This proposal addresses the need for the development of stable, economical protein microarrays for massive parallel screening of protein interactions. Such arrays will be invaluable in determination of protein expression levels, disease diagnosis, drug discovery, and screening the millions of protein interactions generated by the completion of the human genome. The key innovation of this proposal is the use of an electrochemically grown 3-dimensional hydrous matrix to immobilize proteins and its application in a micro-patterning procedure to fabricate economical microarrays. The use of a 3-dimensional immobilization matrix is expected to reduce the denaturing that can occur in traditional arraying methods where proteins are immobilized on 2-dimensional substrates. In addition, it is anticipated that the larger surface area provided by the porous matrix will allow higher protein loadings, and therefore increased sensitivity, compared to planar substrates. In Phase I, microarrays containing immobilized antibodies will be fabricated, their stability evaluated, and their application in fluorescence based parallel immunoassays and determination of protein expression levels will be demonstrated. In Phase II, higher density protein arrays suitable for integration into automated, high-throughput microfluidic delivery systems and fluorescent analyzers will be developed.
