Time-Resolved Fluorescence TRF

An HTS assay typically requires the detection of minute amounts of a probe at nanomolar or lower concentrations in the presence of a 10,000-fold or higher excess of other reagents chemical test compounds from the screening Hbrary at a level of 10-100 pm large amounts of proteins and lipids from cells or crude membranes containing only fractions of the target and a sample compartment (micro-titer plate), which is a disposable made of plastic and generates a totally different situation to a quartz cuvette in a spectroscopic set-up. 

Optical assay methods, including the scintillation readout for radioactive assays, suffer from interference through absorbance by colored test compounds, which give rise to a so-called inner filter effect or color quenching . Additional sources of artefacts in fluorescence assays are auto-fluorescence of compounds, plates, and reagents, as well as scattering of excitation Hght. 

Typical labels used for HTS assays are chelates and cryptates based on lantha-rtide ions such as europium (Eu ) [113-120] and terbium (Tb ) [119, 121, 122]. These ions show long excited-state hfetimes of several 100-1000 ps, which aUow for time delays of 100 ps and the use of a flash lamp as excitation source. The lantharride-based labels are commercially available from CisBio International (Eu cryptates), Perkin Ehner life Sciences (Eu chelates), Amersham (Eu chelates), and Panvera (Tb chelates). Pig. 15 shows examples of a cryptate from CisBio International and a chelate from Perkin Ehner Life Sciences. 

The homogeneous TRF assay formats HTRF ( homogeneous time-resolved fluorescence ) by GisBio International [115, 120, 136, 137] and LANGE ( lantha- 

Note As for TRF, the term fluorescence is in fact misused in TR-FRET. In the spectroscopic Hteratirre, resonance energy transfer from a long-Hfetime probe such as a lanthanide chelate is termed LRET (luminescence RET) [121]. 

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In time-resolved fluorescence (TRF) (Maundrell et al., 1985), europium chelates are excited at 340 nm to emit two types of fluorescence, a shortlived background fluorescence (< 0.1 ms) and a fluorescence due to emitted photons of Eu " lasting up to over 1 ms. This difference in fluorescence decay rate can be exploited by measuring fluorescence only after background fluorescence has completely decayed to obtain a very high signal to noise ratio (detectability down to 10 5 M) as shown in Fig. 7.5. Originally, anti-hapten antibody was labeled with Eu " but in more recent procedures Eu " is directly attached to the nucleic acid (Sections 7.3.2.1 and 7.8.1). 

In another study, 42,000 compounds were screened in three different versions of a farnesoid X receptor (FXR) antagonism assay. AlphaScreen, time resolved fluorescence (TRF), and time resolved FRET (TR-FRET) were used. These identified 104, 23, and 57 actives, respectively. Only 18 compounds were identified by all three methods. Further cellular testing showed that only about 30% of them were functionally active, with AlphaScreen identifying more of the functionally active compounds than the other two methods. This group also found that screening mixtures rather than discrete compounds was more likely to result in both false positives and false negatives. ... 

Scientists at Xen Biosdences and Cambridge Consultants are jointly developing a compact, modem-size device that deploys time-resolved fluorescence (TRF) spectroscopy technology. This device can be used to perform tests for up to 20 different ailments, enabling the physician to make rapid decisions with minimum cost and complexity. According to the device designer, the power requirements will be less than 100 milliwatts, which can be satisfied by low-power Li-ion batteries... 

In Time Resolved Fluorescence (TRF) experiments, depicted schematically in Figure 12, the emission spectrum line shape changes from a peak value of AE(0) to the final AE(oo) peak of the equilibrium emission spectrum as the solvent responds to the new solute electronic state." " " Experimental results in bulk liquids show that the nonequilibrium correlation functions initially exhibit a very fast (less than 50 fs" ) inertial component, which may account for 60-80% of the total relaxation in water. This is followed by a multiexponential relaxation on the subpicosecond to picosecond timescale," corresponding to reorientation and translation of solvent molecules, or, to particular intramolecular solvent modes" " around the solute. Slower dynamics are found in more viscous liquids." ... 

Zeiss have developed a high-throughput reader system (the Plate Vision multimode reader) for microwell plates that is capable of fast time-resolved fluorescence (Fast-TRF) imaging with nanosecond resolution [ 189] (Fig. 28), which has found applications in pharmaceutical screening. This microplate imager is suitable for time-resolved RET assays. Kinase assays, for instance, can be performed with antibodies labeled with a Ru(batho)2bipy complex or... 

FI = fluorescence intensity FP = fluorescent polarization TRF = time-resolved, fluorescence Lum = luminance Abs = absorbance. 

DELFIA or time-resolved fluorometty (TRF) is a well-established technology that exploits the unique fluorescence properties of lanthanide chelates and provides a powerful alternative to radioisotopic assays in many high throughput-screening applications. 

Fig. 3 The outline of fast dynamic processes that proceed at rates comparable with that of the radiative depletion of the excited state and can be studied by time-resolved fluorescence techniques (TRF) the rate of the polymer chain dynamics (vibrational motion and relaxation) strongly overlaps that of electronic relaxation and can be studied by TRF. Adapted from Springer, Self Organized Nanostructures of Amphiphilic Block Copolymers I, 241, 2011, 187-249, figure 3, [2], Copyright 2011. With kind permission from Springer Science and Business Media...

TA transient absorbance TRIR time-resolved infrared resonance TRFS time-resolved fluorescence spectroscopy. 

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