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PubMed2005

Development of a microfluidic biosensor module for pathogen detection.

The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 microL min(-1). The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.

Bacteria↗

PubMed2003

Use of a site-specific recombination-based biosensor for detecting bioavailable toluene and related compounds on roots.

We constructed and characterized a plasmid-based genetic system that reports the expression of a toluene-responsive promoter (PtbuA1) by effecting an irreversible, heritable change in the biosensor cell. Expression of the reporter gene gfp is strongly repressed in the absence of expression from the PtbuA1 promoter, and high level gfp expression in the original cell and its progeny is mediated by the site-specific recombination machinery of bacteriophage P22 to initiate removal of a repressor cassette. The reporter plasmid pTolLHB was functional in two soil saprophytes, Pseudomonas fluorescens A506 and Enterobacter cloacae JL1157, with the efficiency and sensitivity to low toluene concentrations being optimal in P. fluorescens A506. In culture, 80-100% of the A506 (pTolLHB) population expressed gfp following exposure to 0.2 micro m toluene for one to three hours. Compared to the response of A506 containing a plasmid-borne PtbuA1-gfp fusion, the recombination-based biosensor was more sensitive at detecting low toluene and trichloroethylene concentrations. An A506 (pTolLHB) inoculum, which had a background of 2.5% of the cells expressing gfp, was introduced onto barley roots in soil microcosms. If toluene was introduced into the microcosms, after 24 h, 72% of the A506 (pTolLHB) cells recovered from roots expressed gfp, indicating bioavailable toluene to rhizosphere bacteria. When toluene was not introduced, 16.5% of the A506 (pTolLHB) cells recovered from the roots expressed gfp, indicating that natural inducers of the PtbuA1 promoter were present in the barley rhizosphere. When introduced into rhizotrons containing barley plants and toluene vapours, the biosensor allowed localization of the availability of toluene along the seminal roots. In rhizotrons that were not exposed to toluene vapours, the biosensor exhibited high PtbuA1-promoter activity in distinct regions along the seminal roots, indicating spatial heterogeneity plant- or rhizosphere microbial community-derived inducers of the PtbuA1 promoter. This recombination-based toluene biosensor thus was useful in identifying bacterial exposure to transient or low levels of toluene, or related compounds, directly in the environment.

Bacteria↗

PubMed2006

An amperometric biosensor for uric acid determination prepared from uricase immobilized in polypyrrole film.

In order to prepare a biosensor for the determination of uric acid, electropolymerization of pyrrole on Pt surface was carried out with an electrochemical cell containing pyrrole, ferrocene (as a electron mediator) and tetrabutylammonium tetrafluoroborat in acetonitrile by cyclic voltammetry between 0.0 and 1.0 V (vs. Ag/AgCl) at a scan rate of 50 mV/s upon Pt electrode. Uricase was immobilized by a glutaraldehyde/gelatine croslinking procedure on to polypyrrole film after the electropolymerization processes. The response of the biosensor against uric acid was measured after 330 seconds following the application of a constant potential of +0.7 V (vs. Ag/AgCl). The resulting biosensor exhibits excellent electrocatalysis for the uric acid. The amperometric determination is based on the electrochemical detection of H2O2, which is generated in enzymatic reaction of uric acid. The sensor responds to uric acid with a detection limit of 5.0 x 10(-7) M. The sensor remains relatively stable for 5 weeks. Interference effect were investigated on the amperometric response of the biosensor. Determination of uric acid was carried out in the biological fluids by biosensor.

Acetaminophen↗

PubMed1999

Biosensor for the enantioselective analysis of S-perindopril.

Because S-perindopril enantiomer is the eutomer which is responsible for the angiotensin-converting enzyme inhibition activity, it is necessary to develop a reliable method for its assay from its distomer, the R-enantiomer. For this purpose, an amperometric biosensor was developed based on L-amino acid oxidase. The working range of the described biosensor was 20pmol/L-10 micromol/L on the 7.0-7.4 pH range, with a detection limit of 2pmol/L. The low enantioselectivity for R-perindopril, as compared with S-enantiomer, was demonstrated by both mixed solutions and separate solutions methods (amperometric selectivity coefficient is 1.0 x 10(-4)). The biosensor was also selective towards D-proline and polyvinylpyrrolidone. The amperometric biosensor can be used for enantioselective analysis of S-perindopril in raw material, with an RSD < 1%. The life time (t95%) of the biosensor is three weeks.

