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Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy

Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, United Kingdom

Sandra Turconi

Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, United Kingdom

Stephen Ashman

Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, United Kingdom

Martin Ruediger

Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, United Kingdom

Ulrich Haupts

Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, United Kingdom

Victoria Emerick

SmithKline Beecham Pharmaceuticals, Upper Merion, King of Prussia, PA

Andrew J. Pope

Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, United Kingdom

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of -1 fl (10-15 L) such that the signal is insensitive to miniaturization of HTS assays to 1 A1 or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.

Journal of Biomolecular Screening, Vol. 4, No. 6, 335-353 (1999)
DOI: 10.1177/108705719900400609


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