A nanotechnology-enhanced workflow for direct, sensitive detection of viral RNA in exhaled breath

Ultrasensitive detection of airborne viral RNA is achieved at 10 PFU L −1 nominal input under controlled aerosol conditions. • Surface spikes provide morphology-dependent protection of captured RNA from RNase degradation. • Nanotechnology-Enhanced Workflow combined with RT-qPCR enables pathogen detection in clinical exhaled breath samples. • Integration with LAMP-CRISPR/Cas12b enables rapid detection of airborne viruses under controlled conditions. The detection of airborne RNA viruses is critically important for public health but remains technically challenging due to the extremely low ambient concentrations. Here, we present a highly sensitive and integrated detection workflow based on engineered spiky silica nanoparticle variants (SSNPRs). Engineered with tunable spike lengths, the SSNPRs concentrate trace viral RNA and protect it from ambient RNase degradation via a morphology-dependent RNA protection mechanism. Combined with an optimized lysis and elution protocol, this method enhances detection sensitivity by 100-fold compared to commercial RNA extraction kits and enables detection at a nominal airborne input equivalent to 10 PFU L −1 under controlled aerosol conditions. We successfully applied this system to detect SARS-CoV-2 and influenza A viruses in the exhaled breath of febrile patients, suggesting that respiratory viral shedding may not always be synchronized with clinical symptoms. Furthermore, by integrating the workflow with a LAMP-CRISPR/Cas12b assay, we demonstrated equipment-minimal detection of airborne coronaviruses. This work demonstrates the potential of SSNPR-2-based nanotechnology to integrate air sampling and molecular detection, providing a proof-of-concept approach for sensitive detection of low-abundance airborne viral RNA under controlled aerosol conditions. This work introduces an innovative “spiky” nanoparticle-based platform for sensitive detection of airborne viruses. By integrating viral RNA enrichment and protection with molecular detection, the approach provides a proof-of-concept strategy for sensitive detection of viral RNA in environmental and exhaled-breath samples and may support the further development of air-based molecular detection approaches with potential relevance to environmental, clinical, and One Health research. Download: Download high-res image (71KB) Download: Download full-size image