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. (Journal of Biomolecular Screening 2003:19-33)

[1]  A. Pope,et al.  Ligand Binding to Transmembrane Receptors on Intact Cells or Membrane Vesicles Measured in a Homogeneous 1-Microliter Assay Format , 2001, Journal of biomolecular screening.

[2]  W. Webb,et al.  Fluorescence correlation spectroscopy: diagnostics for sparse molecules. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Pope,et al.  Simple Absorbance-Based Assays for Ultra-High Throughput Screening , 2001, Journal of biomolecular screening.

[4]  P E Stanley,et al.  Chemiluminescent and bioluminescent reporter gene assays. , 1994, Analytical biochemistry.

[5]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[6]  Christian Eggeling,et al.  Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy. , 2002, Biophysical journal.

[7]  Ramm,et al.  Imaging systems in assay screening. , 1999, Drug discovery today.

[8]  M. Eigen,et al.  Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[10]  D. E. Wolf,et al.  Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  E Gratton,et al.  The photon counting histogram in fluorescence fluctuation spectroscopy. , 1999, Biophysical journal.

[12]  U. Haupts,et al.  Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein-Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy , 2001, Journal of biomolecular screening.

[13]  J. Jungmann,et al.  Two-dimensional fluorescence intensity distribution analysis: theory and applications. , 2000, Biophysical journal.

[14]  U. Haupts,et al.  Macroscopic versus microscopic fluorescence techniques in (ultra)-high-throughput screening , 2000 .

[15]  M. Eigen,et al.  Sorting single molecules: application to diagnostics and evolutionary biotechnology. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[16]  W. Webb,et al.  Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy. , 2000, Journal of cell science.

[17]  M. Auer,et al.  Enzyme inhibition assays using fluorescence correlation spectroscopy: a new algorithm for the derivation of kcat/KM and Ki values at substrate concentrations much lower than the Michaelis constant. , 2000, Biochemistry.

[18]  R. Rigler,et al.  Fluorescence correlation spectroscopy , 2001 .

[19]  D. Ullmann,et al.  Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  U. Haupts,et al.  Developments in fluorescence lifetime-based analysis for ultra-HTS , 2001 .

[21]  E. Lopez‐Calle,et al.  A Novel and Robust Homogeneous Fluorescence-Based Assay Using Nanoparticles for Pharmaceutical Screening and Diagnostics , 2000, Journal of biomolecular screening.

[22]  Stephen Ashman,et al.  Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy , 1999, Journal of biomolecular screening.

[23]  T. Pederson.  Movement and localization of RNA in the cell nucleus , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[24]  M. Sauer,et al.  Probes for detection of specific DNA sequences at the single-molecule level. , 2000, Analytical chemistry.

[25]  Christian Eggeling,et al.  Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy. , 1998 .

[26]  Ü. Mets,et al.  Fluorescence intensity multiple distributions analysis: concurrent determination of diffusion times and molecular brightness. , 2000, Biophysical journal.

[27]  R. Stocco,et al.  A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture. , 2000, Analytical biochemistry.

[28]  E. Gratton,et al.  Fluorescence fluctuation spectroscopy. , 1999, Methods.

[29]  E. Gratton,et al.  Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy. , 2000, Biophysical journal.

[30]  H. Tanke,et al.  Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy. , 1996, Biophysical journal.

[31]  M. Lohse,et al.  Receptor-Ligand Interactions Studied with Homogeneous Fluorescence-Based Assays Suitable for Miniaturized Screening , 2001, Journal of biomolecular screening.

[32]  R. Rigler,et al.  Resolution of fluorescence correlation measurements. , 1999, Biophysical journal.

[33]  N. Thompson,et al.  Fluorescence Correlation Spectroscopy , 2002 .

[34]  E Gratton,et al.  Resolving heterogeneity on the single molecular level with the photon-counting histogram. , 2000, Biophysical journal.

[35]  U. Haupts,et al.  Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice. , 1999, Drug discovery today.

[36]  D. Schild,et al.  Fluorescence correlation spectroscopy in small cytosolic compartments depends critically on the diffusion model used. , 2000, Biophysical journal.

[37]  W. Webb,et al.  Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. , 1999, Biophysical journal.

[38]  R. Rigler,et al.  Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion , 1993, European Biophysics Journal.