Phase-Resolved Fluorescence Immunoassay

In addition to fluorescence intensity and polarization, fluorescence spectroscopy also includes measurement of the lifetime of the excited state. Recent improvements in the design of fluorescence instrumentation for measuring fluorescence lifetime have permitted additional applications of fluorescence techniques to immunoassays. Fluorescence lifetime measurement can be performed by either phase-resolved or time-resolved fluorescence spectroscopy. 

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F. V. Bright and L. B. McGown, Homogeneous immunoassay of phenobarbital by phase-resolved fluorescence spectroseopy, Talanta 32, 15-18 (1985). 

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

Bright, F. V. and Mcgown, L. B. (1985). Homogeneous Immunoassay of Phenobarbital by Phase-Resolved Fluorescence Spectroscopy. Talanta 32 15-18. 

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. 

P. Helsingius, 1. Hemmila, and T. Lovgren, Solid-phase immunoassay of digoxin by measuring time-resolved fluorescence, Clin. Chem. 32, 1767-1769 (1986). 

Fluorometry is widely used for automated immunoassay. It is approximately 1000 times more sensitive than comparable absorbance spectrophotometry, but background interference caused by fluorescence of native serum can create a major problem. This interference is minimized by careful design of the filters used for spectral isolation, by the selection of a fluorophore with an emission spectrum distinct from those of interfering compounds, or by using time- or phase-resolved fluorometry (see Chapter 3). 

Another technique recently applied for immunoassays is phase-resolved fluorometry (frequency-domain fluorometry). This technique is also based on different fluorescence decay times. The decay time can change upon antigen-antibody binding. Instead of pulsed excitation, in this technique the sample is excited with sinusoidally modulated light. With phase-resolved fluorometry, decay times and decay time differences within the range of subnanoseconds can be measured. The phase-resolved technique can be used also for elimination of background noise. This technique, however, has found only a few applications to immunoassays yet. 

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

S. E. Kakabakos, T. K. Christopoulos, and E. P. Diamandis, Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solid-phase time-resolved fhiorometry, Clin. Chem. 38, 338-342(1992). 

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