Progress in Delayed Fluorescence Immunoassay

The utilization of delayed detection of the emission in fluoroimmunoassays relates to the desire to improve the analytical sensitivity by means of temporal background rejection. Background is present in all fluorometric determinations and is due, among other things, to light scattering, emissions from the samples’ endogenous fluorescence, autofluorescence of cells and tissues, luminescent properties of solid matrixes, cuvettes, test tubes, lenses, etc. Time-resolved fluorometry with pulsed excitation can be used to eliminate the interfering background, provided that the decay time of the specific signal clearly differs from that of the background (Fig. 18.1). The efficient use of time-resolved detection requires the employment of delay times longer than 10 µs, and, accordingly, luminescent probes exhibiting excited state life times longer than 10 µs must be developed for the system.