Nucleic acid-based recognition events in hydrogel array systems

Abstract

Nucleic acids play an important role in the construction of novel biosensing units. Integrating nucleicacid- diagnostic tools with polymeric assemblies can enhance selectivity and sensitivity and protect them from nuclease digestion. In this thesis, DNA-based sensing molecules are incorporated into hydrogel films based on poly(oligoethylene glycol methacrylate) (POEGMA) to fabricate printable DNA hybridization and DNA aptamer microarrays to detect small molecules. Firstly, a printable hydrogel microarray incorporating long-chain-sensing DNA generated via a rolling circle amplification reaction was developed to exploit the benefits of isothermal nucleic acid amplification technology and the non-covalent immobilization potential of POEGMA-hydrogel films on a nitrocellulose substrate. Fluorophore-labelled nucleic acid analytes of varying sizes were spotted on hydrogel-trapped RCA sensing units and assessed on their ability to detect specific DNA hybridization events. Compared to hydrogel-free sensors, the developed sensors provided greater detection potential for small oligonucleotides while also enabling uniform spot-to-spot fluorescence signal distribution from the sensing analytes. The hydrogel’s ability to increase base-pairing affinity in short DNA duplexes was further investigated using fluorescence resonance energy transfer (FRET)-based structure-switching aptamers that bind adenosine triphosphate (ATP). Moderate thermal stabilization was observed in the hydrogel matrices embedding FRET-based structure-switching aptamer constructs (SSAC) assembled with quencher stems having lengths of ≤ 10-bp. The combination of a water-swollen/flexible matrix and the thermal stabilization capabilities of the POEGMA polymeric matrix endowed the hydrogel-immobilized aptamer reporters with greater sensitivity compared to hydrogel-free systems. The developed hydrogel-based aptamer sensor was then translated into a printable nitrocellulosesupported microarray format, with additional modulation of the SSAC configuration. DNA microarrays with even greater sensitivity were produced by rationally positioning and further shortening the quencher-stems to 7-bp using the optimized assay conditions. In addition to effective immobilization and ligand-binding affinity comparable to the native aptamer, the hydrogel interface provided protection against nucleases and supported applications using real biological samples (i.e., in human serum).