Quenching surface plasmon resonance

Direct binding systems. Many biosensors that utilize receptors as a mode of obtaining selectivity depend on the detection of the binding event, using devices such as surface plasmon resonance, surface acoustic waves, and fluorescence quenching [27]. 

Once phage or scFv which specifically bind the antigen of interest have been identified it may be necessary to cany out further screening tests to assess which antibodies fiom the positive population have the highest affinities. There are a variety of wtq of achieving this including surface plasmon resonance (BlAcore) screening, fluorescence quench measmement, and competition ELISAs. Descriptions of these protocols are beyond the scope of this chapter. 

Fluorescence measurements and detection can either be made under steady-state or time-resolved conditions. Some of the commonly used measurement techniques focus on changes in optical properties such as fluorescence intensity quenching, phase fluorometry (lifetime), polarization, surface plasmon resonance (SPR), and evanescent waves. Here, we will present detector systems based for (a) fluorescence intensity quenching and (b) phase fluorometry in detail. A few example references of integrated optical sensor systems based on the various optical measurement techniques are given in Table 1 and the reader is encourage to review those papers if more details are desired. 

Figure 14. FRET (fluorescence resonance energy transfer) experiment between a surface-attached donor dye-labeled probe strand and an acceptor dye-labeled target strand from solution, Shown are the angular scans of the reflectivities and of the fluorescence emission of the donor dye before and after hybridization, as well as, after denaturing the hybrid. While the reflectivities are virtually identical the fluorescence shows a strong enhancement at surface plasmon resonance (—) which is completely quenched after hybridization (—), however, can be fully recovered upon the dissociation of the hybrid (..).

An additional effect to consider for SERRS is that of the fluorescence which can occur simultaneously with a RRS process. This is the so-called delayed fluorescence, " which is a radiative decay process for molecules which have relaxed from their initial excited vibrational state of the excited electronic state to the lowest vibrational level of the excited state. Delayed fluorescence can be additionally damped on a rough Ag surface for the same reasons that the RRS process is additionally damped, i.e., by coupling to the surface plasmon resonances. On the other hand, the electromagnetic enhancement factors L (o)L (Oip), will also cause an enhancement of the fluorescence in a similar manner to the Raman process. However, the decay of excited molecules by the surface channel will tend to mitigate the EM fluorescence enhancement effect. Two cases have been discussed in the literature " (a) the case of a molecule with a fluorescence quantum efficiency near unity in the free state, i.e., QE 1, and (b) the case with QE 1. If QE is low (<0.01), the fluorescent emission is estimated to be enhanced by ca. 10 whereas, for QE 1, a surface quenching of fluorescent emission by ca. 10" has been... 

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