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PubMed1996

Epitope analysis of the Goodpasture antigen using a resonant mirror biosensor.

We have used a new technique for studying molecular interactions-a resonant mirror biosensor-to identify B cell epitopes within the Goodpasture antigen, which has recently been identified as the non-collagenous domain of the alpha 3-chain of type IV collagen (alpha 3(IV)NC1). Recombinant antigen (r-alpha 3) was immobilized onto the sensing surface of a sample cuvette, and the binding of patients' autoantibodies or a MoAb to the Goodpasture antigen was followed in real time. All patients' sera bound r-alpha 3 in this system, while control sera did not bind. A MoAb inhibited the binding of all patients' autoantibodies to r-alpha 3, from 27% to 90% (mean inhibition 60%), and patients' sera cross-inhibited the binding of each other to the antigen. Binding was inhibited by pre-incubation of autoantibody with both native sheep alpha 3(IV)NC1 and purified human alpha 3(IV)NC1 monomers. Inhibition experiments using soluble overlapping peptides from human alpha 3(IV)NC1 identified putative B cell epitopes. These results suggest that there is a major immunodominant epitope on the Goodpasture antigen, and that there is very limited heterogeneity in the autoantibody response in Goodpasture's disease. The resonant mirror biosensor can be successfully used to monitor antibody-antigen binding using polyclonal sera, and to map epitopes on autoantigens.

Amino Acid Sequence↗

PubMed2003

Rice seedling whole exudates and extracted alkylresorcinols induce stress-response in Escherichia coli biosensors.

A set of Escherichia coli sensor strains was used to evaluate the stress exerted on surrounding bacteria by germinating rice seed exudates. These biosensor strains contain Vibrio fischeri luxCDABE genes fused to the promoters of different genes involved in bacterial responses to environmental stresses. They provided clear evidence for a stress exerted by rice exudates, as shown by the induction of the universal stress protein gene uspA as well as genes of the heat shock regulon, grpE, lon and dnaK. The oxidative stress gene katG, and the post-transcriptional ompF regulator encoded by micF were also activated. The lack of derepression of recA, uvrA and alkA indicated that damage to the DNA was not induced in the E. coli strains tested. Interestingly, resorcinolic lipids extracted from rice root seedlings induced the same promoters as whole exudates, suggesting that these compounds may contribute to the stress exerted by seedling exudates. The results obtained with E. coli biosensors thus indicate that, in vivo, exudates may also exert a selective pressure on root-colonizing bacteria.

Bacterial Proteins↗

PubMed2003

High-resolution real-time recording with microelectrode biosensors reveals novel aspects of adenosine release during hypoxia in rat hippocampal slices.

We have used improved miniaturized adenosine biosensors to measure adenosine release during hypoxia from within the CA1 region of rat hippocampal slices. These microelectrode biosensors record from the extracellular space in the vicinity of active synapses as they detect the synaptic field potentials evoked in area CA1 by stimulation of the afferent Schaffer collateral-commissural fibre pathway. Our new measurements demonstrate the rapid production of adenosine during hypoxia that precedes and accompanies depression of excitatory transmission within area CA1. Simultaneous measurement of adenosine release and synaptic transmission gives an estimated IC50 for adenosine on transmission in the low micromolar range. However, on reoxygenation, synaptic transmission recovers in the face of elevated extracellular adenosine and despite a post-hypoxic surge of adenosine release. This may indicate the occurrence of apparent adenosine A1 receptor desensitization during metabolic stress. In addition, adenosine release is unaffected by pharmacological blockade of glutamate receptors and shows depletion on repeated exposure to hypoxia. Our results thus suggest that adenosine release is not a consequence of excitotoxic glutamate release. The potential for adenosine A1 receptor desensitization during metabolic stress implies that its prevention may be beneficial in extending adenosine-mediated neuroprotection in a variety of clinically relevant conditions.

Adenosine↗

PubMed1998

A rapid method for determination of in vitro susceptibility to antibiotics with a bulk acoustic wave bacterial growth biosensor.

