Polymer brush structures functionalized with molecular beacon for point-of-care diagnostics

Abstract

Development of point-of-care (POC) diagnostic tools is an emerging area with significant potential for disease surveillance, monitoring, and diagnosis, especially for underdeveloped or developing countries. Our current research focuses on rapid, POC technologies for DNA or RNA detection that can be deployed to significantly decrease the turnaround time when encountering demands for massive quantities of tests, e.g. during a pandemic. Hairpin-like DNA or molecular beacon (MB) probes can be used as bioreceptors to specifically bind to a pathogen DNA or RNA. In the presence of complementary DNA, the immobilized MBs undergo a conformational change, and the fluorescent signal of 5’-FAM is restored from the internally quenched fluorophore. Here we studyinvestigating 3D polymer brush (PB) structures with antifouling surface properties, functionalized with a particular MB-DNA probe. Patterns of polymer brushes were created on foils of poly(ethylene-co-tetrafluoroethylene) (ETFE) activated through a metal mask using extreme ultraviolet (EUV) radiation, yielding patterns of initiators for the subsequent graft-copolymerization of vinylpyrrolidone (VP) and glycidyl methacrylate (GMA). The successful copolymerization of VP and GMA on the EUV-exposed areas was proved based on characteristic peaks of VP and GMA in ATR-IR spectra. Structure heights in the range of micrometers were achieved, which enables binding of considerably higher densities of probe molecules compared to monolayer systems. The grown polymer brush structures provide both hydrophilicity, beneficial to minimize bio-fouling, and epoxide functional groups for further functionalization. These were biotinylated and functionalized with streptavidin and 3′-biotinylated MBs, resulting in a promising platform for fluorescence-based DNA detection as demonstrated by significant fluorescence increase upon addition of target DNA down to nM concentrations. Finally, embedding of optimized MB/PB structures into a microfluidic channel and coupling to a mobile-phone-based fluorescence microscope for signal detection was demonstrated.