Molecular interaction analysis using a microscopic platform. (A) Schematic drawing of the imaging strategy for detection of the molecular interactions. Proteins, nucleotides, or other biomolecules are immobilized onto the surface of microbeads, and the microbeads are arrayed on a glass coverslip. A solution containing fluorescently labeled target molecules is injected into the microbead array and the fluorescence, which binds to probe molecules on the surface of microbeads, is observed by objective lens-type total internal reflection fluorescence microscopy. (B) The chemical structure of sulfo-SANPAH (sulfosuccinimidyl 6-[4′-azido-2′-nitrophenylamino] hexanate). Bifunctional cross-linking reagent, sulfo-SANPAH, has NHS ester and nitrophenyl azide, which react with amino groups and is activated by the light irradiation, respectively. (C) Schematic diagram of microbead array fabrication. The amino group coated coverslip is conjugated with sulfo-SANPAH by its NHS ester. A microbead is captured by using an optical trap and carried to the fixing position. In the final step, the area where the microbead was carried is activated by light irradiation (300–360 nm) and the microbead is fixed at this position. (D) Microscopic image of a microbead array. The 100 microbeads (10 × 10 pattern) arrayed on a coverslip are shown. The mean diameter of microbeads was 0.95 μm. Scale bar, 20 μm.
