Parallel peptide synthesis on microfluidic microarrays for identification of protein and cell binding ligands.

Abstract

Advances in combinatorial chemistry have enabled the application of small molecule microarrays for study of limitless receptor-ligand interactions. Short synthetic peptides have emerged as popular probes for microarrays due to their stability, diverse range of biomolecular interactions and sequence specific bioactivity. Consequently, there is an increasing potential for spatially addressable peptide libraries, with high synthesis efficiencies and which are routinely producible at low cost and less time. In addition, the potential of peptide microarray applications has not yet been realized; with a relatively unexplored application of identification of synthetic peptides that target tumor cells. In this work, we present the development of an integrated parallel peptide synthesis system using solid phase peptide synthesis and photogenerated acid chemistry. Next we demonstrate the application of arrays synthesized by this system to the identification of cell and protein adhesive ligands. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to non-specific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed, using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells and key residues for binding were identified by deletional analysis. In conclusion, a combination of PEG-based surface passivation techniques and spatially addressable solid phase peptide synthesis was used to develop a highly specific cell-peptide adhesion assay on a microfluidic platform.