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PubMed1996

Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.

The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.

Animals↗

PubMed2004

Development of a biosensor for the detection of tributyltin.

A biosensor (LUMISENS I), based on the inducible bioluminescence of the Escherichia coli strain TBT3 (Ec::luxAB TBT3), was developed for the detection of the biocide tributyltin. LUMISENS I was set up with a minibioreactor and additional equipment for growth monitoring and light acquisition. The 100-mL minibioreactor has allowed us to establish a stable and reproducible environment for the bacteria (regulation of the growth rate, temperature, pH, and oxygenation), as well as for in situ contact with the xenobiotic. The optical components of the transducer were chosen according to the spectral emission of the strain being studied using a highly sensitive spectrophotometer that was initially devoted to Raman scattering. LUMISENS I was patented according to the in situ, automatic, and simultaneous measurement of the cell density and bioluminescence in the bioreactor. The first results showed that cells cultivated in a synthetic glucose medium provided a better detection limit than did those cultivated in a complex Luria-Bertani (LB) medium (0.02 and 1.5 microM of tributyltin, respectively). Cells maintained at a high growth rate (0.9 h(-1)) led to maximum bioluminescence. Moreover, air bubbling was efficient enough to provide suitable quantities of oxygen for both growth and light emission. When the TBT3 strain used the luxAB genes on its own, decanal, a long-chain aldehyde, had to be added to obtain the bioluminescence reaction. We found that the continuous addition of decanal was the most effective means of obtaining this reaction. The monitoring of the bioluminescence after tributyltin induction showed that the aldehyde was not toxic up to 300 microM during a 7-day experiment. Measurement of tributyltin with LUMISENS I was performed, which showed significant response up to 0.125 microM without any effect on optical density. Even though optimization of the performance of LUMISENS I is still under development, because of its original design, this biosensor is already in use as a warning system for the online monitoring of tributyltin.

Biosensing Techniques↗

PubMed1993

An amperometric flow-injection analysis biosensor for glucose based on graphite paste modified with tetracyanoquinodimethane.

A biosensor system using flow injection analysis (FIA) has been developed for the analysis of glucose in human serum. The system consists of the enzyme glucose oxidase incorporated into graphite paste modified with the electroactive material tetracyanoquinodimethane (TCNQ). TCNQ acts as an efficient mediator for oxidation of the reduced enzyme at 200 mV vs Ag/AgCl. The flow injection assay described has detection limits of 2 mM glucose using a 100-microliters sample injection through a 250-microliters sample loop. Data are presented to show the effect of sample injection volume and flow rate on the response of the FIA sensor. The biosensor exhibited excellent reproducibility for 800 injections. The loss of response after 800 injections was due to leaching of TCNQ from the graphite paste. Each assay takes 3 min giving a sample throughput of 20 per hour at a flow rate of 30 ml/h. The sensor was applied to the determination of glucose in human serum. The glucose measurements are in good agreement with those of a commercially available spectrophotometric method. Data showing the effect of interfering substances, ascorbic acid and acetaminophen, on the response of the sensor are also reported.

Acetaminophen↗

PubMed1993

Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor.

Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.

Base Sequence↗

PubMed1995

Real-time monitoring of DNA manipulations using biosensor technology.

The potential of real-time biospecific interaction analysis technology for applications in molecular biology is described. DNA fragments are immobilized onto a biosensor surface using the high-affinity streptavidin-biotin system and subsequently used to monitor different unit operations in molecular biology, e.g., DNA strand separation, DNA hybridization kinetics, and enzymatic modifications. A model system comprising six oligonucleotides was used, which can be assembled into a 69-bp double-stranded DNA fragment. Using this system, the biosensor approach was employed to analyze multistep solid-phase gene assembly and the performance of different enzymes routinely used for the synthesis and manipulation of DNA. In addition, a concept for the determination of single-point mutations in DNA samples is described.

Base Sequence↗

PubMed1995

Interpreting complex binding kinetics from optical biosensors: a comparison of analysis by linearization, the integrated rate equation, and numerical integration.

The binding kinetics recorded for many interactions using BIAcore and IAsys optical biosensors do not fit a simple bimolecular interaction model (A + B<-->AB). Three methods of analysis have been used to derive estimates for kinetic constants from such data:linearization, curve fitting using the integrated rate equation, and curve fitting using numerical integration. To test how well these methods could interpret complex binding kinetics, we generated and analyzed simulated data for two systems, one involving a two-state conformational change (A + B<-->AB<-->(AB)*) and a second involving surface heterogeneity (A + B<-->AB and A + B*<-->AB*). The linearization method assumed a simple bimolecular interaction and was inadequate at interpreting these systems as both produced complex kinetics in the association and dissociation phases. The sum of two integrated rate equations correctly modeled surface heterogeneity; but, when applied nonglobally, it fit the data from the conformational change system equally well and thus provided misleading results. Numerical integration allowed a choice of model for analysis and was therefore the only method capable of returning accurate estimates of rate constants for both complex systems. Global analysis, in combination with numerical integration, provided a stringent test of the assumed model. However, this stringency suggests that its application to experimental systems will require high-quality biosensor data.

