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PubMed1997

Identification of the 4-hydroxyphenylacetate transport gene of Escherichia coli W: construction of a highly sensitive cellular biosensor.

The mechanism of uptake of 4-hydroxyphenylacetate (4-HPA) by Escherichia coli W was investigated. The 4-HPA uptake was induced by 4-HPA, 3-hydroxyphenylacetate (3-HPA) or phenylacetate (PA) and showed saturation kinetics with apparent Kt and Vmax values of 25 microM and 3 nmol/min per 10(9) cells, respectively. Transport of 4-HPA was resistant to N,N'-dimethylcarbodiimide (DCCD), but was completely inhibited by cyanide and 4-nitrophenol, and, to a lower extent, by arsenate and azide, suggesting that energy is required for the uptake process. Competition studies showed that 4-HPA uptake was inhibited by 3-HPA or 3,4-dihydroxyphenylacetate (3,4-DHPA) but not by 2-hydroxyphenylacetate (2-HPA), L-tyrosine or other structural analogues, indicating a narrow specificity of the transport system. We have demonstrated, using two experimental approaches, that the hpaX gene of the 4-HPA catabolic cluster, which encodes a protein of the superfamily of transmembrane facilitators, is responsible for 4-HPA transport. Aside from the aromatic amino acid transport systems, hpaX is the first transport gene for an aromatic compound of enteric bacteria that has been characterized. A highly sensitive cellular biosensor has been constructed by coupling the 4-HPA transport system to a regulatory circuit that controls the production of beta-galactosidase. This biosensor has allowed us to demonstrate that the transport system performs efficiently at very low external concentrations of 4-HPA, similar to levels that would be expected to occur in natural environments.

Binding, Competitive↗

PubMed1999

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.

Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 beta-lactamase and its inhibitory protein as our model protein-ligand system. 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cell biology.

Allosteric Regulation↗

PubMed1997

Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor. A comparison of immobilisation strategies.

The interaction between the humanised A33 monoclonal antibody and the corresponding F(ab)'2 or Fab' fragments with the colonic epithelial A33 antigen, purified by micropreparative HPLC from membrane extracts of the colonic carcinoma cell line LIM 1215, has been studied with the BIAcore 2000 biosensor using surface plasmon resonance detection. The surface orientation of immobilised antibody and the Fab' fragment onto the biosensor surface was controlled using alternative immobilisation chemistries. This resulted in significantly higher molar binding activities compared with the conventional N-hydroxysuccinimide (NHS)/N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC) chemistry. This increase in signal resulted in a concomitant increase in sensitivity of detection, which facilitates analysis of low levels of A33 antigen. The apparent association rate (ka) and dissociation rate (kd) constants obtained with the different immobilisation chemistries were determined. These analyses showed that the kinetic constants obtained for the IgG were not significantly affected by the method of immobilisation. F(ab)'2 and Fab' fragments immobilised using NHS/EDC chemistry showed significantly lower apparent affinity. By contrast the use of the thiol coupling chemistry with the Fab' fragment gave a five fold increase in observed KA, resulting in a similar affinity to that observed with the intact IgG molecule.

Antibodies, Monoclonal↗

PubMed2000

Micropreparative ligand fishing with a cuvette-based optical mirror resonance biosensor.

We have previously demonstrated the role of an optical biosensor (BIAcore 2000) as a specific detector to monitor chromatographic fractions during the purification and characterisation of ligands for orphan biomolecules. We have now extended this application to perform micropreparative ligand fishing directly on the sensor surface using an automated cuvette-based optical biosensor (Iasys Auto+) equipped with a high-capacity carboxymethyldextran surface (surface area 16 mm2). Using a F(ab)2' fragment of the A33 monoclonal antibody as bait, we have recovered microgram quantities of essentially homogeneous A33 ligand from the sensor surface in a form suitable for subsequent sensitive and specific down stream analysis (micropreparative HPLC, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting). The design of the cuvette-based system facilitates recovery of desorbed material from the constrained workspace in small volumes at high concentration. The use of on-surface detection allows the surface viability to be continuously monitored and permits direct quantitation of both bound and recovered material.

Biosensing Techniques↗

PubMed1993

Mapping of the antibody- and receptor-binding domains of granulocyte colony-stimulating factor using an optical biosensor. Comparison with enzyme-linked immunosorbent assay competition studies.

