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PubMed1993

A combined cellobiose oxidase/glucose oxidase biosensor for HPLC determination on-line of glucose and soluble cellodextrines.

Highly specific biosensors can be prepared by immobilizing flavin-containing oxidases in a redox polymer on an electrode surface. By combining one glucose oxidase and one cellobiose oxidase electrode in a flow cell we have made a sensor for flow-injection analysis, or postcolumn quantification, of glucose, cellobiose, and higher cellodextrines in an HPLC system. Samples of different concentrations of glucose and cellobiose, separately or mixed, were injected into the mobile phase and the current response was recorded simultaneously from both electrodes. The recorded response peak heights could be used for calibration curves. The usable measuring ranges were roughly 50 microM-50 mM for glucose and 5 microM-80 mM for cellobiose. Soluble cellodextrines, Glc1-6, could be separated on a C18 column by isocratic elution and detected by the sensor.

Biosensing Techniques↗

PubMed1995

Kinetics of protein-protein interactions at the surface of an optical biosensor.

Methods based on the use of optical biosensors have recently become available to provide a convenient means of determining the rate and equilibrium constants for bimolecular interactions between immobilized ligands and soluble ligate molecules. However, the association data that these methods provide are not always accurately described by the expected pseudo-first-order reaction mechanism, particularly when the ligand is immobilized on a dextran matrix. We show that a better description of the association data, especially at higher ligate concentrations, is achieved with a double exponential function, indicating that at least two rate-limiting processes are involved. Various models are considered in order to explain these observations: the presence of two (or more) distinct populations of immobilized ligand; a change, possibly conformational, in the immobilized ligand before or after ligate binding; or the hindrance of ligate binding to immobilized ligand. We suggest that steric hindrance caused by ligate binding to the dextran-coated sensor surface seems the most likely explanation for the observed biphasic association kinetics and that the faster initial phase should be used in oder to determine association constants that can be compared to those in solution.

Antigen-Antibody Reactions↗

PubMed1996

Oriented binding of a lipid-anchored cell adhesion protein onto a biosensor surface using hydrophobic immobilization and photoactive crosslinking.

The carboxymethyl-dextran surface of a biosensor instrument was modified to couple, in an active state, the lipid-anchored contact site A (csA) glycoprotein, a homophilic adhesion molecule of aggregating cells of Dictyostelium discoideum. The carboxy groups were modified by heptyl residues for hydrophobic binding of the molecule with its lipid anchor to the dextran matrix. Alternatively, the protein was fixed in a similar orientation by covalent linkage through a perfluorophenylazide-derived hydrophobic crosslinker. Titration of the bound csA molecules with antibodies that recognize either the native or the denatured glycoprotein verified that the csA molecules were coupled in a native state to the sensor surface. Interaction of the immobilized csA protein with csA in solution established that the bound molecules are capable of taking part in homophilic interactions.

Animals↗

PubMed1997

Studies of protein interactions by biosensor technology: an alternative approach to the analysis of sensorgrams deviating from pseudo-first-order kinetic behavior.

A procedure for evaluating the thermodynamic equilibrium constant by kinetic analysis of sensorgrams which deviate from the pseudo-first-order kinetic behavior predicted for 1:1 interactions between ligate and affinity sites on the sensor surface is described. This analysis employs quantitative expressions that are used in conventional kinetic characterization of protein interactions by biosensor technology, but with the equilibrium sensorgram response fixed at a predetermined magnitude. Simulated sensorgrams for situations in which the aberrant kinetic behavior reflects (i) heterogeneity of affinity sites and (ii) isomerization of the complex between ligate and affinity sites are used to explore the feasibility of the approach. Its application is then illustrated with BIAcore studies of the interaction between the Fab fragment of an anti-paraquat monoclonal antibody and immobilized antigen in the form of a paraquat analog attached covalently to the sensor surface. Studies with an extremely high degree of antigen substitution on the sensor surface yielded sensorgrams that deviated markedly from pseudo-first-order kinetic behavior. However, they yielded the same binding constant (3 x 10(6) M-1) as the value deduced by conventional analysis of sensorgrams that conformed with pseudo-first-order kinetics because of a much lower concentration of immobilized antigen on the sensor surface. Such identity of binding constants eliminates heterogeneity of immobilized paraquat sites as the likely source of the aberrant kinetic behavior.

