We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.
Small-molecule microarrays composed of tens of thousands of distinct synthetic molecules, natural products, and their
combinations/modifications provide a high-throughput platform for studying protein-ligand interactions. Immobilization
of small molecule compounds on solid supports remains a challenge as widely varied small molecules generally lack
unique chemical groups that readily react with singly or even multiply functionalized solid support. We explored two
strategies for immobilizing small molecule compounds on
epoxy-functionalized glass surface using primary-aminecontaining
macromolecular scaffolds: bovine serum albumin (BSA) and
amine-modified poly-vinyl alcohol (PVA).
Small molecules with N-hydroxysuccinimide (NHS) groups were conjugated to BSA or amine-modified PVA.
Small-molecule-BSA conjugates and small-molecule-PVA conjugates were subsequently immobilized on epoxy-functionalized
glass slides through amine-epoxy reactions. Using an
oblique-incidence reflectivity difference (OI-RD) scanning
microscope as a label-free detector, we performed a comparative study of the effectiveness of BSA and PVA as
macromolecular scaffolds for anchoring small molecule compounds in terms of conjugation efficiency, surface
immobilization efficiency, effect of the scaffold on end-point and kinetics of subsequent binding reactions with protein
probes.
We describe a new oblique-incidence reflectivity difference (OI-RD) scanning microscope for high-throughput
screening, in microarray format on functionalized glass slides, small-molecule compound libraries for protein ligands.
The microscope employs a combination of scan mirror for y-scan and single-axis translation stage for x-scan. For a
printed microarray with over 10,000 features, each of 100 μm in diameter and distinct small molecule targets, we can
acquire an end-point image of the microarray in 90 minutes with pixel resolution of 20 μm × 20 μm. The microscope is
also capable of measuring binding kinetics of over 10,000
protein-ligand reactions simultaneously. We also describe a
number of strategies for immobilizing small molecule compounds on functionalized glass slides: (1) conjugating the
compounds (through a chemically inert linker) with a lysine residue so that the primary amine on the lysine serves as the
anchor to epoxy-functionalized glass surface; (2) conjugating the compounds (through a linker) with a biotin residue so
that the biotin serves as the anchor to streptavidin-functionalized glass surface; (3) immobilizing small molecule
compounds without modification on isocyanate-functionalized glass surface through non-specific reaction of
nucleophilic molecular motifs on most bioactive compounds with isocyanate groups. We present preliminary
measurements of protein-small molecule binding reactions using the new microscope and the surface immobilization
strategies.
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