Receptor-analyte binding

Despite their extensive use, assays employing labeled analytes suffer from several disadvantages. First, analytes need to be conjugated to an enzyme, fluorophore or a radioactive moiety, which increases the cost of the assay. Second, assays that employ reporters typically require multiple washing and incubation steps and in most cases are end-point assays. Finally, in many instances, conjugation of reporters near the active site of the receptor can interfere with receptor-analyte binding. 

The value of dn/dC depends on the properties of the material, e.g., in case of proteins this coefficient is 0.188 ml/g35 and in case of glucose dissolved in water (used for calibration measurements as discussed later in this chapter) this coefficient is 0.069 ml/g36. In the second mode of operation, analytes bind to the sensor surface (e.g., mediated by a receptor layer). In this case a thin layer with thickness w and refractive index nw is formed by the adsorbed analytes. Because the value of nw (e.g., 1.45 for proteins) is usually different than the refractive index of the solution (e.g., 1.33 for water) that contains the analyte molecules, a phase change is induced. The average layer growth (Aw) on the sensor surface can be related to the mass change (Am) per surface area (A) ... 

In an immunosensor the core-cover interface of an optical waveguide structure is coated with a chemo-optical transducer receptor layer, which can selectively bind to specific analyte molecules present in the cover medium. The receptor-analyte reaction obeys the law of mass action, which states that the rate of a reaction is proportional to the concentration of the reactants. At equilibrium, the rate of formation of the receptor-analyte complex is equal to the rate of breaking, and the equilibrium constant, K. can be written as... 

The separation of binding site and fluorophore by a nonconjugating spacer opens the path to other mechanisms of communication, most prominently ET and exci-mer/exciplex formation. In the first case, the electronic nature of both fluorophore and receptor unit and the steric nature of the spacer are the important parameters for signal generation. In the second case, for most systems the electronic nature of the fluorophores and the steric nature of the receptor as well as its change upon analyte binding determine the signal. 

Consider the two hypothetical situations illustrated in Figure 2. On the top is pictured a collection of individual fluorescent receptors, while on the bottom is pictured the same number of receptors, yet they are connected via a molecular wire conjugated polymer. In addition, suppose it is known that binding of the analyte paraquat (PQ) into a receptor causes a complete quenching ofits fluorescence. In the case of individual receptors, the binding of one analyte gives only a small diminution in the total intensity of emission observed. This is because each exciton is fixed on one molecule and cannot communicate with other receptor sites. 

In fields such as biosensing, analyte binding often relies on very specific molecular recognition interactions that nature has supplied, such as antibody-antigen interactions or strands of complimentary DNA forming double hefices. Unfortunately, because versatile and highly selective receptors for TNT or other explosive molecules are not available, chemists are left to rely on less specific interactions. 

NPs provide highly efficient catalysts and sensors due to their unique chemical and physical properties. NPs can be used as exo-active surfaces where a multitude of molecular receptors can bind analytes and generate a signal. Alternatively, NPs can be used as core-based materials in which biocatalytic processes can activate their core or they provide a biologically inert electrochemical label. As catalysts, NPs utilize their large surface area to volume ratio and enhance either electrochemical reactions or electron transfer at an electrode. The use of NPs in catalysts and sensors will continue as these functional materials serve as active units within these applications. 

Lipodex E [octakis(3-0-butanoyl-2,6-di-0- -pentyl)-y-cyclodextrin] dissolved in a polysiloxane matrix (SE-54) was used as a chiral receptor. The binding strength of the analyte molecules depends on the interaction mech-... 

Three basic types of relay mechanisms are relevant to the chemosensors of Sec. IV a conformational change, energy transfer, and electron transfer. Operating alone or in concert, these relay mechanisms communicate the molecular recognition event of analyte binding at the receptor site to the luminescent reporter site. We now briefly discuss each of these relay mechanisms. 

Figure 11.24 A silicon wafer array, with micromachined pyramidal wells (detail shown right) for holding receptor derivatised beads. Fluid containing the experimental solution is added to the top of the array and flows through the bead matrix, and out of the bottom of the pyramidal wells holding the beads. Analyte binding by the differential receptors anchored to the beads gives a recognition pattern unique to each analyte mixture (reproduced with permission from [34] 2001 American Chemical Society).

The electrochemical biosensor method has shown high potential in microarray usage for its sensitivity, reproducibility, selectivity, and reversibility. Unlike other methods, the biosensor method depends on the electrochemical property of the coating receptors and binding analytes. It is prepared by electrochemical synthesis of polymers in situ on platinum electrodes. This technology, however, will require further... 

Fig. 8 Enhanced receptor effect on analyte binding affinity. Upon chelation, the electronegative ancillary ligand causes a polarization of the Ln + ion, inducing an increased positive charge at the binding site. This anisotropy results in an increased binding affinity for the anionic analyte. Limit of detection (LOD) values shown are for the Tb/D02A/ dipicolinate system.

Despite the differences in generation of the evanescent field, the basic binding experiment is basically the same for all the optical biosensors (see Fig. 5.3). One of the interacting partners, the receptor, is attached to the sensor surface while the analyte binds to the receptor from free solution. As the sensor monitors refractive index changes occturing in real time, the amount of receptor, analyte and the rate of binding can be determined. Indeed, the estimation of the interaction kinetics is one of the key advantage of this technique. 

Figure 6-4 Principle of affinity chromatography.The analyte (enzyme, antibody, antigen, tissue receptor, etc.) binds to the support-bound ligand. Subsequently, it is eluted with a general eluent (such as a chaotropic agent), pH change, or biospecific eluent (such as an inhibitor or substrate).

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