Antibodies, Monoclonal↗

PubMed1997

Theoretical analysis of protein concentration determination using biosensor technology under conditions of partial mass transport limitation.

The theory of a method for determining active concentration of nonpurified protein samples using the BIAcore biosensor technology has been developed. The method relies on change in binding rate with varying flow rate at high ligand concentration where mass transport from bulk to surface with immobilized ligand becomes partially rate limiting. Prior study of binding kinetics is not necessary. If the molecular weight and the diffusion coefficient of the analyte protein are known, no standard with a known concentration is required. From numeric computer simulations a simple analytical expression for the mass transport coefficient derived at totally mass transport limiting conditions is shown to be applicable for conditions of partial mass transport limitation. Simple one to one association is assumed in deriving the method, but it is applicable even with more complex kinetics due to a bivalent analyte or heterogeneity of analyte or ligand.

Algorithms↗

PubMed1997

Theoretical and experimental considerations of the pseudo-first-order approximation in conventional kinetic analysis of IAsys biosensor data.

The validity of the conventional interpretation of IAsys biosensor profiles in terms of pseudo-first-order kinetic behavior is subjected to closer scrutiny by its application to simulated data for low- and high-affinity interactions between ligate and immobilized ligand. As might reasonably have been expected, analysis of the simulated data for the low-affinity system (association equilibrium constant of 10(5) M-1) in such terms returned the input association and dissociation rate constants (10(3) M-1 s-1 and 10(-2) s-1, respectively)-a consequence of essential compliance with the assumed constancy of ligate concentration in the liquid phase. For the high-affinity interaction (ka = 10(5) M-1 s-1, kd = 10(-2) s-1, KAX = 10(7) M-1) the ligate concentration was depleted by up to 35%, and hence its assumed constancy was clearly an untenable approximation. Whereas no symptomatic evidence of such violation (apart from the return of incorrect estimates of ka and kd) was evident from pseudo-first-order kinetic analysis of the adsorption profiles, the corresponding analysis of desorption profiles was more informative in that the data deviated demonstrably from pseudo-first-order kinetic behavior. A second-order kinetic analysis was therefore developed and shown to be applicable to adsorption and desorption profiles, irrespective of the validity or otherwise of the pseudo-first-order kinetic approximation. Experimental results obtained for the interaction of histidine-rich glycoprotein with immobilized IgG were then used to illustrate various features of the pseudo-first-order and second-order kinetic analyses, and to determine from the second-order analysis an association equilibrium constant of 2 x 10(8) M-1, which is 20-fold greater than the value obtained by interpretation of the profiles in terms of pseudo-first-order kinetic behavior.

Adsorption↗

PubMed1998

Characterization of Helicobacter pylori interactions with sialylglycoconjugates using a resonant mirror biosensor.

A new optical biosensor technique based on the resonant mirror was used to characterize Helicobacter pylori strains according to their sialic acid binding, demonstrating the suitability of using intact bacteria in real-time measurements and classifying strains based on their binding abilities. Results obtained from both competition and displacement assays using different glycoconjugates confirmed that several, but not all, H.pylori strains express sialic acid-binding adhesin(s), specific for alpha-2,3-sialyllactose. The adhesin, removable from the bacterial surface by water extraction, is not related to other reported H.pylori cell surface proteins with binding ability to sialylated compounds such as sialylglycoceramides.

Biosensing Techniques↗

PubMed1998

Second-order kinetic analysis of IAsys biosensor data: its use and applicability.

The kinetic analysis of IAsys biosensor association data usually relies upon the assumption of constant ligate concentration. In certain circumstances this assumption may no longer be valid. In a similar vein, the analysis of the dissociation phase assumes the concentration of ligate to be negligible in the liquid phase-an assumption that may not be sustainable for high-affinity interactions. In this paper we derive analytical solutions of the second-order differential kinetic equations for the association and dissociation phases, together with a binding isotherm that also allows for changes in concentration of both the ligand and the ligate. Using these equations it is possible to determine the conditions under which the pseudo-first-order assumption ceases to be valid. It is found that the effect of ligate depletion on the association rate constant becomes significant only when binding low ligate concentrations to ligand on surfaces with high binding capacities or high affinities. Similarly, the rebinding in the dissociation phase is dependent upon the affinity and the ligand capacity together with the starting dissociation response compared to the capacity. Finally, depletion also affects the form of the binding isotherm, particularly in situations involving high matrix capacities for ligate and high-affinity interactions.