Biosensing Techniques↗

PubMed1995

A fiber-optic cocaine biosensor.

A fiber-optic biosensor was developed for detection of cocaine, its metabolites, and other coca alkaloids, using a monoclonal antibody (mAb) against a derivatized benzoylecgonine (BE). The mAb was immobilized noncovalently on quartz fibers and a flow fluorometer was used to detect changes in evanescent wave fluorescence. A fluorescein (FL) conjugate of BE bound to the mAb specifically in a saturable manner and with high affinity (Kd = 7.6 nM). Cocaine or other test compounds competed with FL-BE for binding to the mAb in a concentration-dependent manner, thereby reducing the initial rate or steady-state fluorescence. Addition of cocaine to the flow buffer after reaching steady-state fluorescence enhanced the dissociation of bound FL-BE, and cocaine removal allowed fiber regeneration for multiple measurements. The detection limits for cocaine, cocaethylene, norcocaine, and BE were 5, 5, 29, and 30 ng/ml, respectively, but for ecgonine it was 4600 ng/ml and for methylecgonine it was 2000 ng/ml. Tropacocaine was detected at 10 ng/ml, but atropine was detected at 2900 ng/ml. The biosensor discriminated by 833-fold between cocaine and its stereoisomer pseudococaine. Structural features necessary for high-affinity recognition by this mAb are benzoate and 3 beta configuration, both of which are found in BE, cocaine, norcocaine, and cocaethylene.

Antibodies, Monoclonal↗

PubMed1996

Analysis of mass transport-limited binding kinetics in evanescent wave biosensors.

It is shown that currently used methods for analyzing surface plasmon resonance or resonant mirror biosensor data do not adequately take into account the effects of mass transport on the kinetics of ligand association and dissociation. Conventional analyses may yield arbitrary apparent reaction rate constants lying between the mass transport rate constant and the true intrinsic chemical binding rate constants, depending on the choice of ligand concentrations used in the experiments. A new kinetic analysis of biosensor data, based upon a phenomenological two-compartment approximate description of transport, is presented and tested on experimental data and on simulated data generated with a computer model for combined mass transport and reversible binding to a single class of immobilized sites. Results of the analysis indicate the extent to which the experimental binding progress curve is transport controlled and whether or not values of chemical rate constants may be validly extracted from the data. The new analysis is independent of the details of the transport process, simple in its application, and in favorable cases permits determination of the correct values of chemical rate constants that are 10- to 100-fold greater than those that can be correctly evaluated by previous analyses.

Biological Transport↗

PubMed1997

Determination of association rate constants by an optical biosensor using initial rate analysis.

We show that initial rate analysis can be successfully applied to analyze experimental binding data generated by an optical biosensor. The initial rates of binding obtained from linear regression are concentration dependent, and plots of initial rate against ligate concentration yield a straight line that passes through the origin. The slope of this graph is the product of the association constant times the maximal binding capacity of the immobilized ligand. This latter parameter is easily obtained from a single binding curve at high ligate concentration, allowing rapid determination of the association rate constant. The association rate constant obtained in this manner is found to be in good agreement with that obtained by the more customary method of nonlinear regression analysis of the entire binding profile. Initial rate analysis is more simple than fitting the full association profile and needs less data collection time. It also requires fewer assumptions about the functional form of the association profile. This can be advantageous when fitting biosensor-derived data, which often show complex association kinetics. Furthermore, it avoids the potential complication of second-order kinetics which may be found at low ligate concentrations with high-affinity interactions.

Biosensing Techniques↗

PubMed1997

Thermodynamic analysis of antigen-antibody binding using biosensor measurements at different temperatures.

The thermodynamic parameters of the interaction between hen egg white lysozyme and Fab D1.3 were determined by measuring the temperature dependence of the ratio of its kinetic association and dissociation rate constants. Biosensor technology (BIAcore 2000) was used to measure the rate constants at temperatures ranging from 5 to 40 degrees C. The value of DeltaG degrees at 25 degrees C (-49 kJ M-1) calculated by this method was very close to that obtained previously from fluorescence quenching measurements (-48.5 kJ M-1). However, the value of DeltaH degrees measured at 25 degrees C by biosensor technology (-35 kJ M-1) was smaller than that determined previously by microcalorimetry (-90 kJ M-1). Another difference was the limited variation of ln K and DeltaG with temperature observed with BIAcore compared to the steady decrease of ln K with temperature found by calorimetry. Our data showed that the binding reaction was driven only by enthalpy below 23 degrees C, by enthalpy and entropy between 23 and 35 degrees C, and only by entropy above 35 degrees C. This suggests, inter alia, that the contribution from the enthalpy of hydration due to the water molecules present at the interface in the lysozyme-antibody complex is progressively eliminated as the temperature increases. Whereas calorimetric data pertain to all the components present in the sample, including solvent molecules, BIAcore measurements monitor only the physical association and dissociation of the two macromolecular species. The difference between the two sets of data may also reflect the complexity of the binding mechanism between lysozyme and Fab D1.3.

