Phase-modulation immunoassay

J. R. Lakowicz and B. P. Maliwal, Fluorescence lifetime energy transfer immunoassay quantified by phase-modulation fluorometry, Sensors andActuators B 12, 65-70 (1993). 

A. J. Ozinskas, H. Malak, J. Joshi, H. Szmacinski, J. Britz, R. B. Thompson, P. A. Koen, and J. R. Lakowicz, Homogeneous model immunoassay of thyroxine by phase-modulation fluorescence spectroscopy, Anal. Biochem. 213, 264-270(1993). 

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. 

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-modulation immunoassay measurements are made with sinusoidally modulated light. Since the emission is a forced response to the excitation, the emitted light has the same periodicity as the excitation. Due to the time lag between absorption and emission, the emission is delayed in comparison with the excitation. The time delay between the zero crossing of one period of the excitation and of the emission is measured as the phase angle (Figure 14.11). The emission is also demodulated, due to a decrease in the alternating current (AC) component of the AC to direct current (DC) ratio. 

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... 

Figure 14.16. Dose-response curves for a phase fluorescence immunoassay of thyroxine at modulation frequencies of 49.5 MHz ( ), 64.8 MHz (a), and 99.1 MHz (o). (From Ref. 34 with permission.)...

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. 

---