On-chip Nanoplasmonic RT-RPA and CRISPR/Cas12a Assay for Point-of-care Molecular Diagnostics

Rapid and accurate nucleic acid detection at point-of-care (POC) is essential for advancing effective disease diagnosis and management. Here, we report a handheld nanoplasmonic all-in-one setup for on-chip recombinase polymerase amplification (RPA) and real-time fluorescence detection by CRISPR/Cas12a reaction. The all-in-one setup consists of AuNIs-based nanoplasmonic cavity (AuNIs-NC), a disposable plastic-on-polymer (PoP) cartridge, and fluorescence microlens array (FMLA) camera. The AuNIs-NC allows uniform and efficient photothermal heating under white LED illumination due to strong broadband light absorption and internal reflection by randomly distributed AuNIs and thin Al film. This setup allows the RPA and CRISPR/Cas 12a reactions in a single chamber of PoP cartridge, with fluorescence signals monitored by a FMLA camera. The experimental result demonstrates rapid SARS-CoV-2 E gene plasmid DNA detection within 20 min, achieving a detection sensitivity of 10 copies/ul. Testing with 16 clinical samples shows a linear trend with RT-qPCR, indicating the platform's reliable sensitivity and specificity. This compact platform offers affordable and reliable molecular diagnosis, facilitating rapid and scalable POC testing for a range of infectious diseases.Clinical Relevance- This on-chip real-time RT-RPA and CRISPR/Cas12a assay provides rapid and precise molecular diagnostics at POC using fully integrated plasmonic system.