehensive analysis on various types and subtypes of biosensors are given. The fields of interest within the scope of this review are label-free electrical, mechanical and optical biosensors as well as other emerging and popular technologies. Especially, the latter half of the last decade is reviewed for the types, methods and results of the most prominently researched detection mechanisms. Tables are provided for comparison of various competing technologies in the literature. The conclusion part summarises the noteworthy advantages and disadvantages of all biosensors reviewed in this study. Furthermore, future directions that the micro- and nanoscale biosensing technologies are expected to take are provided along with the immediate outlook.

  19. 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.

  20. Fundamental Design Principles for Transcription-Factor-Based Metabolite Biosensors.

    Science.gov (United States)

    Mannan, Ahmad A; Liu, Di; Zhang, Fuzhong; Oyarzún, Diego A

    2017-10-20

    Metabolite biosensors are central to current efforts toward precision engineering of metabolism. Although most research has focused on building new biosensors, their tunability remains poorly understood and is fundamental for their broad applicability. Here we asked how genetic modifications shape the dose-response curve of biosensors based on metabolite-responsive transcription factors. Using the lac system in Escherichia coli as a model system, we built promoter libraries with variable operator sites that reveal interdependencies between biosensor dynamic range and response threshold. We developed a phenomenological theory to quantify such design constraints in biosensors with various architectures and tunable parameters. Our theory reveals a maximal achievable dynamic range and exposes tunable parameters for orthogonal control of dynamic range and response threshold. Our work sheds light on fundamental limits of synthetic biology designs and provides quantitative guidelines for biosensor design in applications such as dynamic pathway control, strain optimization, and real-time monitoring of metabolism.