Biosensing Techniques↗

PubMed1998

Screening of compounds interacting with HIV-1 proteinase using optical biosensor technology.

A high-resolution optical biosensor assay for screening of low-molecular-weight compounds, using an immobilized protein target, has been developed. HIV-1 proteinase was immobilized on the sensor surface by direct amine coupling and a variety of inhibitors and noninteracting reference drugs were applied to the sensor surface in a continuous flow of buffer. The procedure did not require intrinsic reporter groups, substrates, inhibitors, or other ligands for detection. By using a reference protein, the signal could be corrected for the relatively large background signal caused by differences in dimethyl sulfoxide concentration between running and sample buffers. Substances binding with high affinity (Ki in nM range) required efficient regeneration of the sensor surface and washing of the injection system between sample cycles to get consistent results. Analysis was simplified by using report points, extracted during both association and dissociation phases, and a simple graphical display of data. The optimized assay could correctly distinguish HIV-1 inhibitors from other compounds in a randomized series, indicate differences in their interaction kinetics, and reveal artifacts due to nonspecific signals, incomplete regeneration, or carryover. The method is expected to be generally applicable to secondary screening of low-molecular-weight compound libraries with proteins as targets.

Biosensing Techniques↗

PubMed1999

Kinetic analysis of analyte binding by optical biosensors: hydrodynamic penetration of the analyte flow into the polymer matrix reduces the influence of mass transport.

I examined the penetration of the hydrodynamic flow into a polymer matrix immobilized by grafting to a surface, such as used in optical biosensors designed to measure binding reactions in real time. I show that the flow penetrates with an appreciable velocity into a region located at the tip of such a polymer brush, corresponding to about 10 to 15% of the total mass of the grafted polymer. Furthermore, under the conditions recommended for kinetic measurements, the concentrations of both polymer and immobilized ligands are low in these regions of the matrix, where crowding effects are negligible. Under such conditions, the hydrodynamic flow penetrating into the dextran matrix flow will bring the analytes close to their targets, thus considerably reducing transport problems.

Biosensing Techniques↗

PubMed1999

Analysis of the binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase from tern and whale using the BIAcore biosensor: effect of immobilization level and flow rate on kinetic analysis.

The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 microl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (K(A) = 6.3 +/- 1.3 x 10(7) M(-1)) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (K(A) = 4.3 +/- 0.7 x 10(6) M(-1)). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn(200), which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.

Animals↗

PubMed2000

Detection of DNA via an ion channel switch biosensor.

Detection of DNA by an ion channel switch biosensor has been demonstrated in a model system, using single-stranded oligonucleotide sequences of 52-84 bases in length. Two different biotinylated probes are bound, via streptavidin, either to the outer region of a gramicidin ion channel dimer or to an immobilized membrane component. The ion channels are switched off upon detection of DNA containing complementary epitopes to these probes, separated by a nonbinding region, at nanomolar levels. The DNA cross-links the ion channel to the immobilized species, preventing ions passing through the channel. Addition of DNase I after the target DNA has been added switches the ion channels on. The DNA response is dependent on the rate of hybridization of the individual probes to their complementary epitopes, as shown by using a single probe against DNA containing a repeat of the complementary epitope. These results were correlated with hybridization rates determined using surface plasmon resonance (BIAcore 2000), and with free energies of dimer formation for the probes.

Anti-Bacterial Agents↗

PubMed2000

A ganglioside-based assay for cholera toxin using an array biosensor.