A novel bulk acoustic wave (BAW) bacterial growth biosensor was developed to study in vitro susceptibility by continuous monitoring of disturbances of bacterial growth at low antibiotic concentrations, followed by the accurate and rapid estimation of growth kinetic parameters and minimum inhibitory concentrations (MICs). The susceptibilities of bacteria, e.g. Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pr. morganii and Pr. mirabilis, to various antibiotics, e.g. penicillin, streptomycin, gentamicin and cefotaxime, were investigated, respectively, and the MICs were rapidly determined with a higher reproducibility than the conventional broth micro-dilution technique (BMDT). The effects of cell constant of conductivity electrode, pH and temperature on bacterial growth and biosensor signals were discussed in detail. The proposed method offers an effective alternative to the conventional methods.

Acoustics↗

PubMed1994

Clinical usefulness of the glucose concentration in the subcutaneous tissue--properties and pitfalls of electrochemical biosensors.

Biosensors are miniaturized analytical tools which comprise a biological detection element providing specificity to the analyte, and a physical transducer which guarantees an output signal, e.g. an electric current the size of which is proportional to the concentration of the analyte. They provide the unique possibility of continuous in vivo monitoring. Glucosensors were in fact the first biosensors under study. Among them, the most advanced devices are measuring amperometrically the hydrogen peroxide generated in a stoichiometric relation to the prevailing glucose concentration during glucose oxidase-mediated glucose oxidation. They proved useful in commercially available glucose analyzers and in experimental subcutaneous monitoring. Here it is shown (a) that under steady state conditions the s.c. glucose concentration is nearly identical to that in blood, (b) that s.c. inserted glucose electrodes do mirror the intracorporal glucose concentration both under hypo-, normo-, and hyperglycaemic conditions with a clinically relevant accuracy, (c) that even stable feedback control of intracorporal glucose concentration is possible employing s.c. glucosensor signal as an input to automated insulin pump controller, and (d) that stable function of s.c. sensor is usually accomplished over intervals up to one day but in some cases applications over up to ten days could be realized. The underlying problem consists in an insufficient functional biostability which is a function of biocompatibility and size of the sensor, of its sterility, and of the permanent skin penetration. The latter is still required to get the device in place, to keep it in function, and to make use of the data under any condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

Optical modeling of liquid crystal biosensors.

Optical simulations of a liquid crystal biosensor device are performed using an integrated optical/textural model based on the equations of nematodynamics and two optical methods: the Berreman optical matrix method [J. Opt. Soc. Am. 62, 502 (1972)] and the discretization of the Maxwell equations based on the finite difference time domain (FDTD) method. Testing the two optical methods with liquid crystal films of different degrees of orientational heterogeneities demonstrates that only the FDTD method is suitable to model this device. Basic substrate-induced texturing process due to protein adsorption gives rise to an orientation correlation function that is nearly linear with the transmitted light intensity, providing a basis to calibrate the device. The sensitivity of transmitted light to film thickness, protein surface coverage, and wavelength is established. A crossover incident light wavelength close to lambda(co) approximately 500 nm is found, such that when lambda>lambda(co) thinner films are more sensitive to the amount of protein surface coverage, while for lambda<lambda(co) the reverse holds. In addition it is found that for all wavelengths the sensitivity increases with the amount of protein coverage. The integrated device model based on FDTD optical simulations in conjunction with the Landau-de Gennes nematodynamics model provides a rational basis for further progress in liquid crystal biosensor devices.

Adsorption↗

PubMed2004

Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse.

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca(2+)](i) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.

Animals↗

PubMed1996

Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

Amino Acid Sequence↗

PubMed2005

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution.

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

Age Factors↗

PubMed2006

Ecotoxicological screening of Kenyan tannery dust using a luminescent-based bacterial biosensor.

