International Carl Zeiss Workshop on International Carl Zeiss ...

<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on Fluorescence Correlation Spectroscopy and Related Methods
March, 30 to March 31, Jena / Germany
16 ABSTRACTS
Single Molecule Detection in ultra-High Throughput Screening
Steve Ashman
Molecular Interactions & New Assay Technologies, SmithKline Beecham, New Frontiers Science Park (North),
Third Avenue, Harlow, Essex CM19 5AW, UK
Homogeneous fluorescence techniques are now established as the most important
readout strategy for miniaturised HTS in the future 1 . However, this encompasses a wide
range of different approaches, each with particular advantages, limitations and
applications. In this talk, we discuss a mixture of theoretical and practical aspects aimed
at identifying the techniques of choice for robust miniaturised (e.g. 1536-well of greater
densities) screening of different classes of drug targets. Where possible the single
molecule detection techniques explored are compared to a macroscopic (ensemble)
fluorescent equivalent. The latter monitor the time and assay volume weighted-average
ensemble signal from the well and include most of those techniques currently used in
HTS 1 (fluorescence intensity, prompt/time-resolved energy transfer, anisotropy).
3,4
Single molecule detection techniques (e.g FCS ) monitor the optical/biophysical
properties of individual molecules within the assay well in a stochastic fashion by using a
tightly focussed (typically 10 -15
L) volume element. Traditionally, FCS has been used to
analyse binding reactions via differences in the diffusion rates of fluorescently-labelled
analytes undergoing binding reactions 4 . However, a number of recent developments
have opened the way to considerably broader applications including the discrimination of
molecular brightnesses ("FIDA", ref. 5, 6), distance-independent formation of labelled
complexes 7,8
(analogous to energy-transfer techniques) and a range of 2-dimensional
techniques in which a combination of biophysical properties are analysed simultaneously
(e.g. brightness and diffusion rates ("FIMDA") or anisotropy).
Cont.
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