Assays
Figure 1: Perception of FLT as a screening technology
N/A – know too little to be able to assess the technique 21%
Early stage technology, have big concerns over applicability and instrumentation 10%
Promising analytical technique that needs to mature 34%
Interesting but largely unproven assay technology 22%
© HTStec 2010
Excellent research and screening analytical technique 13%
What is known about FLT? Of the different approaches to FLT measurement survey respondents were most aware and had greatest understanding of time-correlated single photon counting (TCSPC), as enabled by Tecan and Edinburgh Instruments. Least awareness and understanding was shown for time-gated integra- tion, as enabled by IOM-Berthold and CyBio (Figure 2). Overall, less than 1 in 5 of all survey respondents had a high understanding of the dif- ferent approaches to FLT measurement, which is suggestive that more needs to be done to educate screeners about the principles and inherent advan- tages of FLT measurements (see box). More recent- ly a third alternative method for measuring FLT has been developed by Fluorescence Innovations, based on microchip laser excitation sources and proprietary digitisers (see below).
Figure 2: Awareness/understanding of approaches to FLT measurement
Time-gated Integration (eg IOM & CyBio) Frequency Domain Technique (eg LJL)
Time-Correlated Single Photon Counting (TCSPC) (eg Tecan & Edinburgh Instruments)
0% 20% 40% 60% 80% High
Medium © HTStec 2010 Low None 100%
What are main problems encountered in screening?
The screening problems of greatest importance to survey respondents are shown in Figure 3. False positives were ranked by far the most problematic, this was followed by compound autofluorescence, poor data quality (%CV and Z’) and then back- ground fluorescence. Photobleaching was ranked of least importance. It should be pointed out here that FLT has the potential to impact nearly all of these screening problems, which probably explains the latent appetite within the screening community.
Figure 3: Screening problems of greatest importance
Compound quenching (inner filter effect) Labelling chemistry Background fluorescence Poor data quality (%CV and Z’) Compound autofluorescence False negatives False positives
Inability to reduce fluorophore concentration Turbidity and light scattering Radioactive assay format
Variability in assay volume Inability to miniaturise Photobleaching 0% © HTStec 2010
1% 2% 2%
5% 10% 15% 20% 25% % Selecting Problem
6% 4% 3% 22% 13% 12% 10% 9% 8% 7%
What has hitherto restricted the widespread use of FLT?
Feedback on the reasons that have prevented or restricted the use of FLT technology is given in Figure 4. Cost of new instrumentation was rated the main reason, and still poses a major barrier to the adoption of any new technology, particularly in the current economic climate with constraints on capex budgets. It calls for more inventive strategies (eg free loan, lease, lease-to-buy etc) by vendors regarding instrument placement to nurture initial FLT adoption. The next most important restriction was insufficient in-house expertise in FLT assay development. Again this calls for new efforts by vendors and early adopters to encourage new investigations. The next four reasons were all sim- ilarly rated (ie lack of relevant applications; lack of suitable dyes (eg covering wide range of fluorescent lifetimes); lack of publications validating tech- nique; and lack of standard toolbox reagents and dye derivatives) all have been subject to much improvement over the past few years (see the updates below).
72 Drug Discovery World Summer 2010
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