Extended strand-mediated target unmasking synergized with lyophilized hairpin probes for one-step viral RNA detection

This method pioneers a synergistic strategy combining ‘extended-strand unmasking with freeze-drying pre-packaging’. By designing unequal-length double-strand replacement strands that competitively bind to regions flanking the target site, it forces the unfolding of hairpin/cloverleaf conformations, thereby physically exposing concealed targets. Concurrently, a five-step lyophilization process (pre-folding-anchoring-sealing-encapsulation-freezing) employs graphene oxide (GO) π-π stacking to suppress >90 % of DNA breathing effects. Combining 0.02 % (w/v) low-melting-point agarose achieves in situ encapsulation and probe conformation fixation, forming microchannels for water molecules. The final lyophilized product forms a porous solid matrix (integrable into nitrocellulose test strips or 2-mm diameter lyophilized spheres), enabling stable probe storage at ambient temperature. This technology overcomes the ‘impossible triangle’ challenge in molecular diagnostics: achieving high room-temperature stability (>6 months, compared to <7 days for traditional liquid-phase CHA), ultra-high sensitivity (0.01 pM), and a simple, rapid testing process. This solution provides a “sample-in, result-out” point-of-care testing approach for home health management (no cold chain required), primary care clinics (no equipment dependency), and pandemic emergency response.