Abstract
Drug discovery begins by identifying protein-small molecule binding pairs. Afterwards, binding kinetics and biofunctional assays are performed, to reduce candidates for further development. High-throughput screening, typically employing fluorescence, is widely used to find protein ligands in small-molecule libraries, but is rarely used for binding kinetics measurement because: (1) attaching fluorophores to proteins can alter kinetics and (2) most label-free technologies for kinetics measurement are inherently low-throughput and consume expensive sensing surfaces. We addressed this need with polarization-modulated ellipsometric scanning microscopes, called oblique-incidence reflectivity difference (OI-RD). Label-free ligand screening and kinetics measurement are performed simultaneously on small-molecule microarrays printed on relatively inexpensive isocyanate-functionalized glass slides. As a microarray is reacted, an OI-RD microscope tracks the change in surface-bound macromolecule density in real-time at every spot. We report progress applying OI-RD to screen purified proteins and virus particles against a 51,200-compound library from the National Cancer Institute. Four microarrays, each containing 12,800 library compounds, are installed in four flow cells in an automated OI-RD microscope. The slides are reacted serially, each giving 12,800 binding curves with ∼30 sec time resolution. The entire library is kinetically screened against a single probe in ∼14 hours and multiple probes can be reacted sequentially under automation. Real-time binding detection identifies both high-affinity and low-affinity (transient binding) interactions; fluorescence endpoint images miss the latter. OI-RD and microarrays together is a powerful high-throughput tool for early stage drug discovery and development. The platform also has great potential for downstream steps such as in vitro inhibition assays.
Original language | English (US) |
---|---|
Title of host publication | Progress in Biomedical Optics and Imaging - Proceedings of SPIE |
Volume | 8587 |
DOIs | |
State | Published - 2013 |
Event | Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI - San Francisco, CA, United States Duration: Feb 2 2013 → Feb 5 2013 |
Other
Other | Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI |
---|---|
Country | United States |
City | San Francisco, CA |
Period | 2/2/13 → 2/5/13 |
Keywords
- biomolecular interaction analysis
- chemical microarrays
- drug screening
- ellipsometry
- high-throughput
- label-free biosensing
- oblique incidence reflectivity difference (OI-RD)
- small-molecule microarrays
ASJC Scopus subject areas
- Atomic and Molecular Physics, and Optics
- Electronic, Optical and Magnetic Materials
- Biomaterials
- Radiology Nuclear Medicine and imaging