期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：4
页码：1375-1378
DOI：10.1073/pnas.96.4.1375
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Fluorescence-based assay technologies play an increasing role in high-throughput screening. They can be classified into different categories: fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. In this work we present an alternative analytical technique for high-throughput screening, which we call confocal fluorescence coincidence analysis. Confocal fluorescence coincidence analysis extracts fluorescence fluctuations that occur coincidently in two different spectral ranges from a tiny observation volume of below 1 fl. This procedure makes it possible to monitor whether an association between molecular fragments that are labeled with different fluorophores is established or broken. Therefore, it provides access to the characterization of a variety of cleavage and ligation reactions in biochemistry. Confocal fluorescence coincidence analysis is a very sensitive and ultrafast technique with readout times of 100 ms and below. This feature is demonstrated by means of a homogeneous assay for restriction endonuclease EcoRI. The presented achievements break ground for throughput rates as high as 106 samples per day with using only small amounts of sample substance and therefore constitute a solid base for screening applications in drug discovery and evolutionary biotechnology.
