B TRF and HTRF

Separation or wash steps may not be acceptable for miniaturized assays. Ideal assays for miniaturized formats will be in a simple mix-and-read, homogeneous format. Recently developed FRET-based approaches combine the benefits of a highly sensitive fluorescent label and a homogeneous assay protocol under a variety of HTS experimental circumstances. One of these systems, called homogeneous time-resolved fluorescence (HTRF), involves fluorescence resonance energy transfer between a slow-decay fluor and a short-lived fluor [9,40-42].

Processes involving light emission do not occur on the same time scale. When excitation is performed in a repetitive timed fashion, fluorescence from any long-lived (or slow decay) species can then be detected selectively by delaying measurement until after such time that all short-lived species have decayed. Various lanthanide ion cryptates (particularly Eu3+, Tb3+, Sm3+, and Ru2+) are a source of long-lived fluorescence. Organic chromophores (the acceptor), when in proximity to a lanthanide donor, emit long-lived fluorescence resulting from the energy transfer from the lanthanide. Free acceptor, distant from the donor, emits only short-lived fluorescence, that is not measured in a time-resolved fashion (Fig. 6).

The HTRF principle has been demonstrated with the donor-acceptor pair europium cryptate (EuK) and allphycocyanin (APC) [9,40]. APC has an absorption band overlapping the EuK emission. APC has a typical broadband organic emission monitored at 665 nm. Because of the energy transfer process, the APC is continuously reexcited, and the apparent emission decay is delayed to match the lifetime of EuK; consequently, the APC emission can be measured after a 50 |s time delay. The emission of an unbound APC-labeled biomolecule at 665 nm can be disregarded because of its fast decay. Free EuK-labeled biomolecule, that is also present during the measurement, emits a slow-decay fluorescence at 620 nm and can be captured in a time-resolved fashion as well, and used as an internal reference. This reference intensity, as well as the bound APC emission, may vary due to interfering absorption by the serum component or turbidity. However, the ratio (intensity at 665 nm/620 nm) is unaffected and depends only on the concentration of analytes.

Typical background emission in biological samples is due to organic fluoro-phores and is short-lived. The long-lived fluorescence from the energy transfer to APC allows removal of essentially all background fluorescence by time resolution. Labeling of biological reagents with either APC or cryptate can be achieved either directly or by using labeled antibodies directed toward a nonobtrusive site in the target. The development of APC- and cryptate-labeled secondary antibod

10 usee

50-400 usee

Figure 6 Time-resolved fluorescence. This figure shows the rapid excitation of the ''background'' fluorescence and its rapid decay. It also shows the rapid excitation of the donor fluor and its long lifetime (slow decay). Readings are taken after the rapid decay of the background but before the complete decay of the sample.

10 usee

50-400 usee

Time

Figure 6 Time-resolved fluorescence. This figure shows the rapid excitation of the ''background'' fluorescence and its rapid decay. It also shows the rapid excitation of the donor fluor and its long lifetime (slow decay). Readings are taken after the rapid decay of the background but before the complete decay of the sample.

ies for HTS will not only allow a more generic approach but also further reduce the cost of this technology regardless of specific application.

So far, FRET- and HTRF-based assays have been demonstrated in 96-, 384-, and 1536-well microplates. Since conventional plate readers for fluorescence typically read one well at a time, for a miniaturized format (1536 or higher), it would take at least sixteen times longer to read, which is unacceptably slow for high/ultrahigh-throughput requirements. A more promising approach is to use fluorescence imaging technology to collect data from all the wells at once. This detection system, similar to FLIPR (fluorescence imaging plate reader) [12], should permit parallel laser pulses to each well and capture dual-wavelength fluorescence intensity imaging simultaneously.

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