Animals↗

PubMed1997

Concentration measurement of unpurified proteins using biosensor technology under conditions of partial mass transport limitation.

Using biosensor technology, it is possible to measure protein concentration when the binding of the protein to an appropriate ligand immobilized on the sensor surface is totally limited by diffusion and mass transport, a condition difficult to achieve in practice. In such a case, the observed binding rate does not reflect the intrinsic binding capacity of the molecular partners, but is simply proportional to the concentration of the protein analyte that is introduced in a continuous flow over the ligand. We describe here a more general biosensor method for measuring protein concentration which is applicable to conditions where mass transport is not totally but only partially rate limiting. The proposed method, which is based on measurements at different flow rates, does not require a standard of known protein concentration and can be used with unpurified proteins. The method is applicable to ligand-analyte pairs with an association rate constant as low as 10(3) M-1 s-1 and requires only knowledge of the molecular weight and diffusion coefficient of the analyte. The method was used successfully to measure the concentration of monoclonal antibodies, monoclonal antibody fragments (Fab) obtained by papain cleavage, and recombinant Fab fragments of widely different affinities in crude Escherichia coli extracts.

Amino Acid Sequence↗

PubMed1997

Peroxide biosensors and mediated electrochemical regeneration of redox enzymes.

This article describes the research investigations on the development of the amperometric biosensors based on mediated bioelectrochemistry. The mediated bioelectrochemistry involving horseradish peroxidase and glucose oxidase within the graphite paste is reported. The enzyme horseradish peroxidase together with electrochemical mediator was incorporated within the graphite paste electrode. The amperometric response is based on the mediated electrochemical regeneration of peroxidase within the paste. The mediated electrochemical regeneration of peroxidase and glucose oxidase was studied and compared using three different electron transfer mediators-tetracyanoquinodimethane (TCNQ), tetrathiafulvalene (TTF), and dimethyl ferrocene (dmFc). The mediated electrochemistry involving these three mediators was studied based on the cyclic voltammetry. The electrochemical measurements show that TTF is better mediator for the development of peroxide biosensor compared to TCNQ and dmFc. On the other hand, TCNQ is better mediator for the development of glucose sensor. The response curves for peroxide have been reported using these three mediators. A comparative study on the amperometric response based on the mediated electrochemical oxidation of peroxidase and glucose oxidase has also been made using these three mediators. The various parameters, i.e., background current, stability of the mediator within the graphite paste during the electrochemical measurements, and reproducibility of the amperometric response, are discussed.

Biosensing Techniques↗

PubMed1998

Ethanol biosensors and electrochemical oxidation of NADH.

Comparative studies of the electrochemical oxidation of reduced nicotinamide coenzyme (NADH) at the surfaces of chemically modified graphite paste electrodes (CMEs) are reported. Three different electroactive materials, tetracyanoquinodimethane (TCNQ), tetrathiafulvalene (TTF), and dimethyl ferrocene (dmFc), were used to construct three different chemically modified paste electrodes. The oxidation of NADH was examined on the basis of cyclic voltammetric measurements. The results show that all three mediators (TCNQ, TTF, and dmFc) behave as efficient mediators of the oxidation of NADH. The typical response curves of NADH at the CMEs surfaces are reported. Incorporating alcohol dehydrogenase and electroactive materials (TCNQ, TTF, and dmFc) within the graphite paste electrodes has led to the development of ethanol biosensors. Typical response curves for the ethanol analysis are reported. Comparative studies on the mediated electrochemical responses of the biosensors to ethanol are discussed.

Biosensing Techniques↗

PubMed1998

Interpreting kinetic rate constants from optical biosensor data recorded on a decaying surface.

A capturing assay was used to monitor a Fab-antigen interaction using a BIACORE optical biosensor. The antigen, a truncated single-site mutant (F43V) version of the CD4 receptor, was captured onto the sensor surface using an immobilized nonneutralizing monoclonal antibody. While this assay design created an oriented antigen surface, the antigen slowly dissociated during subsequent binding of the Fab, thus complicating the binding responses. In this paper, we illustrate how binding events occurring on a decaying surface can be accurately described by globally fitting the response data to a model that accounts for the background surface decay. Support for the method was obtained by showing the equilibrium dissociation constant calculated from the kinetic rate constants (Kd = 2.20 +/- 0.01 nM) was similar to the value measured in solution using titration calorimetry (Kd = 2.6 +/- 0.5 nM). The ability to interpret rate constants from decaying surfaces significantly extends the types of experimental systems that can be quantitatively studied on optical biosensors.

