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PubMed2007

A folic acid-based functionalized surface for biosensor systems.

The performance of a biosensor depends largely on its interface with the biological system. This interface imparts a biologically relevant function to the device and provides a measure of specificity towards the biological analyte of interest. This paper documents the choice of folic acid as the functional component of a cantilever sensor to recognize nasopharyngeal (KB) cancer cells. A conjugation chemistry protocol has been outlined to deploy folic acid onto a titanium-coated sensor surface using a silane linker. The presence and biological activity of the sensor was verified by means of an immunospecific (ELISA) procedure. The overall performance of the folic acid-based cantilever sensor was measured using cancerous KB cell-binding experiments.

Biosensing Techniques↗

PubMed1990

A fiberoptic cholesterol biosensor with an oxygen optrode as the transducer.

A biosensor for the continuous optical determination of cholesterol is presented. Cholesterol oxidase is immobilized covalently on a nylon membrane and the consumption of oxygen is measured by following, via fiberoptic bundles, the changes in fluorescence of an oxygen-sensitive dye whose fluorescence is dynamically quenched by molecular oxygen. The dye is dissolved in a very thin silicone membrane placed beneath the enzyme layer. During interaction of the enzyme with cholesterol, oxygen is consumed, which is indicated by the fluorescent dye. At pH 7.25, the analytical range of the sensor is 0.2 to 3 mM and the time to reach a full steady state in a flowing solution ranges from 7 to 12 min.

Biosensing Techniques↗

PubMed1992

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Animals↗

PubMed1990

Regeneration of immunosorbent surfaces used in clinical, industrial and environmental biosensors. Role of covalent and non-covalent interactions.

The durability and regeneration of antibodies immobilized to commercial immunosorbents were investigated by monitoring Ag-Ab dissociation. Solutions consisting of 0.01 M hydrochloric acid (HCl), 10% propionic acid, 50% ethylene glycol and 10% SDS in 6 M urea were used in the evaluation of antigen dissociation from antibody covalently immobilized to glass and polystyrene beads, microtiter plates and Immobilon filters. RAH-IgG, used as a model antibody, bound strongly to all covalent surfaces. However, on adsorption to Nunc-1 microtiter plates, 25-60% of RAH-IgG was removed by all dissociating solutions. Covalent binding to Sanger beads was weakest relative to other covalent surfaces, exhibiting 30% and 65% detachment with ethylene glycol and SDS in urea, respectively. Although all four solutions dissociated antigen from surface-bound antibody, HCl and propionic acid were more effective on most surfaces. The antibody remained functional following antigen dissociation and reassociated to nearly 100% on all surfaces except Sanger beads and Nunc-1 microtiter plates. This study was initiated to evaluate regeneration and reuse of microelisa plates and emerging biosensors as a means of reducing routine laboratory analysis costs. Data are presented to demonstrate the reusability of microtiter plates in ELISAs following antigen dissociation from covalently bound antibody.

Antigen-Antibody Complex↗

PubMed1991

Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system.

An automated biosensor system for measuring molecular interactions has been used to study the kinetics of monoclonal antibody-antigen reactions. The system combines a microfluidic unit in contact with a sensor surface for surface plasmon resonance detection. The specificity of the surface is determined by the operator. Antibody or antigen is immobilised in a dextran matrix attached to the sensor surface. The interaction of matrix bound antibody or antigen with the corresponding partner in solution is monitored in real time. None of the interacting molecules needs to be labelled and it is not necessary to determine the concentration of the the matrix bound component in advance. Two systems were studied: matrix bound monoclonal antibodies (MAbs) interacting with HIV-1 core protein p24 and immobilised aminotheophylline reacting with MAbs. Control of the amount of immobilised ligand and reusable sensor surfaces permits the comparison of different MAbs reacting with antigen under almost identical conditions. Differences in affinity and reaction rates are immediately apparent. The calculated association rate constants for p24 MAbs ranged from 3 x 10(4) - 7.4 x 10(5) M-1 s-1 and for theophylline MAbs association rate constants as high as 1 x 10(6) M-1 s-1 were encountered. The calculated dissociation rate constants were in the region 2 x 10(-4) s-1 to 2 x 10(-2) s-1.

Antibodies, Monoclonal↗

PubMed1993

Measurement of kinetic binding constants of viral antibodies using a new biosensor technology.

Association (ka) and dissociation (kd) rate constants of three monoclonal antibodies raised against tobacco mosaic virus were determined using a biosensor technique based on surface plasmon resonance (BIAcore, Pharmacia). Dissociation rates were constant over the 4-400 nM antibody concentration range whereas apparent association rates decreased over this range probably due to an increased saturation level of the antigen. Affinity constants K calculated from the ratio of ka/kd were in reasonable agreement with values obtained under equilibrium conditions by two standard methods based on enzyme immunoassay.