Ecotoxicological screening of dust sampled throughout a Kenyan tannery was conducted using a luminescence (lux)-based bacterial biosensor for both solid and liquid assays. This was complemented by chemical analysis in an attempt to identify possible causative toxic components. The biosensor results showed a highly significant (p < 0.001) difference in both solid and liquid phase toxicity in samples collected from various identified sampling points in the tannery. A positive correlation was observed between results of the solid and liquid phase techniques, for most of the sampling points indicating that the toxic contaminants were bioavailable both in the solid and liquid state. However, the results generally indicated toxicity associated with liquid phase except certain areas in solid phase such as chemical handling, buffing area and weighing. The most toxic tannery area identified was the weighing area (p < 0.001), showing the lowest bioluminescence for both the solid (0.38 +/- 2.21) and liquid phases (0.01 +/- 0.001). Chromium was the metal present in the highest concentration indicating levels higher than the stipulated regulatory requirement of 0.5 mg Cr/m3 for total Cr (highest Cr concentration was at chemical handling at 209.24 mg l(-1)) in all dust samples. The weighing area had the highest Ni concentration (1.87 mg l(-1)) and the chemical handling area showed the highest Zn concentration (31.9 mg l(-1)). These results raise environmental health concerns, as occupational exposure to dust samples from this site has been shown to give rise to elevated concentrations (above the stipulated levels) of chromium in blood, urine and some body tissues, with inhalation being the main route. Health and Safety Executive (HSE), UK, and American Conference of Governmental Industrial Hygienist (ACGIH) and National Institute for Occupational Safety and Health (NIOSH), USA stipulates an occupational exposure limit of 0.5 mg Cr/m3 (8 h TWA) for total chromium. However, schedule 1 of Controls of substances hazardous to health (COSHH) regulations developed by HSE, indicate 0.05 mg m3 (8 h TWA reference periods) to be the limit for Cr (VI) exposure. The exposure limit for individual (e.g., Cr, Zn, Ni etc.) contaminants (homogeneity) was not exceeded, but potential impact of heterogeneity (multi-element synergistic effect) on toxicity requires application of the precautionary principle.

Air Pollutants, Occupational↗

PubMed1998

Enalapril microbial biosensor.

Enalapril maleate (EMa) belongs to a new class of antihypertensive agents known as angiotensin converting enzyme (ACE) inhibitors. This paper describes the development of a microbial biosensor for EMa using induced Bacillus subitilis cells. This biosensor measures the acceleration of respiration during specific metabolic pathways of this drug. It has been applied, with good results, for determination of the active ingredient in the pharmaceutical tablet formulations.

Antihypertensive Agents↗

PubMed1996

Characterisation of lipid-protein interactions using a surface plasmon resonance biosensor.

The binding of proteins to lipid surfaces is of fundamental importance when considering the mechanisms of lipases and lipid transfer proteins. Current procedures for determining the binding constants suffer from problems related to poor sensitivity, poor reproducibility and to the use of restrictive assumptions in the analysis. We demonstrate here the use of the surface plasmon resonance biosensor to determine the association and dissociation kinetics of binding of cholesteryl ester transfer protein to the surface of emulsion particles of defined size and composition. Uncertainty about the emulsion valency, that is, the number of sites on the lipid particle occupied by the immobilised transfer protein on the biosensor surface, means that the association kinetics can only be characterised in terms of the product of the association rate constant and the concentration of sites, whereas the dissociation rate constant can be obtained unambiguously. The technique will prove useful in characterising the binding of proteins to lipid surfaces.

Apolipoproteins↗

PubMedDate not supplied

Sensitivity of the neuronal network biosensor to environmental threats.

It is widely acknowledged that there is a critical need for broad-spectrum environmental threat detection. While cells/tissue-based biosensors have been discussed for many years as a means of meeting this critical need, these kinds of systems have met with logistic concerns, in particular with regard to stability. Our group has been working with cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds and are sufficiently robust to be shipped to end users. The basis of operation involves extracellular recording using thin-film microelectrode arrays where spontaneous bioelectrical activity, that is, spike firing, can be monitored in a noninvasive manner conducive for potentially long-term measurements. This work describes the current status of our efforts for the fabrication of a portable biosensor that incorporates cultured neuronal networks grown over standardized microelectrode arrays. Based on our protocol for aqueous phase sample introduction under constant flow conditions, minimal variation in mean spike rate is observed, consistent with temporal stability, such that changes of > 10% are readily distinguished. To demonstrate the capability of this system, changes are reported in mean spike rate and network synchronization resulting from exposure to different model environmental threats, cadmium and strychnine. The sensitivity of this assay approach and implications of the experimental findings for environmental threat detection are discussed.

Animals↗

PubMed1995

A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells.

Phosphorylation of the regulatory light chain by myosin light chain kinase (MLCK) regulates the motor activity of smooth muscle and nonmuscle myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin II regulatory light chain phosphorylation (FRLC-Rmyosin II) is described here. The biosensor depends upon energy transfer from fluorescein-labeled regulatory light chains to rhodamine-labeled essential and/or heavy chains. The energy transfer ratio increases by up to 26% when the regulatory light chain is phosphorylated by MLCK. The majority of the change in energy transfer is from regulatory light chain phosphorylation by MLCK (versus phosphorylation by protein kinase C). Folding/unfolding, filament assembly, and actin binding do not have a large effect on the energy transfer ratio. FRLC-Rmyosin II has been microinjected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing FRLC-Rmyosin II with the kinase inhibitor staurosporine produced a lower ratio of rhodamine/fluorescein emission, which corresponds to a lower level of myosin II regulatory light chain phosphorylation. Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers.

