The conformation of a three-dimensional polymeric coating (copoly(DMA-NAS-MAPS)) and immobilization and hybridization of DNA strands on the polymer coated surface are investigated. A conformational change, specifically the swelling of the surface adsorbed polymer upon hydration, is quantified in conjunction with the application of this polymer coating for DNA microarray applications. Fluorescently labeled short DNA strands (23mers) covalently linked to the functional groups on the adsorbed polymer are used as probes to measure the swelling of the polymer. A fluorescence microscopy technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), is utilized to directly measure the change in axial position of fluorophores due to swelling with subnanometer accuracy. Additionally, immobilization characteristics of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) probes, as well as hybridization of ssDNA with target strands have been studied. The results show that ssDNA further away from the surface is hybridized more efficiently, which strengthens the earlier analysis of this polymeric coating as a simple but highly efficient and robust DNA microarray surface chemistry.