title: Single-molecule detection technologies in miniaturized high-throughput screening: fluorescence intensity distribution analysis.
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.
authors.affiliation: GlaxoSmithKline, Direvo, Cologne, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Haupts
authors.forename: Martin
authors.initials: M
authors.lastname: Rüdiger
authors.forename: Stephen
authors.initials: S
authors.lastname: Ashman
authors.forename: Sandra
authors.initials: S
authors.lastname: Turconi
authors.forename: Ryan
authors.initials: R
authors.lastname: Bingham
authors.forename: Charlotte
authors.initials: C
authors.lastname: Wharton
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hutchinson
authors.forename: Charlotte
authors.initials: C
authors.lastname: Carey
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Moore
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Pope
mesh_terms: D000426:Alcohol Dehydrogenase / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis; D016824:Antigens, Human Platelet / Q000032:analysis / Q000378:metabolism; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism; D005453:Fluorescence; D008024:Ligands; D018613:Microscopy, Confocal; D012313:RNA / Q000378:metabolism
chemical_list: D016824:Antigens, Human Platelet; D008024:Ligands; C083451:human platelet antigen 1b; D012313:RNA; D000426:Alcohol Dehydrogenase; D000469:Alkaline Phosphatase; D010450:Endopeptidases