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FIGURE 1.8 Different forms of immunoassays (Lukosz, 1991). Immobilized molecules shown at left; the addition of the analyte in the middle; resulting adlayer on the surface on the right, (a) Direct binding; (b) sandwich assay; (c) displacement assay; (d) replacement assay. Reprinted from Biosensors and Bioelectronics, vol 6, W. Lukosz, pp. 215-225, 1991, with permission from Elsevier Science.

binding of the hapten to the primary antibody (Abi) in this case induces only insignificant or very small changes in the signal to be measured (such as the change in refractive index when using an evanescent fiber-optic biosensor). A secondary antibody (Ab2) is simultaneously added to the analyte (antigen in our case) in solution, or soon thereafter. The complex formed (Ab2-Abi-Ag) is then continuously monitored.

(c) Displacement assay Here an analogue of the analyte to be measured is immobilized on the biosensor surface. Then, the corresponding antibody is bound. On the addition of the analyte in solution to be measured, antibodies are displaced from the surface and bind to the free antigens. In this case, one observes and measures a negative change (for example, a negative change in the refractive index).

(d) Replacement assay In this case the surface is coated with the antibody molecules. An analogue of the antigen (conjugated to a larger molecule) is bound to the antibody immobilized on the sensor surface. This is the refractive index probe (Drake et ah, 1988). On the addition of the analyte in solution to be measured to the biosensor, the analyte partially replaces the bound conjugated antigen and a negative index change (for example, refractive index occurs) and is measured.

The understanding of biological processes at the molecular level is becoming increasingly important, especially for medical purposes. Two basic approaches may be employed: structural and functional analysis. Ramakrish-nan (2000) indicates that under ideal conditions these should complement each other and provide a complete and a comprehensive picture of the molecular process. Some of the structural techniques routinely employed are electron microscopy, sequence analysis, mass spectrometry, and x-ray, and electron diffraction studies. These techniques are quite effective and have provided information about the atomic organization of individual as well as interacting molecules, but have a major drawback in that they are static and frozen in time. Functional investigation techniques such as affinity chromatography and immunological and spectrometric techniques give valuable information on the conditions and the specificity of the interaction. However, these techniques are either unable to follow a process in time or are too slow to be rendered suitable for most biospecific interactions. Moreover, these techniques demand some kind of labeling of interactants, which is undesirable as it may interfere with the interaction and necessitate purification of the expensive interactants in large quantities. Another difficulty encountered while monitoring biomolecular interactions is that a number of experimental artifacts such as surface-imposed heterogeneity, mass transport, aggregation, avidity, crowding, matrix effects, and nonspecific binding complicate binding responses (Morton and Myszka, 1998).

Ekins and Chu (1994) have reviewed the techniques to develop multianalyte assays. In discussing the need for and importance of developing miniaturized assays, the authors emphasize the decrease in traumatic effects in taking blood (fingertip), especially for young children. Also, the cost of using miniaturized assays at the point of application (for example, in the medical doctor's laboratory itself) should be less than the cost of using large centralized analyzers in hospital laboratories. These authors state that the real advantage of miniaturization is that it permits multianalyte assays, which is especially helpful in medical diagnostics where the trend is to detect and determine more and more analytes to provide a better perspective for diagnosis. This is especially true for complex diseases where multiple criteria need to be satisfied to help discriminate between closely related diseases with similar symptoms.

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