A rapid assay for cholera toxin (CT) has been developed using a fluorescence-based biosensor. This sensor was capable of analyzing six samples simultaneously for CT in 20 min with few manipulations required by the operator. The biochemical assays utilized a ganglioside-"capture" format: ganglioside GM1, utilized for capture of analyte, was immobilized in discrete locations on the surface of the optical waveguide. Binding of CT to immobilized GM1 was demonstrated with direct assays (using fluorescently labeled CT) and "sandwich" immunoassays (using fluorescently labeled tracer antibodies). Limits of detection for CT were 200 ng/ml in direct assays and 40 ng/ml and 1 microg/ml in sandwich-type assays performed using rabbit and goat tracer antibodies. Binding of CT to other glycolipid capture reagents was also observed. While significant CT binding was observed to loci patterned with GD1b, Gb3, and Gb4, CT did not bind significantly to immobilized GT1b at the concentrations tested. This is the first description of such a non-antibody-based recognition system in a multi-specific planar array sensor.

Antibodies, Monoclonal↗

PubMed2000

A fiber-optic microarray biosensor using aptamers as receptors.

A fiber-optic biosensor using an aptamer receptor has been developed for the measurement of thrombin. An antithrombin DNA aptamer was immobilized on the surface of silica microspheres, and these aptamer beads were distributed in microwells on the distal tip of an imaging fiber. A different oligonucleotide bead type prepared using the same method as the aptamer beads was also included in the microwells to measure the degree of nonspecific binding. The imaging fiber was coupled to a modified epifluorescence microscope system, and the distal end of the fiber was incubated with a fluorescein-labeled thrombin (F-thrombin) solution. Nonlabeled thrombin could be detected using a competitive binding assay with F-thrombin. The aptamer beads selectively bound to the target and could be reused without any sensitivity change. The fiber-optic microarray system has a detection limit of 1 nM for nonlabeled thrombin, and each test can be performed in ca. 15 min including the regeneration time.

Animals↗

PubMed2000

A protein biosensor for lactate.

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.

Anilino Naphthalenesulfonates↗

PubMed2000

Flow cytometry-based biosensor for detection of multivalent proteins.

Microsphere-based flow cytometric detection of cholera toxin (CT) through distance-dependent fluorescence resonant energy transfer (FRET) has been developed. Simultaneous double-fluorescence changes induced by multivalent interactions between CT and fluorophore (both fluorescence donor and acceptor)-labeled ganglioside GM1 on a biomimetic membrane surface (supported bilayers of phospholipids) can be measured by a commercial flow cytometer, providing a convenient and sensitive detection method for CT. The flow cytometry-based biosensor is capable of detecting less than 10 pM CT within 30 min. The signal generation strategy coupled with flow cytometry also provides a convenient method for kinetic studies of multivalent interactions. The surface density and the ratio of donor/acceptor-labeled GM1 on the surfaces of phospholipid bilayers are optimized to achieve high sensitivity.

Biosensing Techniques↗

PubMed2001

Use of optical biosensors for the study of mechanistically concerted surface adsorption processes.

The advent of commercial optical biosensors, such as the BIAcore from Pharmacia and IAsys from Affinity Sensors, has made available to the biochemist a powerful means to examine and characterize the interaction of biological macromolecules with a binding surface. By analysis of the kinetic and equilibrium aspects of the observed experimental adsorption isotherms, rate and affinity constants can be determined. This Review focuses on pertinent aspects of the technology and its use for the performance and quantitative characterization of some various types of mechanistically concerted adsorption behavior.

Adsorption↗

PubMed2001

A kinetic study of analyte-receptor binding and dissociation, and dissociation alone, for biosensor applications: a fractal analysis.

A fractal analysis is presented for (a) analyte-receptor binding and dissociation kinetics and (b) dissociation kinetics alone for biosensor applications. Emphasis is placed on dissociation kinetics. Data taken from the literature may be modeled, in the case of binding, using a single-fractal analysis or a dual-fractal analysis. The dual-fractal analysis represents a change in the binding mechanism as the reaction progresses on the surface. A single-fractal analysis is adequate to model the dissociation kinetics in the examples presented. Predictive relationships developed for the dissociation rate coefficient(s) as a function of the analyte concentration are of particular value since they provide a means by which the dissociation rate coefficients may be manipulated. Relationships are also presented for the binding and dissociation rate coefficients as a function of their corresponding fractal dimension, D(f), or the degree of heterogeneity that exists on the surface. When analyte-receptor binding or dissociation is involved, an increase in the heterogeneity on the surface (increase in D(f)) leads to an increase in the binding and in the dissociation rate coefficient.

Animals↗