Amino Acid Oxidoreductases↗

PubMed2005

Urea biosensors based on PVC membrane containing palmitic acid.

A new urea biosensor was prepared by immobilizing urease with four different procedures on poly(vinylchloride) (PVC) ammonium membrane electrode containing palmitic acid by using nonactine as an ammonium-ionophore. The analytical characteristics were investigated and were compared those of the biosensor prepared by using carboxylated PVC. The effect of pH, buffer concentration, temperature, urease concentration, stirring rate and enzyme immobilization procedures on the response to urea of the enzyme electrode were investigated. The linear working range and sensitivity of the biosensor were also determined. The urea biosensor prepared by using the PVC membranes containing palmitic acid showed more effective performance than those of the carboxylated PVC based biosensors. Additionally, urea assay in serum was successfully carried out by using the standard addition method.

Biosensing Techniques↗

PubMed2003

Biosensors for the determination of ortho-acetyl-L-carnitine. Their utilization as detectors in a sequential injection analysis system.

In order to determine ortho-acetyl-L-carnitine, two biosensors were proposed. The biosensors were designed using physical immobilization of L-amino acid oxidase (L-AAOD) and horseradish peroxidase (HRP). Electrode characteristics were obtained and compared for the two carbon paste (graphite powder and paraffin oil) biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol/L to nmol/L, magnitude order with low limits of detection. Due to their reliability, the biosensors were used as detectors in a sequential injection analysis system, and gave reliable results for on-line assay of ortho-acetyl-L-carnitine in synthesis process control with a frequency of 75 samples per hour.

Acetylcarnitine↗

PubMed1999

Biosensor for enantioselective analysis of S-cilazapril, S-trandolapril, and S-pentopril.

The purpose of this research was to construct an amperometric biosensor on L-amino acid oxidase (L-AAOD) supported on a carbon paste for the enantioselective analysis of S-cilazapril (I), S-trandolapril (II), and S-pentopril (III). A chemically modified carbon paste was constructed with L-AAOD. The proposed amperometric biosensor proved reliable for the purity of I, II, and III. The linear working ranges obtained for drug assay were 0.001-100 mumol/l (I), 0.02-10 mumol/l (II), 0.08-50 mumol/l (III) over the pH ranges 7.0-7.4 (I), 6.8-7.4 (II), and 7.0-7.4 (III). The low limits of detection obtained were 5 pmol/l (I), 15 mumol/l (II), and 5 mumol/l (III), respectively. The selectivity of the biosensors was checked by both mixed and separate solution methods. Polyvinylpyrolidone and D-proline did not interfere with the assay of the studied drugs, whereas L-proline presented a potent interfering species. The relative standard deviation values (< 0.2%) make the biosensors suitable for direct amperometric assay of these angiotensin-converting enzyme (ACE) inhibitors. The proposed amperometric biosensor that was based on L-AAOD proved reliable in the analysis of the above-mentioned ACE inhibitors and can be used for the quality control of these drugs.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2006

Label-free cell-based assays with optical biosensors in drug discovery.

Once viewed solely as a tool for low throughput and kinetic analysis of biomolecular interactions, optical biosensors are gaining widespread uses in drug discovery because of recent advances in instrumentation and experimental design. These advances have expanded the capabilities of optical biosensors to meet the needs at many points in the drug discovery process. Concurrent shifts in drug discovery paradigms have seen the growing use of whole cell systems for drug screens, thus creating both a need in drug discovery and a solution in optical biosensors. This article reviews important advances in optical biosensor instrumentation, and highlights the potential of optical biosensors for drug discovery with an emphasis on whole cell sensing in both high throughput and high content fashions.

Biological Assay↗

PubMed2005

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions.

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.

Biological Transport↗

PubMed1998

Development and testing of a bacterial biosensor for toluene-based environmental contaminants.

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator xylR from the TOL plasmid of Pseudomonas putida mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, Pu. A reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of XylR and Pu. When Escherichia coli cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited K1/2 values ranging from 39.0 +/- 3.8 microM for 3-xylene to 2,690 +/- 160 microM for 3-methylbenzylalcohol (means +/- standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.

