A single-molecule biosensor based on multiple fluorophore nucleic acid probe and STORM imaging for detection of Hantaan virus.

Hemorrhagic fever with renal syndrome (HFRS), caused by Hantaan virus, poses a serious public health threat. Current diagnostic methods remain limited by low sensitivity, complex procedures, and high sample requirements. To address this, we developed a highly sensitive single-molecule biosensor using multi-fluorophore nucleic acid probes and STORM imaging for the detection of Hantaan virus RNA. The probe was synthesized via PCR incorporating EdUTP, enabling site-specific coupling of multiple Cy5 fluorophores through copper-catalyzed click chemistry. This multi-fluorophore probe, combined with magnetic beads and a capture sequence, specifically targeted viral RNA and enabled quantification by super-resolution imaging. Compared to conventional single-fluorophore probes, our system exhibited a significantly lower detection limit of 57.54 aM. Notably, this is the first application of STORM to a single-molecule viral RNA detection platform. The method offers a broadly applicable, ultrasensitive strategy for early clinical diagnostics of Hantaan virus and potentially other pathogens.