An automated optical biosensor instrument for measuring molecular interactions (Pharmacia BIAcore) has been used to characterise the epitopes recognised by 15 monoclonal antibodies raised against recombinant human granulocyte colony-stimulating factor (G-CSF). The BIAcore combines an autosampler and integrated microfluidic cartridge for the introduction and transportation of samples to the sensor chip surface, with surface plasmon resonance to detect binding events. A rabbit anti-mouse Fc antibody, coupled to the sensor surface in situ using conventional protein chemistry techniques, was used to capture an anti-G-CSF monoclonal antibody. G-CSF was bound to this antibody by injection over the sensor surface. Multi-site binding experiments were then performed in which other anti-G-CSF monoclonal antibodies were injected sequentially over the surface, and their ability to bind to the G-CSF in a multimolecular complex monitored in real time. Results obtained using the biosensor have been compared with data obtained by cross competition studies using biotinylated antibodies or antibody binding studies using chemically or enzymatically derived G-CSF peptide fragments or synthetic peptides. The results of these studies are in excellent agreement with the data from the BIAcore, although modification of the antibody or G-CSF occasionally altered the epitope affinity.

Animals↗

PubMed2003

A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental effects.

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ER) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. Both cases are analyzed: unliganded as well as liganded. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate signaling and signaling deleterious to health) may be better controlled. The fractal approach is applied to analyte-ER interaction data available in the literature. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D(f)) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. For example, the binding rate coefficient, k, exhibits a 4.60 order of dependence on the fractal dimension, D(f), for the binding of unliganded and liganded VDR mixed with GST-RXR in solution to Spp-1 VDRE (1,25-dihydroxyvitamin D(3) receptor element) DNA immobilized on a sensor chip surface (Cheskis and Freedman, Biochemistry 35 (1996) 3300-3318). A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. A predictive relationship is also presented for the ratio K(A)(=k/k(d)) as a function of the ratio of the fractal dimensions (D(f)/D(fd)). This has biomedical and environmental implications in that the dissociation and binding rate coefficients may be used to alleviate deleterious effects or enhance beneficial effects by selective modulation of the surface. The K(A) exhibits a 112-order dependence on the ratio of the fractal dimensions for the ligand effects on VDR-RXR interaction with specific DNA.

Biophysical Phenomena↗

PubMed2002

Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor.

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.

Animals↗

PubMed1997

Validation parameters for a novel biosensor assay which simultaneously measures serum concentrations of a humanized monoclonal antibody and detects induced antibodies.

This report documents the validation of an assay using BIAcore 2000 that, with a single injection of mouse serum, allows the quantitation of a humanized monoclonal antibody and can also detect the presence of antibodies directed against this humanized antibody. The important components required for the validation of biosensor assays including precision, accuracy, linearity, stability of the immobilized ligand, specificity and sensitivity are addressed. The tandem assay that is used as a model for biosensor validations is accomplished by flowing each sample across the surface of two flowcells in sequence. The first flowcell has the antigen that the humanized mAb was generated against immobilized while the humanized mAb itself is immobilized on the second flowcell. The quantitation component of this assay is precise and accurate with a limit of quantitation of 1 microg/ml in mouse serum samples. Any antibodies directed against the humanized mAb can be detected and also characterized as to isotype. This unique assay can be performed with as little as 10 microl of serum which is then diluted ten-fold prior to analysis. The small sample requirement allows analysis of individual mouse serum samples rather than requiring the use of pooled samples from multiple mice. A further advantage of this assay is the automation capability which allows unattended operation.

Animals↗

PubMed1999

Effective rate models for the analysis of transport-dependent biosensor data.

Optical biosensors, including the BIACORE, provide an increasingly popular method for determining reaction rates of biomolecules. In a flow chamber, with one reactant immobilized on a chip on the sensor surface, a solution containing the other reactant (the analyte) flows through the chamber. The time course of binding of the reactants is monitored. Scientists using the BIACORE to understand biomolecular reactions need to be able to separate intrinsic reaction rates from the effects of transport in the biosensor. For a model to provide a useful basis for such an analysis, it must reflect transport accurately, while remaining simple enough to couple with a routine for estimating reaction rates from BIACORE data. Models have been proposed previously for this purpose, consisting of an ordinary differential equation with 'effective rate coefficients' incorporating reaction and transport parameters. In this paper we investigate both the theoretical basis and numerical accuracy of these and related models.