Antibodies, Monoclonal↗

PubMed1995

Mapping of viral conformational epitopes using biosensor measurements.

Earlier electron microscopy studies of the location of various antigenic sites in tobacco mosaic virus indicated that epitopes specific for the quaternary structure and absent in dissociated viral subunits (so-called neotopes) were present along the entire length of the virus particle. In contrast, epitopes expressed in both intact particles and dissociated subunits (so-called metatopes) were found only at the one extremity of the particle containing the 5' end of the RNA. In the present study, the binding properties of antibodies to neotopes and metatopes were studied with the BIAcore. From the results of capture assays with viral subunits and on the basis of binding stoichiometry calculations, it was possible to demonstrate the presence of neotope and metatope specificities on additional parts of the viral surface where they had not been identified before by classical immunoassays. In two site binding assays it was also found that a neotope specificity could be induced in dissociated viral subunits by the binding of a first antimetatope antibody. The results clearly demonstrated the superiority of the biosensor technology for mapping conformational epitopes in viral proteins.

Antibodies, Monoclonal↗

PubMed1995

Measurement of kinetic binding constants of a panel of anti-saporin antibodies using a resonant mirror biosensor.

We have used a resonant mirror biosensor to determine the kinetics of binding of four antibodies, and their Fab' fragments, to their antigen, the plant-derived ribosome-inactivating protein (RIP) saporin. The analysis of the affinity of the antibodies was in reasonable agreement with values obtained by conventional techniques. However, the kinetic data showed that all four antibodies have a high dissociation rate constant (kdiss). These antibodies have been used in the construction of bispecific antibodies used to deliver saporin to tumour cells, and it is highly probable that the in vivo efficacy of the bispecific antibodies is limited by the high rate of dissociation of antibody-toxin complexes.

Allergy and Immunology↗

PubMed1995

Colloidal gold conjugated monoclonal antibodies, studied in the BIAcore biosensor and in the Nycocard immunoassay format.

Interactions between immobilized capture monoclonal antibodies (mAbs), analyte molecules and colloidal gold conjugated second monoclonal antibodies have been investigated in the BIAcore biosensor and in the Nycocard immunoassay format. This report focuses on six monoclonal antibodies against human heart myoglobin, although, results with other antigens are also discussed. The BIAcore was used to screen monoclonal antibodies as antigen capture reagents, and for their function as colloidal gold conjugated second antibodies in the Nycocard. Some antibodies with low affinity caused by a rapid antigen dissociation rate, showed high affinity kinetics when used unlabelled or as gold conjugated detector reagents. One gold conjugated mAb with excellent properties in the Nycocard, showed double binding to one epitope, when tested in the BIAcore. The real time visualization of association and dissociation rates was a unique tool in the elucidation of antigen-antibody interactions. Our study confirmed that good antibody candidates selected with the BIAcore must always be tested in their actual conjugation situation before final optimization.

Antibodies, Monoclonal↗

PubMed1991

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.

Alanine Transaminase↗

PubMed1994

Assay of dehydrogenases with an O2-consuming biosensor.

Coimmobilization of L-lactate oxidase (LOD) and L-lactate dehydrogenase (LDH) on a film bound to an oxygen electrode makes it possible, by recycling L-lactate and pyruvate, to lower their detection limit to 10 nM. With LOD immobilized alone and LDH in solution, easy LDH measurements were performed. The detection limit was 1 IUl-1. Similarly, this mixed biosensor, using an LDH sensitive to mercurials, was used for p-chloromercuribenzoate (PCMB) detection. Coimmobilization of LOD + LDH with an NAD(+)-dependent dehydrogenase allows the measurements of all dehydrogenases. NAD+ is recycled. The detection limit was 50 nM. Applications can be found in medicine, the food industry, and the environment.

Animals↗

PubMed1991

Preliminary in vivo biocompatibility studies on perfluorosulphonic acid polymer membranes for biosensor applications.

The first biocompatibility studies on the DuPont perfluorosulphonic acid polymer (Nafion) are presented. Presterilized samples of commercially cast and solution cast Nafion membranes were implanted subcutaneously, intraperitoneally and intravenously in male Sprague-Dawley rats. Scanning electron microscopy and histological examination of explanted samples and surrounding tissues reveal little, if any, evidence of acute or chronic foreign body inflammatory response. The fibrous capsules surrounding the implant remain nominally thin (less than 100 microns) after more than 3 months in situ, while the surrounding tissue remains well vascularized. Nafion polymer exhibits sufficient biocompatibility to make it a viable candidate for some implantable biosensor applications. It may, however, be necessary to compensate for the effect of the progression of fibrous encapsulation on sensor performance, particularly during the acute response stage.

