This Article Full Text (PDF) All Versions of this Article: 1087057106291541v1 11/7/765    most recent References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Moger, J. Articles by Winlove, C. P. Search for Related Content PubMed PubMed Citation Articles by Moger, J. Articles by Winlove, C. P. Social Bookmarking                   What's this?

The Application of Fluorescence Lifetime Readouts in High-Throughput Screening

School of Physics, University of Exeter, Exeter, UK

Philip Gribbon

Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent, UK, GlaxoSmithKline Research and Development Limited, Medicines’ Research Centre, Stevenage, Herts, UK

Andreas Sewing

Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent, UK

C. Peter Winlove

School of Physics, University of Exeter, Exeter, UK

Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10², but the same accuracy with a 2-component decay requires a peak photon count of 10³. When a 3rd component is introduced, the minimum peak count increases to 10⁴. The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods.

Key Words: high-throughput screening • fluorescence lifetime • FIDA • fluorescence polarization

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. S. Lebakken, Hee Chol Kang, and K. W. Vogel A Fluorescence Lifetime Based Binding Assay to Characterize Kinase Inhibitors J Biomol Screen, September 1, 2007; 12(6): 828 - 841. [Abstract] [PDF]