Phase-Modulation Fluoroimmunoassays

Immunoassays based on phase-modulation spectroscopy have been implemented by two distinctly different approaches. Phase-resolved immunoassays rely on fluorescence intensity measurements, in which the emission of one fluorescent species in a mixture is suppressed, and the remainder is quantitated. Phase fluorescence immunoassays utilize measurements of the phase angle and modulation, which change in response to fluorescence lifetime changes. Common aspects of the theory and instrumentation are discussed in this section, followed by individual discussions of the different approaches. 

Phase-resolved, or phase-suppression technology was investigated for FIA development by McGown and co-workers(49, 53, 105, 106) to test the prediction that phase fluorometry would be useful for monitoring two-state equilibria in the analysis of macromolecular binding interactions)31,104) Fluorophores with different fluorescence 

For a single fluorophore with an exponential decay, the phase and modulation are related to the fluorescence lifetime by the expressions 

It is possible to resolve the emission from one species in the presence of another by phase-suppression techniques. The emission from a single fluorophore excited by sinusoidally modulated light is described by 

PFIAs and fluorescence lifetime immunoassays (FLIAs) are uniquely based on measurement of probe emission properties other than the intensity. The phase and modulation are measured, and they directly reflect the fluorescence lifetime of the fluorophore. This provides a major advantage, since the intensity can vary over a broad range, with only minor effects on the results. Phase-modulation measurements can be 

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