Engineered microarrayed surfaces for the detection of biomolecules

Abstract

DNA microarrays have become an increasingly important tool for genomic investigations. This work is directed toward establishing methods and surface architectures that allow the monitoring of DNA hybridization and dehybridization at microarrayed surfaces in real time. For this purpose, a method for generating a surface architecture that allows end-immobilization of DNA probes with high sequence fidelity is provided. This method combines the advantages of solid-phase oligonucleotide synthesis chemistry and conventional microarray spotting for generating end-immobilized oligonucleotide structures at surfaces. Total internal reflection fluorescence (TIRF) imaging provided in situ measurements of the hybridization kinetics between target DNA molecules and surface oligonucleotides on a homogeneous surface. A transport limited reaction model that included the effects of diffusion and solution depletion yielded estimates of surface hybridization rate constants, equilibrium constants, and the surface probe densities. Non-equilibrium desorption melting curves for DNA probe-target duplexes on both homogeneous and patterned surfaces were obtained in situ by TIRF. Redhead theory was used to simulate the melting curves. Single base pair mismatch was discerned from the melting curves.

Imaging mass spectrometry (IMS) has emerged as a powerful technology for examining the relative abundance and spatial localization of biomolecules in a thin tissue section. Current methods for IMS are time-consuming and require expensive equipment for sample preparation. This work employs pre-coated surfaces as a way to address these issues. Such surfaces were patterned by microcontact printing to form 100~200 µm hydrophilic spots surrounded by a continuous hydrophobic surface. Matrix crystals were regioselectively deposited onto the hydrophilic areas, forming a matrix crystal microarray. Tissue samples were placed directly onto the patterned matrix surfaces and then analyzed by IMS. This approach decreases sample preparation time from a few hours to tens of minutes, avoids the need for expensive spotters, and allows high-throughput imaging mass spectrometry at high resolution for tissue sample analysis.