Modulation of Photophysical Properties to Enhance Quantum Yield in Fluorogenic Nanoprobes for Multiplexed Detection and Monitoring of Airborne Infectious

Airborne viruses, notably COVID-19 and influenza virus, continue to present significant public health challenges due to their prolonged airborne survivability and rapid transmission. Accurate and timely diagnosis of these viruses is essential to preemptive misdiagnosis, inappropriate treatment, and further community spread. While qPCR remains the gold standard for viral detection, its complexity limits large-scale field use, making fluorescent signal-based immunoassays a promising alternative for multiplexed virus diagnosis without nucleic acid extraction. Our study introduces a multiplexed immunoassay leveraging self-quenching nanoprobes integrated with a portable fluorescence reader for efficient field diagnostics against respiratory viruses. Especially multiplex virus predictive immunoassay (shortened MVP assay), comprising magnetic separation and an amplified fluorogenic probing system, achieves a 90% recovery rate of target viruses, high accuracy, and sensitivity with a detection limit of 10¹ TCID₅₀/mL in under 40 minutes. The stability of the fluorophore, coupled with the significant amplification achieved through the self-quenching effect within the nanoprobe, generates a signal independent of biofluid type, thereby enabling reliable analysis with R² values of 0.97 even with portable detectors. Furthermore, the test accurately detected the presence or absence of viruses in nasal and saliva samples, demonstrating performance comparable to PCR assays and surpassing commercially available rapid kits in diagnostic accuracy. This underscores the potential of the integrated diagnostic system for rapid and sensitive monitoring of large-scale field tests.