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Fluorescence lifetime immunoassay

Although not strictly a homogeneous method, fluorescence lifetime immunoassay is a useful technique with particular applications in the clinical [Pg.161]

The fluorescence associated with the proteins of blood serum typically has a decay time of 10 ns. Accordingly, this emission may be allowed to decay prior [Pg.162]

A commercial immunoassay kit (the Fluoromatic System, Biorad, Richmond, California, USA) also utilizes a method with potential applications in immunoassay development using sensors. In this assay polyacrylamide microbeads having a refractive index equal to the assay medium, making them transparent in the assay medium, are used. The method uses a dedicated automatic fluorimeter with photon counting techniques for increased sensitivity (8). [Pg.163]


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... [Pg.477]

Guo XQ, Castellano FN, Li L, Lakowicz JR (1998) Use of a long lifetime Re(I) complex in fluorescence polarization immunoassays of high-molecular weight analytes. Anal Chem 70 632-637... [Pg.23]

Fluorescence lifetime-based applications require probes and labels with environment-sensitive lifetimes, while immunoassays or hybridization-based analysis require fluorescent tracers preferably labeled with a single, mono-reactive fluorescent label. [Pg.66]

J. R. Lakowicz and B. P. Maliwal, Fluorescence lifetime energy transfer immunoassay quantified by phase-modulation fluorometry, Sensors andActuators B 12, 65-70 (1993). [Pg.19]

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. [Pg.452]

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. [Pg.473]

Figure 14.14. Competitive Uuorescence lifetime immunoassay dose-response curve ( ), in which the analyte, human IgG, competes with eosin-human IgG for fluorescein-anti-human IgG binding sites. Fluorescence lifetime changes of fluorescein-human IgG donor in the presence of increasing amounts of eosin-human IgG acceptor are also shown (o). (From Ref. 107 with permission.)... Figure 14.14. Competitive Uuorescence lifetime immunoassay dose-response curve ( ), in which the analyte, human IgG, competes with eosin-human IgG for fluorescein-anti-human IgG binding sites. Fluorescence lifetime changes of fluorescein-human IgG donor in the presence of increasing amounts of eosin-human IgG acceptor are also shown (o). (From Ref. 107 with permission.)...
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. [Pg.285]

There have been several new developments in fluorescence polarization immunoassay (FPIA). Lackowicz and Terpetschnig reviewed the use of long-lifetime metal-ligand complexes in fluorescence polarization assays [154, 155]. New complexes with Re(l) and Ru(II) were described for the highly sensitive detection of high-molecular weight analytes by FPIA. Laser-induced fluorescence polarisation detection has been used by Yatscoff and coworkers in capillary electrophoresis detection (CE-LIFP) [156]. For the analyte cyclosporin picomolar detection limits were attained. [Pg.653]


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