Biological Transport↗

PubMed1996

MutS interaction with mismatch and alkylated base containing DNA molecules detected by optical biosensor.

An optical biosensor was used to monitor interactions between the Escherichia coli DNA mismatch repair molecule MutS and various immobilized oligonucleotides. While associating poorly with single-stranded DNA, MutS was capable of rapid association/dissociation from homoduplex DNA. The interaction of MutS with oligonucleotide 30-mers containing single site mismatches demonstrated that during the dissociation phase, MutS binding was greatest to a G-G mismatch, followed by G-T > A-A > C-T, A-C. Binding to A-G, T-T and C-C mispairs was marginally higher than that seen between MutS and homoduplex DNA. The ability of MutS to interact with 30-mers containing alkylated bases was also tested. While binding to O6-methyl-G-C, or to O4-methyl-T-A base pairs was similar to that of homoduplex DNA, strong binding was seen to a O6-methyl-G-T mispair. O4-methyl-T-G, however, was poorly recognized by MutS, with relative binding affinity similar to homoduplex DNA, predicting poor in vivo recognition of O4-methyl-T-G by MutS. Interestingly, MutS demonstrated a relatively high affinity for an 1,N6-etheno-A-T containing homoduplex. Thus, in allowing rapid evaluation of interactions between such molecules, the biosensor will be useful to structure-function analyses.

Adenosine Triphosphatases↗

PubMed1998

Use of a channel biosensor for the assay of paralytic shellfish toxins.

Gonyautoxin (GTX), saxitoxin (STX) and tetrodotoxin (TTX), also known as paralytic shellfish poisons (PSP), block Na+ channels, including those in the frog bladder membrane. A tissue biosensor has been developed, consisting of a Na+ electrode covered with a frog bladder membrane integrated within a flow cell. The direction of Na+ transfer, investigated in the absence of Na+ channel blockers, established that active transport of Na+ occurs across the frogs bladder membrane from the internal to the external face. Transfer was shown to be TTX sensitive. The tissue sensor response to each of the different PSP was recorded and the results compared with toxicities determined by the standard mouse bio-assay. Using high concentrations of TTX from the puffer fish Takifugu niphobles, a linear correlation was found between the results from the two assay systems. However, the tissue biosensor system was also able to detect very low concentrations of TTX in samples from two species of puffer fish (Takifugu niphobles and Takifugu pardalis) at concentrations below the detection limit of the mouse bio-assay.

Animals↗

PubMed2000

Use of a lux-marked rhizobacterium as a biosensor to assess changes in rhizosphere C flow due to pollutant stress.

The flow of carbon from plant roots into soil supports a range of microbial processes and is therefore critical to ecosystem function and health. Pollution-induced stress, which influences rhizosphere C flow is of considerable potential importance, and therefore needs to be evaluated. This paper reports on a method, based on reporter gene technology, for quantifying pollutant effects on rhizosphere C flow. The method uses the lux-marked rhizobacterium Pseudomonas fluorescens, where bioluminescence output of this biosensor is directly correlated with the metabolic activity and reports on C flow in root exudate. Plantago lanceolata was treated with paraquat (representing a model pollutant stress) in a simple microcosm system. The lux-biosensor response correlated closely with C concentrations in the exudate and demonstrated that the pollutant stress increased the C flow from the plantago roots, 24 h after application of the herbicide. The lux-reporter system therefore potentially offers a technique for use in assessing the impact of pollutant stress on rhizosphere C flow through the soil microbial biomass.

Carbon↗

PubMed2002

Oxidation of organophosphorus pesticides for the sensitive detection by a cholinesterase-based biosensor.

A potentiometric flow injection-type biosensor developed in our laboratory was used for the determination of organophosphorus pesticides (OPs). The principle of the biosensor is that the degree of inhibition of a sensor enzyme by an OP is dependent on the concentration of the pesticide. The sensor system consisted of a reactor with acetylcholinesterase (AChE) immobilized on a controlled pore glass and a detector with a tubular H(+)-selective membrane electrode. In order to examine the possibility of enhancing the sensitivity of the sensor by converting OPs to oxidized forms (stronger inhibitors), a comparison of the degree of enzyme inhibition by OPs at 10(-6) M before and after their oxidation was made. All of the ten pesticides tested exhibited greater inhibitory power toward the sensor enzyme following oxidation. All of the oxidized pesticides at 10(-6) M inhibited the sensor enzyme to a considerable degree, demonstrating the utility of the developed method for the class-specific determination of OPs. A calibration curve for diazinon, over the concentration range of 10(-11)-10(-4) M, was obtained. The lower detection limit was 2 x 10(-10) M. Treatment of the inhibited enzyme with pyridine-2-aldoxime restored the enzyme to near full activity, allowing repeated use of the sensor.

