• Abstract
• Table of Contents
• Figures
• Literature Cited

Abstract

In recent years, optical evanescent wave biosensors have been used to characterize protein?protein interactions, including determination of equilibrium binding constants and bimolecular rate constants. This surface binding technique can provide information about chemical on?rate constants, the lifetimes of complexes formed, and the time course of the signal. This unit provides a thorough, well?illustrated discussion of the principles of optical biosensors, experimental design, ligand immobilization, experimental data analysis, and common obstacles and possible solutions.

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• Strategic Planning
• Immobilization Protocols
• Binding Experiments and Data Analysis
• Common Experimental Obstacles
• Summary
• Acknowledgment
• Literature Cited
• Figures
• Tables

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• Figure 17.6.1 Schematic presentation of a typical optical biosensor experiment. Light is coupled into a structure that allows generation of surface‐confined electromagnetic waves (e.g., surface plasmons in a gold film, or modes of a planar waveguide), which are sensitive to the refractive index of the solution in the range of the evanescent field, n surf . Typical penetration depths of the sensitive volume into the solution are in the order of 100 nm. Ligands are attached to the sensor surface, as indicated by half‐circles. (A ) When analytes (full circles) are introduced into the solution above the surface, reversible interactions with the ligand lead to association events at a rate governed by K on c A c L,free (solid arrows), and dissociation events at a rate c complex K off (dashed arrows). In the association phase, association events outnumber dissociation events until a steady state is reached. (B ) When the surface is washed with running buffer in the absence of analyte, only dissociation events take place. (C ) Signal obtained from probing the refractive index n surf during the sequential application of the configurations depicted in A and B, given in arbitrary units. Following the association phase (A) and the dissociation phase (B), usually a regeneration procedure is applied for removing all remaining analyte from the surface before a new experimental cycle takes place.

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• Figure 17.6.2 (A ) The binding of a bivalent analyte can take place through the interaction with a single ligand molecule (left), or with two ligand molecules (right), depending on the local ligand density. If an unoccupied immobilized ligand molecule is within an accessible distance to an already singly bound analyte molecule, then the on‐rate constant of the second interaction is strongly enhanced because of the entropic colocalization constraints. The overall dissociation rate constant of the double attached analyte is substantially reduced (usually by orders of magnitude), because it requires the virtually simultaneous dissociation of both interactions, which is much more improbable than a single dissociation event. The overall binding kinetics obtained from multivalent analytes is highly complex. (B ) If the configuration is reversed, with the bivalent binding partner immobilized to the sensor surface, then both valencies act independently (left), and (in the absence of cooperativity of the sites) lead to binding kinetics that are identical to that of the monovalent interactions (right).

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• Figure 17.6.3 Schematic presentation of an ideal 1:1 pseudo first‐order reaction. Shown is a superposition of the binding progress curves that should be obtained in a series of experiments at different analyte concentrations. The data are given in percent of the signal at maximal binding, but the units of the signal do not affect the results of the data analysis. The arrows on the curves at the higher concentrations indicate that steady‐state binding is attained, whereas, for the lower curves, longer association times would be required as part of a steady‐state analysis.

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• Figure 17.6.4 (A ) Time course of an equilibrium titration experiment. Arrows indicate the time‐points of the step‐wise increase in the analyte concentration. (B ) Steady‐state binding analysis according to Equation 17.6.5. In a plot of the steady‐state signal versus the base‐10 logarithm of the analyte concentration, the binding isotherm exhibits a typical sigmoid shape. The inflection point at 50% saturation determines K D , and the width of the curve is characterized by ∼10% saturation at 1/10 K D and ∼90% saturation at 10 K D (dotted lines). Visible inspection of the data in this representation already allows a robust parameter estimation.

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• Figure 17.6.5 Mass transport effects on the surface binding process. (A ) In the association phase, insufficient transport does not fully replenish the free analyte in a zone near the sensor surface (depletion zone). The inability to maintain the concentration c A in the depletion zone leads to a limited surface binding rate. (B ) The corresponding effect of mass transport limitation in the dissociation phase is that the surface is insufficiently washed and dissociated analyte is insufficiently removed. This generates a zone near the surface in which free analyte (after dissociation from the ligand) can rebind to empty ligand sites before diffusing into the bulk flow. This zone is indicated as retention zone. (C ) Introduction of an excess of the soluble form of the ligand as a competitor into the buffer of the dissociation phase helps prevent rebinding to the surface. Soluble ligand can bind to dissociated analyte before rebinding takes place, and the soluble complex can diffuse into the buffer flow. This can allow the measurement of K off free of mass transport effects. (D ) Time course of dissociation during a mass transport induced rebinding situation (as depicted in panel B), and after introduction of a soluble competitor into the washing buffer (arrow). In the presence of the competitor, the signal reflects the chemical off‐rate constant K off , while in the rebinding situation, the apparent rate governing the signal is reduced.

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• Figure 17.6.6 Exemplary data of the interaction of TCR with superantigen (for details of this interaction see Andersen et al., ). (A ) Elution profile of the TCR from size‐exclusion chromatography, indicating the fractions used for kinetic analysis of the interaction with immobilized superantigen, as shown in (B ). It should be noted that fraction 1 (bold solid line), fraction 2 (dashed line), and fraction 3 (solid line) all exhibit multiphasic binding, with different relative magnitudes of the slower component. It should also be noted that despite the lower concentration of fraction 1, which results in the lowest response in the association phase, the signal in the dissociation phase is highest. This slower kinetic component in the binding progress curve reflects different amounts of aggregates bound to the sensor surface. The aggregates have a slower dissociation because of their multivalent attachment. For comparison, the same interaction is shown in the reverse orientation, with immobilized TCR and soluble superantigen (dotted line). (C ) Sequence of association‐dissociation curves at different superantigen concentrations, binding to immobilized TCR. In this orientation, the association and the dissociation is much faster, virtually monophasic, and the binding is completely reversible, which provides further evidence that the slow kinetic components introduced in the different fractions of the soluble TCR sample in Panel B are artifacts. For quantitative analysis, the signal measured at a nonfunctionalized surface (dotted line) is subtracted in order to remove the bulk refractive index contribution of the analyte. (D ) A plot of the net steady‐state binding signal versus superantigen concentration allows the measurement of the affinity of the interaction free from artifacts due to TCR aggregation that would be introduced in the configuration of Panel B.

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• Figure 17.6.7 Effects of the ligand density on the binding kinetics for interactions with relative low affinity (A ) and medium affinity (B ). Each panel shows the interaction of immobilized superantigen with single‐chain TCR, observed at high ligand surface density (1700 RU, solid lines) and low ligand surface density (700 RU, dashed lines). For easier comparison, the signal obtained at the lower density surface was scaled proportionally. In panel A, a slow phase of binding in the association phase and a residual binding (possible slow dissociation of multivalently bound aggregates) is introduced at high ligand density, under otherwise identical conditions. For the interaction in panel B, the chemical off‐rate constant is smaller than for the low‐affinity interaction shown above. Nevertheless, from comparison of the binding curves at different ligand surface densities (under otherwise identical conditions) it is obvious that an increased ligand density has significant effects on the surface binding kinetics. This observation could be due to aggregation or to mass transport limitations, both of which are more likely at higher ligand densities.

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