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