Biosensing Techniques↗

PubMed2002

Development and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compounds.

A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K(app) values that ranged from 3.3 +/- 1.8 microM for toluene to 35.6 +/- 16.6 microM for trichloroethylene (means +/- standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

Benzene↗

PubMed2002

Characterization of two novel yeast strains used in mediated biosensors for wastewater.

After isolation from a pulp mill wastewater treatment facility, two yeast strains, designated SPT1 and SPT2, were characterized and used in the development of mediated biochemical oxygen demand (BOD) biosensors for wastewater. 18S rRNA gene sequence analysis revealed a one nucleotide difference between the sequence of SPT1 and those of Candida sojae and Candida viswanthii. While SPT2 had the highest overall homology to Pichia norvegensis, at only 73.5%, it is clearly an ascomycete, based on BLAST comparisons and phylogenetic analyses. Neighbor-joining dendrograms indicated that SPT1 clustered with several Candida spp., and that SPT2 clustered with Starmera spp., albeit as a very deep branch. Physiological tests, microscopic observations, and fatty acid analysis confirmed that SPT1 and SPT2 are novel yeast strains. Physiological tests also indicated that both strains had potential for use in mediated biosensors for estimation of BOD in wastewater. The lower detection limits of SPT1- and SPT2-based K3Fe(CN)6-mediated biosensors for a pulp-mill effluent were 2 and 1 mg BOD/L, respectively. Biosensor-response times for effluents from eight different pulp mills were in the range of 5 min. Reliability and sensitivity of the SPT1- and SPT2-based biosensors were good, but varied with the wastewater.

Ascomycota↗

PubMed1985

Non-electrode biosensors in clinical biochemistry.

This review covers biosensors based on piezoelectric crystals, optical systems, field effect transistors and thermistors. Piezoelectric crystal or microgravimetric biosensors have been used for immunoassay. Optical biosensors are described in which waveguides are used to transmit changes in optical characteristics or, in an innovative mode, the evanescent wave component of a completely internally-reflected light beam is used to study optical changes. Optical biosensors have been used in immuno and in enzyme-based assays. Field effect transistors detect changes in ion concentrations and have been applied to the detection of biochemical reactions which involve a change in concentration of a specific ion. Thermistors are used to monitor the heat produced as the result of an exothermic enzymatic reaction and this has been applied to the assay of compounds or enzymes of interest. Biosensors may find a role in clinical biochemistry in low volume testing, patient self-testing and in vivo monitoring.

Adsorption↗

PubMed2004

The potential for biosensors in cardiac surgery.

Biosensor technology has been used in portable medical diagnostic instrumentation since the 1980s, mainly for the home monitoring of blood glucose in diabetics. Numerous types of sensor have been developed and reported in the literature and instrumentation which incorporates biosensors is now available for use in the emergency rooms and intensive care units for some critical parameters. However, there are still many opportunities for the development of rapid assays using biosensors which could be of assistance in cardiac surgery. This paper discusses the biosensor concept and where the key opportunities for biosensor development arise in current and future cardiac surgery.

Biosensing Techniques↗

PubMed2006

[Use of biosensors for detecting organophosphorus agents].

The present paper reviews the use of electrochemical biosensors for detecting organophosphorus pesticides and nerve agents. Acetylcholine esterase (AChE)-immobilized electrodes have been used for detecting AChE inhibitors including organophosphorus and carbamate pesticides. The sensors are composed of AChE and choline oxidase (ChOx) for converting the AChE-generated choline into betaine and hydrogen peroxide (H(2)O(2)), which is electrochemically oxidized at the electrode surface to produce the output signal of the sensor. In the presence of AChE inhibitors, the suppressed output signal of the sensor can be observed. If the sensors are operated in the presence of acetylthiocholine as a substrate of AChE, one can eliminate ChOx from the sensor design because enzymatically generated thiocholine is electrochemically active and thus directly oxidized at the electrode without using ChOx. Electron-transfer mediators such as tetracyanoquinodimethane have often been used for catalytically oxidizing thiocholine at the electrode set at less positive potential, which is effective in circumventing possible interference arising from oxidizing compounds in the sample solution. One of the drawbacks of the AChE-based biosensors in detecting organophosphorus pesticides and nerve agents arises from the fact that the sensors indirectly detect the signal based on the inhibition of the AChE-catalyzed reaction. On the other hand, for directly obtaining the output signal, organophosphorus hydrolase (OPH) is immobilized on the electrode surface to prepare amperometric biosensors. OPH catalyzes the hydrolysis reaction of organophosphorus compounds to produce electrochemically active compounds such as p-nitrophenol and thiols from parathion and VX, respectively. Thus OPH-based sensors can be used for detecting these compounds directly. These biosensors would be useful for in-site measurements of organophosphorus pesticides and nerve agents because portable-type biosensors are easily fabricated at relatively low cost.