3T3 Cells↗

PubMed1995

A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor.

We have demonstrated a simple yet direct method for determining the kinetic parameters in DNA-DNA interactions using biosensor technology based on the surface plasmon resonance phenomenon; a technique that does not require complex DNA labeling. To determine the effect of mismatches on the kinetics involved in DNA-DNA interactions, DNA hybridization kinetics were monitored in real time using synthetic oligonucleotides less than 20 bases in length which contained either a complementary sequence or mismatched bases. Upon analysis of the kinetic parameters obtained in oligonucleotide hybridization, we found that they were significantly affected by the presence of mismatches as well as by their number and location in a DNA duplex. In addition, the presented biosensor method is sensitive enough to detect kinetic effects caused by the presence of a single-mismatched base pair. Our findings strongly suggest that analysis of kinetic parameters involved in DNA-DNA interactions is advantageous for detecting the presence of mismatch base pairs in a DNA duplex.

Base Sequence↗

PubMed2002

Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin.

Enhanced green fluorescent protein (EGFP) was selected as a signalling scaffold protein for design of a fluorescent biosensor for bacterial endotoxin [or lipopolysaccharide (LPS)]. Virtual mutagenesis was utilized to model EGFP variants containing binding sites for LPS and lipid A (LA), the bioactive component of LPS. Cationic amphipathic sequences of five alternating basic and hydrophobic residues were introduced to beta-sheets located on the surface of EGFP barrel, in the vicinity of the chromophore. Computational methods were employed to predict binding affinity of Escherichia coli LA, to the models of virtual EGFP mutants. DNA mutant constructs of five predicted best binding EGFP variants were expressed in COS-1 cells. The EGFP-mutant proteins exhibited differential expression and variable degrees of fluorescence yield at 508 nm. The EGFP mutants showed a range of LA binding affinities that corresponded to the computational predictions. LPS/LA binding to the mutants caused concentration-dependent fluorescence quenching. The EGFP mutant, G10 bearing LPS/LA amphipathic binding motif in the vicinity of the chromophore (YLSTQ(200-204)-->KLKTK) captured LA with a dissociation constant of 8.5 microm. G10 yielded the highest attenuation of fluorescence intensity in the presence of LPS/LA and demonstrated capability in fluorescence-mediated quantitative detection of LPS in endotoxin-contaminated samples. Thus, the EGFP mutant can form the basis of a novel fluorescent biosensor for bacterial endotoxin.

Biosensing Techniques↗

PubMed1987

Optical biosensors for immunoassays: the fluorescence capillary-fill device.

This paper reports, for the first time, details of a novel type of optical biosensor for immunoassays, the fluorescence capillary-fill device (FCFD). This is based on a straightforward adaptation of the technology used to mass manufacture liquid-crystal display (LCD) cells to give cheap disposable immunosensors. These merely require contact by the sample to give a result in about a minute, and use certain principles of optical fibres and waveguides to avoid the need for operator attention, for physical separation methods or for washing steps. After a very brief introductory review and classification of optical biosensors, the main features of the FCFD and its associated instrumentation are described. The optical characteristics of the FCFD are then described, followed by accounts of the immunoassay method, the measurement system used in the experiments, the fabrication of FCFD sensors and a detailed description of the design of a competitive immunoassay for human immunoglobulin G (hIgG). The experimental details and the results of a first attempt at such an assay are then presented and discussed. It is concluded that the demonstration of this assay is a significant achievement, because the format of the FCFD, its manufacturing process and its instrumentation are completely novel. Certain problem areas have been identified and quantified; intended further work on these is outlined.

Animals↗

PubMed1987

Biosensors in process control.

Improvement in bioprocess control will require development of monitors for a wide range of cell-growth and downstream-process parameters. These requirements are examined in terms of the development and application of biosensor devices and physical measurement techniques. Acoustic, dielectric and laser light scattering techniques are discussed primarily as monitors and analysers for biomass parameters. Developments with biosensor devices are discussed in terms of their application in membrane sampling-flow analysis probes and the potential of more direct biosensing principles.

Animals↗