High-Sensitivity Molecular Detection of Viral Bioaerosols Using a Composite Collection System and Optimized RPA-CRISPR/Cas12a.

Highly pathogenic airborne microorganisms pose a significant public health threat, particularly because their small particle size can support prolonged suspension and long-range transport. Here, we present an integrated platform that combines a composite bioaerosol collection system with a three-stage enrichment mechanism and recombinase polymerase amplification-CRISPR/Cas12a (RPA-CRISPR/Cas12a) detection. The platform incorporates model-based constant-temperature control, stacked optical filters, and multichannel fluorescence imaging. The temperature-control model accurately described the thermal response of the reaction module, while the optical and imaging subsystems supported stable fluorescence readout. Using aerosolized monkeypox virus (MPXV) pseudovirus as a controlled test target, the platform detected target nucleic acid in collection liquid down to 10 copies/μL under the tested conditions. These results support the feasibility of integrating aerosol enrichment with isothermal molecular detection in a compact prototype. The main contributions lie in innovations and the engineering integration of bioaerosol collection and enrichment, thermal control of biochemical reactions in molecular detection, and precision fluorescence detection techniques for molecular detection. Regarding molecular detection methods, the biochemical reaction parameters were experimentally optimized rather than introducing new biochemical reaction mechanisms. Validation with authentic clinical or environmental viral aerosols remains necessary before diagnostic or field-use claims can be made.