Acetylcholinesterase↗

PubMed2006

Novel biosensor-based analytic device for the detection of anti-double-stranded DNA antibodies.

BACKGROUND: Patients with systemic lupus erythematosus (SLE) develop a wide variety of serologic manifestations, including double-stranded DNA autoantibodies (anti-dsDNA). The determination of the potentially pathogenic autoantibodies is diagnostically relevant. METHODS: We developed a novel surface plasmon resonance (SPR) biosensor chip for studies of dsDNA and anti-dsDNA binding. A synthetic oligonucleotide was coupled to biotinylated human transferrin, hybridized with the complementary antistrand, and ligated with a human recombinant dsDNA fragment 233 bp in length. After surface immobilization of this antigenic construct, diluted sera from SLE patients and healthy donors were analyzed with the resulting SPR biosensor system. RESULTS: This SPR biosensor allowed specific detection of anti-dsDNA. In pilot experiments, sera from SLE patients were distinguished from control sera. We also confirmed the specificity of this biosensor by supplementing anti-dsDNA-positive sera with salmon sperm DNA, which blocked the surface binding of anti-dsDNA in a concentration-dependent manner. CONCLUSIONS: An SPR biosensor monitors interactions in real time under homogeneous conditions, providing information about binding kinetics and affinities. Its applicability critically depends on the design of the solid-state surface of the sensor chips. Covalently immobilizing dsDNA as the antigen to the surface in a flow-through cell assured maximal stability for multiple serum injections and regeneration cycles. This technique, which adds a new analytic quality to existing methods, may be beneficial in the diagnosis and clinical monitoring of SLE.

Antibodies, Antinuclear↗

PubMed2006

Enzymatic biosensors.

The biosensor field has grown enormously since the first demonstration of the biosensor concept by Leland C. Clark, Jr. in 1962. Today's biosensor market is dominated by glucose biosensors, mass-produced enzyme electrodes for the rapid self-diagnosis of blood glucose levels by diabetes sufferers. Here we take a historical look at the inception, growth, and development of the enzyme biosensor field from a commercial viewpoint. The current status of the technology is evaluated and future trends in this dynamic and fast-moving field are also anticipated.

Biosensing Techniques↗

PubMed2006

Resonant waveguide grating biosensor for living cell sensing.

This article presents theoretical analysis and experimental data for the use of resonant waveguide grating (RWG) biosensors to characterize stimulation-mediated cell responses including signaling. The biosensor is capable of detecting redistribution of cellular contents in both directions that are perpendicular and parallel to the sensor surface. This capability relies on online monitoring cell responses with multiple optical output parameters, including the changes in incident angle and the shape of the resonant peaks. Although the changes in peak shape are mainly contributed to stimulation-modulated inhomogeneous redistribution of cellular contents parallel to the sensor surface, the shift in incident angle primarily reflects the stimulation-triggered dynamic mass redistribution (DMR) perpendicular to the sensor surface. The optical signatures are obtained and used to characterize several cellular processes including cell adhesion and spreading, detachment and signaling by trypsinization, and signaling through either epidermal growth factor receptor or bradykinin B2 receptor. A mathematical model is developed to link the bradykinin-mediated DMR signals to the dynamic relocation of intracellular proteins and the receptor internalization during B2 receptor signaling cycle. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in four different states of B2 receptors, diffusion of proteins and receptor-protein complexes, and the DMR responses. Classical analysis shows that the system converges to a unique optical signature, whose dynamics (amplitudes, transition time, and kinetics) is dependent on the bradykinin signal input, and consistent with those observed using the RWG biosensors. This study provides fundamentals for probing living cells with the RWG biosensors, in general, optical biosensors.

Animals↗