Spot arrays on modified glass surfaces for efficient SPOT synthesis and on-chip bioassay of peptides

To make SPOT synthesis of peptides and their assays on glass surfaces more convenient, a simple method for making spot arrays on a slide glass was designed through patterning with a photoresist and perfluorination followed by amination with various silane compounds and polymers. With these spot-arrayed glass surfaces, we could measure the coupling completion of each Fmoc amino acid on the glass surface by direct fluorescence analysis after fluorescence-labeling the amino groups on the surface of each spot. Then we synthesized several types of decapeptides and HPQ-pentapeptides on the spot-arrayed glasses and identified the optimal surface condition for stepwise peptide coupling and on-chip bioassay. After optimizing the surface conditions, we synthesized a model library of biotin-Gly-Ala-P(1)-Gly (P(1): one of 19 amino acids) and successfully replicated the well-known alpha-chymotrypsin subsite specificities through Cy5-streptavidin binding to the remaining biotin on the surface after the enzymatic digestion.