The overall goal of this proposal is to develop a robust, sensitive and reliable label-free detector that can evaluate the real-time binding kinetics of 10,000 spot small molecule chemical microarrays against various single component or multi-component analytes. In 1991, we first reported the one-bead one-compound dcombinatorial library (OBOC) method which is highly efficient and we have successfully used it to identify ligands to a large number of biological targets. Although the OBOC library method is highly versatile, the amount of compounds contained in one single bead is small (-100 pico-mol) and is inadequate for many solution phase assays. Last year, we reported on the development of an encoded """"""""one-aggregate one-compound"""""""" library method, in which the powerful split-synthesis method is used for the construction of the library, and compounds at l-10mmol range can be efficiently prepared. Very recently, we reported on the development and application of a novel chemical microarray method, in which small organic molecules or short peptides are chemo-selectively ligated to a polymer (e.g. agarose or protein) and then printed on glass, plastic microscope slides or PVDF membranes. We envision that by combining our highly efficient """"""""one-aggregate one-compound"""""""" library method with this novel chemical microarray technique, we easily can print thousands of replicates of high density small molecule microarrays (10,000 spots/slide) and use them to probe a variety of biological analytes such as serum, cell extracts or pure proteins. Our hypothesis is that by combining our novel combinatorial chemistry and microarray platforms with a highly sensitive and reliable 2-D label-free optical detector that can efficiently measure real-time binding kinetics, we will be able to rapidly and accurately study the binding kinetics of a large number of analytes (e.g., individual protein or complex analytes such as whole serum) against a large number of immobilized small molecules, peptides, oligonucleotides or proteins.
Specific aims of this proposal include the design and construction of a prototype detector that can measure real-time binding kinetics of analytes (dissolved in the mobile phase) against 100- and 400-spot chemical microarrays. The next phase will be to develop a detector that can analyze 10,000 spot microarrays in one single run. Five 10,000 small molecule encoded bead-aggregate libraries will be prepared and printed on glass slide replicates as microarrays, and these microarrays will be analyzed by the optical detector to be developed in this proposed research.
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