Antigen-Antibody Reactions↗

PubMed1998

Determination of binding constants by equilibrium titration with circulating sample in a surface plasmon resonance biosensor.

A commercial surface plasmon resonance biosensor, BIACORE X, is employed as a detector in a closed loop of a small sample volume. The sample is continuously circulated by an external syringe pump over two sensor spots, one functionalized with immobilized binding sites to a soluble binding partner in the mobile phase and one serving as a reference surface. A binding isotherm for the interacting macromolecules can be obtained by a stepwise titration of the soluble reactant into the circulating loop, each step followed by observation of the signal increase until equilibrium is attained. Binding constants can be measured under conditions free of mass transport artifacts and without the requirement for regeneration of the immobilized binding sites. This procedure is similar to the stepwise titration procedure described for the cuvette-based sensor design (D. R. Hall and D. J. Winzor, 1997, Anal. Biochem. 244, 152-160). In the presented configuration, the high baseline stability of the instrument combined with the availability of a reference surface for the detection of nonspecific binding permits refractive index changes upon addition of the aliquots to be measured, as well as accounting for temperature or instrumental drifts, and allows for a very long experimental time. This feature extends the applicability of equilibrium titration to systems with higher affinity or slower dissociation rate constants. Furthermore a solution competition titration is described that avoids artifacts from the immobilization procedure to provide a method for measurement of binding constants in solution. Kinetic information on the complex dissociation can also be obtained by combination of sample delivery via the external pump with the injection of competitor via the microfluidics of the biosensor. The rapid injection of high concentrations of competitor allows the observation of fast dissociation processes under conditions minimizing rebinding.

Animals↗

PubMed1999

Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction.

A heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of the monoclonal antibodies F4, F5, and LT1 directed against mouse polyomavirus large T-antigen. Phage selected by biopanning was cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay. In phage reacting with the F5 antibody the deduced amino acid sequence of the displayed peptides corresponded to a segment of large T-antigen. In phage reacting with the antibodies F4 and LT1, no such similarity was observed. The kinetics of phage particle-monoclonal antibody complex formation and dissociation was analyzed in an optical biosensor instrument. Sensor chips of standard quality were useful for binding analysis of T7 phage in crude lysates of infected Escherichia coli. We synthesized peptides corresponding to selected consensus sequences and showed by biosensor analysis that these peptides (linear NH3-CPNSLTPADPTMDY-COOH and NH3-NSLTPCNNKPSNRC-COOH with an intramolecular S--S bridge) were able to compete with large T-antigen in binding to the corresponding antibodies (LT1 and F4). These synthetic peptides were also used for gentle and specific dissociation of large T-antigen-antibody complexes. The results demonstrate the potential of phage T7 for display of peptides and for rapid analysis of interactions of these peptides with ligands.

Amino Acid Sequence↗

PubMed2000

Kinetic analysis of the interaction between HIV-1 protease and inhibitors using optical biosensor technology.

The interaction between HIV-1 protease and reversible inhibitors was studied by surface plasmon resonance biosensor technology. The steady-state binding level and the time course of association and dissociation could be observed by measuring the binding of inhibitors injected in a continuous flow of buffer to the immobilized enzyme. Fourteen low molecular weight inhibitors (500-700 Da), including the four clinically used HIV-1 protease inhibitors (indinavir, nelfinavir, ritonavir, and saquinavir), were analyzed. Affinities were estimated as B(50) values from a series of sensorgrams at different concentrations of inhibitors. These values were found to be correlated with inhibition constants (K(i)) determined by an enzyme inhibition assay (r(2) = 0.84, logarithmic values). Dissociation rates were estimated at a single saturating concentration of the inhibitors as t(1/2,obs), but these values did not correlate with K(i) (r(2) = 0.26, logarithmic values). Indinavir had the highest affinity (B(50) = 11 nM) and the fastest dissociation (t(1/2,obs) = 500 s) among the clinically used inhibitors while saquinavir had a lower affinity (B(50) = 25 nM) and the slowest dissociation rate (t(1/2,obs) = 6500 s). Since these two inhibitors have similar K(i) values, the differences in dissociation rates reveal important characteristics in the interaction that cannot be obtained by the inhibition studies. The biosensor data are expected to be of greater in vivo relevance since the experiments were performed in a buffer more similar to physiological conditions.

Drug Design↗

PubMed2000

Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode.

We have developed an affinity biosensor system based on avidin-biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 x 10(-12) mol/cm(2) on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.

Animals↗