Enzyme labels and detection by fluorescence
The basis of most nucleic acid assays is exploitation of the specificity of base recognition (e.g., adenine for thymine) and the high binding constant of resulting duplexes. Competitive and noncompetitive amperometric immunoassays have been developed with enzymes as labels. The sensitivity of assays can be improved by using an enzyme label such as acetate kinase, which can be detected with higher sensitivity. Such an enzyme label can be detected down to a zeptomole level using a bioluminescent assay. ATP formed by the action of the enzyme label on the acetylphosphatase substrate is measured using a firefly luciferase reaction. The expressed enzyme and the gene for the bioluminescent luciferase class of enzymes are promising labels for nucleic acid detection. The gene can be manipulated to produce mutants that catalyze light at different wavelengths. The gene for the enzyme luciferase itself may be used rather than the product of the gene. This enables many enzyme molecules to be transcribed from an individual gene label, thus introducing a high amplification factor into the assay.
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