Encapsulation of rolling circle amplification product in hydrogel systems for applications in biosensing

Abstract

The development of easily fabricated, highly stable DNA-based microarray and continuous flow concentrating devices is vital for several biomedical and environmental applications. Nucleic acid biosensors can be used for genetic analysis, disease diagnosis, drug discovery, food and water quality control and more, however methods of fabrication are tedious, and the longevity of sensors is compromised by the fragility of the sensing component. In this report, the fabrication and characterization of two biosensing modalities – microarrays and microgels – composed of Rolling Circle Amplification (RCA) product in poly(oligoethylene glycol methacrylate) (POEGMA) hydrogels are investigated. RCA product microarrays were developed by the sequential printing of aldehyde and hydrazide functionalized POEGMA precursors on nitrocellulose paper, exploiting rapid gelling via hydrazone crosslinking to generate thin film hydrogel sensing arrays. POEGMA/RCA product microgels for affinity column applications were synthesized using an inverse emulsion polymerization technique. Inkjet printing evenly deposited RCA product in all wells, with POEGMA effectively stabilizing DNA on the cellulose substrate. Hybridization of complementary probe to the encapsulated RCA product was optimized, yielding a signal to noise ratio of ~4 for a large range of probe concentrations. Microgels were successfully synthesized in the size range of 10-60 μm diameter, and a linear model that can accurately predict size based on initiator and emulsifier concentration was developed. The encapsulation efficiency of RCA product in different sized microgels was explored, with larger microgels entrapping more product and the highest encapsulation efficiency calculated at 56%. These results demonstrate that POEGMA hydrogels can be utilized to encapsulate and stabilize RCA product in two distinct structures, providing a basis for the development of easily fabricated biosensors for more specific applications.