Single-molecule detection technologies in miniaturized high-throughput screening: fluorescence intensity distribution analysis |
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Authors: | Haupts Ulrich Rüdiger Martin Ashman Stephen Turconi Sandra Bingham Ryan Wharton Charlotte Hutchinson Jonathan Carey Charlotte Moore Keith J Pope Andrew J |
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Institution: | GlaxoSmithKline, Direvo, Cologne, Germany. |
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Abstract: | Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence. |
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