Abdominal Muscles↗

PubMed1993

Monoclonal antipeptide antibodies: affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology.

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Amino Acid Sequence↗

PubMedDate not supplied

Analysis of the interaction between a synthetic peptide of influenza virus hemagglutinin and monoclonal antibodies using an optical biosensor.

The interaction between two monoclonal antibodies and their corresponding Fab' fragments with a synthetic peptide, corresponding to the C-terminal 23 residues of the HA1 chain of influenza virus hemagglutinin against which they were generated, has been examined using an optical biosensor employing the detection principal of surface plasmon resonance (Pharmacia BIAcore). The data obtained has been analysed in detail by linear transformation of the primary data and nonlinear regression analysis, as well as by analysis of equilibrium binding data. The 2/1 antibodies and their Fab' fragments displayed higher affinity than the corresponding 1/1 proteins. The IgGs were found to have equilibrium association constants (KA) 10-20-fold higher than the corresponding Fab' fragments. This appears largely to be due to differences in the dissociation rate constant (kd) and probably reflects increased avidity due to bivalent binding.

Amino Acid Sequence↗

PubMed1993

Enhancing biosensor performance using multienzyme systems.

Enhancing the performance of biosensors, in terms of increasing the range of analytes that may be detected, and the sensitivity and specificity of the detection event, would improve the prospects for commercializing this technology. Coupling the catalytic activities of several enzymes is one approach being used to address these issues. Sequences of enzymes, where ligand binding triggers the activation of enzymes, or where biocatalytic pre-concentration of intermediates permits augmentation of the signal, may be used. In addition, enzymatic recycling of the analyte can be used to increase the sensitivity by several orders of magnitude.

Animals↗

PubMed1993

Two FIA-based biosensor systems studied for bioprocess monitoring.

In this paper, two different FIA-based biosensor systems are described for application to different biotechnologically relevant purposes. In the first system, single fiber optodes were used to determine the pH, urea and penicillin V concentrations. A two-channel system was developed for the simultaneous monitoring of different variables to increase the analysis accuracy. This system was used for monitoring the penicillin V concentration during a cultivation of Penicillium chrysogenum. The second system described is a calorimetric immunoassay based on the use of an enzyme thermistor. A sandwich assay with protein A immobilized on a solid support for the determination of various IgGs was established. A fusion protein of protein A and beta-galactosidase obtained from a recombinant E. coli strain was used in the labelling and detection reaction. This system is designed for future application in bioprocess monitoring.

Biosensing Techniques↗

PubMed1995

Development of a thin membrane glucose sensor using beta-type crystalline chitin for implantable biosensor.

A biocompatible thin membrane glucose sensor has been developed using beta-type crystalline chitin as a supporting material for the purpose of implantable biosensor. Gold electrode was formed by vapor deposition of the surface of glucose oxidase-immobilized beta-chitin membrane. The sensor showed rapid responses to glucose and H2O2 (< 45 s in batch operation), and addition of 2,6-dichlorophenolindophenol as a mediator caused expansion of the measuring range. To investigate the possible uses for the implantable, the sensor sterilization and toxicity were studied. Sterilization with ethylene oxide was achieved by exposing samples, and a decrease in activity was permissible for measurements of body fluid. From the results of the toxicity assay using mouse fibroblast cells, beta-chitin sensor, constructed with chitin, glucose oxidase and gold electrode, showed no particular toxicity.

Animals↗

PubMed1989

Transducer mechanisms for optical biosensors. Part 1: The chemistry of transduction.

The first stage of optical biosensor transduction involves a chemical interaction between the analyte and an indicator phase to produce an optically detectable signal. This stage is critical because it determines stability, selectivity and sensitivity as well as establishing the wavelengths required for the optical measurement. Several types of analyte/indicator interaction are possible. Direct indicators such as those used in optical pH sensors are in equilibrium with the analyte in the sample. They may be used for continuous measurements and can be coupled to the sensing of other analytes such as acidic and basic gases like carbon dioxide and ammonia. Integrating reagents react irreversibly with analyte and require that the rate of product formation be measured. Sensors based on catalysis by an immobilised enzyme involve a steady-state measurement of optically detectable substrate or product. High selectivity can be achieved using antibodies as reagents and basing sensing on competitive binding; however, response time is a serious problem. A variety of methods have been employed to immobilize the indicator phase including adsorption on solid substrates, covalent bonding to a substrate and confinement by membranes permeable to analyte but not to indicator.

Blood Chemical Analysis↗