Surface Functionalization by Tosylation for Digital Detection of Nucleic Acids with Photonic Resonator Absorption Microscopy and Target Recycling.

Biosensors for nucleic acid sequences frequently rely on surface functionalization of sensing materials with DNA capture sequences. For ultrasensitive sensing, this DNA surface layer must be stable, high-density, and uniform. Here, we present a novel method for attaching amine-terminated capture sequences directly to titanium dioxide surfaces using a tosylate-mediated reaction. This new tosylate method was to previous DNA functionalization methods that rely on silanes like APTES or GPTMS, with characterization through atomic force microscopy, surface wettability, and scanning electron microscopy. This process was demonstrated by attaching DNA to one-dimensional photonic crystal grating structures, then using photonic resonator absorption microscopy (PRAM) to digitally detect nanoparticles with a complementary probe sequence. We compared this novel method with APTES and GPTMS to examine DNA surface stability and density. Finally, we used the tosylate method for the digital detection of microRNA using PRAM with a target recycling amplification process (TRAP) assay, which uses toehold-mediated strand displacement reactions to create room temperature linear amplification without requiring thermocycling or enzymatic amplification. New TRAP sequences were designed and validated for miR-148-3p, a biomarker of certain cancers. Using these methods, we achieved attomolar detection levels in under thirty minutes at room temperature, without requiring PEG linkers or silanes, which can be unstable or expensive. These surface functionalization methods could be more broadly applied to other titanium dioxide-based biosensors.