Acetylcholinesterase↗

PubMed1997

Novel biosensoric devices based on molecular protein hetero-multilayer films.

We have developed a novel concept for the modification of technical surfaces with molecularly well-organized layers of bioorganic components. A molecular construction set has been used to implement this concept which is based on molecularly stratified polyelectrolyte films as a structure decoupling protein layers from solid substrates. Utilizing this technology, one can start from a number of different substrates to obtain the same surface structures, on which protein hetero-multilayer films can be prepared to functionalize the interface for (potentially very different) purposes. We have demonstrated the viability of this concept by constructing a biosensor surface that was characterized by x-ray, spectroscopic, and immunological techniques. For the preparation of this functionalized interface, a cascade of biospecific recognition processes has been used, in which a layer of SpA was supplanted on a dense SA monolayer (binding stoichiometry, approximately 0.2 SpA/SA). On the SpA, IgG has been immobilized (binding stoichiometry, approximately 0.5 IgG/ SpA). The resultant biosensor displays extremely low unspecific background reactivity, high sensitivity, and good response linearity over more than 3 orders of magnitude in antigen concentration.

Adsorption↗

PubMed2000

Bacterial metal-resistance proteins and their use in biosensors for the detection of bioavailable heavy metals.

We have expressed and purified metal-resistance and metal regulatory proteins from the bacterial determinants of resistance to heavy metals and utilised these in the development of biosensors for heavy metals. Both the metallothionein from the cyanobacterium Synechococcus PCC 7942 and the MerR regulatory protein from transposon Tn501 allow the detection of non-specific metal binding down to 10(-15) M concentrations of Hg(II), Cu(II), Zn(II) and Cd(II) in pure solution. Differential effects of the metals can be detected at both low and high concentrations, and the shape of the capacitance curves may reflect biologically relevant responses of the proteins to metals. Further work is required to establish the relationship between the detected binding of metal and the biological response of the protein, or to provide biosensors of use in the natural environment.

Bacterial Proteins↗

PubMed2002

Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide.

Biosensors based on acetyl cholinesterase (AChE) inhibition have been known for monitoring of pesticides in food and water samples. However, strong inhibition of the enzyme is a major drawback in practical application of the biosensor which can be overcome by reactivation of the enzyme for repeated use. In the present study, enzyme reactivation by oximes was explored for this purpose. Two oximes viz., 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were compared for the reactivation of the immobilized AChE. TMB-4 was found to be a more efficient reactivator under repeated use, retaining more than 60% of initial activity after 11 reuses, whereas in the case of 2-PAM, the activity retention dropped to less than 50% after only 6 reuses. Investigations also showed that reactivation must be effected within 10 min after each analysis to eliminate the ageing effect, which reduces the efficiency of reactivation.

Acetylcholinesterase↗

PubMed2003

Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection.

Many rapid tests have been developed for the detection of Escherichia coli O157:H7 from complex matrices such as food and water. However, many of these methods rely on traditional culture steps for confirmation, which can take an extra 24-48 h. The fiber optic biosensor has been used to rapidly detect pathogens from complex matrices. In this paper, we demonstrate a method using a rapid biosensor assay, recovery through a short enrichment, and PCR to detect and confirm the presence of at least 10(3) CFU/ml of E. coli O157:H7 in a sample in less than 10 h.

Biosensing Techniques↗

PubMed1997

Developments and applications of biosensors in food analysis.

The food industry needs suitable analytical methods for process and quality control; that is, methods that are rapid, reliable, specific and cost-effective in their provision of information about physical and chemical characteristics of food. Apart from a few important analytes, such as sugars, alcohols, amino acids, flavours and sweeteners, food applications mainly focus on the determination of contaminants. However, very few biosensors play a prominent role in food processing or quality control. Considerable effort must be made to develop biosensors that are inexpensive, reliable, and robust enough to operate under realistic conditions.

Animals↗