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25d

Parallel Peptide Synthesis on Microfluidic Microarrays for Epitope Mapping and Cell Adhesion Assays

Suparna Mandal and Erdogan Gulari. Chemical Engineering, University of Michigan, Ann Arbor, 2300 Hayward, 3074 H.H. Dow, Ann Arbor, MI 48109

Short peptides with greater stability than proteins are popular probes for studying receptor-ligand interactions on microarrays. Spotted peptide microarrays have been used before for high throughput analysis of biomolecule interactions in form of epitope and kinase activity mapping but the technology is limited by difficult synthesis and purification methods. In situ synthesis eliminates the need for individual synthesis and purification of a large number of peptides, thereby enabling routine synthesis of bioactive peptides.

This work demonstrates the synthesis and application of in-situ synthetic peptide microarrays for a high-throughput parallel study of peptide-antibody interactions at amino acid level. A combination of solid phase peptide synthesis, photogenerated reagent chemistry and fluorescence imaging techniques was used to generate spatially addressable peptide microarrays with up to 12mer sequences. The synthesis substrate was a microfluidic microarray of individual reactors connected by channels, converging into a solution inlet and outlet, etched on a flat silicon surface sealed by a glass cover. A constant synthesis efficiency of >98% was observed for sequences of up to 9 amino acids. Peptide sequences from Respiratory Synctial Virus (RSV) were synthesized and used to identify binding sites to the antibody MAb 19 by performing on-chip, high throughput ELISA. Key residues for binding were identified by mutational and deletional analysis on these sequences. To further elucidate the biological applications of the synthetic peptides, the on-chip binding specificity of a 8mer peptide to a murine B lymphoma cell line was demonstrated.