Fluorescence signal generation

Several approaches have been reported for the screening of polymerase activity, for example radioisotope assays such as scintillation proximity assays [56, 57] or fluorescence-based assays [58-61], Most of these assays, however, suffer from use of tedious procedures, the use of radioisotopes, or use of expensive reagents for fluorescence signal generation. A convenient means of online monitoring of DNA polymerase activity has recently been presented by Andreas Marx and Daniel Summerer [62]. Their technique involves a DNA template that forms a stable hairpin structure labeled at two positions ... 

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

As with the UV absorption detector, the sample compartment consists of a special cell for measuring a flowing, rather than static, solution. The fluorescence detector thus individually measures the fluorescence intensities of the mixture components as they elute from the column (see Figure 13.10). The electronic signal generated at the phototube is recorded on the chromatogram. 

Reppy MA (2002) Signal generation from switchable polydiacetylene fluorescence. Mater Res Soc Symp Proc 723 05 9 l-05 9 6... 

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