Fluorescent labels quenching effects

Utilization of resonance effects can facilitate unenhanced Raman measurement of surfaces and make the technique more versatile. For instance, a fluorescein derivative and another dye were used as resonantly Raman scattering labels for hydroxyl and carbonyl groups on glassy carbon surfaces. The labels were covalently bonded to the surface, their fluorescence was quenched by the carbon surface, and their resonance Raman spectra could be observed at surface coverages of approximately 1%. These labels enabled assess to changes in surface coverage by C-OH and C=0 with acidic or alkaline pretreatment [4.293]. 

In the substrate, the fluorescence of the donor is quenched by the quenching label in close proximity. Upon cleavage of the substrate by the protease, this proximity is lost and a strong increase in fluorescence can be recorded. Table 2.3 summarizes a selection of fluorescence donor and acceptor pairs frequently used for protease activity assays based on a FRET quench readout. In principle, the FRET quench effect can be achieved by labeling the substrate with two different dyes with overlapping spectra (hetero-double labeling). 

The data listed in Table 2.1 follow the same general trends irrespective of the label. The lifetime is shorter at a higher pH when the polyelectrolyte is in the expanded water-swollen conformation, which exposes the excited state to the quenching effects of the aqueous phase. In addition, the labels reside in a largely homogeneous environment, which results in fluorescence, which can be successfully described by a monoexponential function of the form of Equation 2.6. 

In this method the effects of specific components of an assay matrix can be utilized to a positive (modulator) effect. These components, for example an excess of serum proteins or detergents, will modulate the fluorescence of the chelate in the unbound (labelled ligand) fraction. On binding of the fluorescent ligand to a receptor or an antibody the fluorescence is quenched whereas any free ligand remains fluorescent. 

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