Oligonucleotide surface attachment by tosylation for digital detection of microRNA using photonic resonator absorption microscopy

DNA capture probes attached to surfaces are necessary for many biosensing assays for the specific detection of nucleic acid target sequences. In this work, we present a chemically mediated direct oligonucleotide-titanium dioxide bond for stable DNA surface immobilization on photonic crystal (PC) biosensors. We validated the DNA-TiO₂ functionalization using photonic resonator absorption microscopy (PRAM) to digitally detect hybridization-bound nanoparticles, then compared the tosylate-mediated chemical binding method to commonly used silanes (3-aminopropyl)triethoxysilane and (3-glycidyloxypropyl)trimethoxysilane. This surface functionalization process was then applied to the digital detection of nanoparticles for the ultrasensitive detection of microRNA (miRNA) sequences on PRAM. By immobilizing a single-stranded capture DNA sequence onto a titanium dioxide PC surface through a reaction with <i>p</i>-toluenesulfonic anhydride, we demonstrate the target recycling amplification process for digital detection of miRNA using PRAM through toehold-mediated strand displacement reactions with linear signal amplification. Using this method of DNA surface functionalization, we achieved attomolar levels of detection of target miRNA-148a-3p, a potential biomarker for certain cancers, without requiring silanes or polyethylene glycol linkers, which can be unstable or expensive. While demonstrated here using PCs for the detection of miRNA, this functionalization approach could be broadly applied to any biosensor with a titanium dioxide surface.