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Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

GlaxoSmithKline, Direvo, Cologne, Germany

Martin Rüdiger

GlaxoSmithKline, Third Ave., New Frontiers Science Park North, Harlow, CM19 5AW, UK. Martin.P.Ruediger@gsk.com

Stephen Ashman

GlaxoSmithKline, Harlow, UK

Sandra Turconi

GlaxoSmithKline, Devgen, Ghent, Belgium

Ryan Bingham

GlaxoSmithKline, Harlow, UK

Charlotte Wharton

GlaxoSmithKline, Harlow, UK

Jonathan Hutchinson

GlaxoSmithKline, Harlow, UK

Charlotte Carey

GlaxoSmithKline, Harlow, UK

Keith J. Moore

GlaxoSmithKline, Stevage, UK

Andrew J. Pope

GlaxoSmithKline, King Of Prussia, PA

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. (Journal of Biomolecular Screening 2003: 19-33)

Key Words: Single molecule • confocal • fluorescence • miniaturization • uHTS

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