Development of Novel Multiplex Detection Platforms Based on Multiepitope Nanobody Pairs for Rapid Screening of Waterborne Pathogens

The rapid and multiplexed detection of waterborne viruses is crucial for infection prevention. However, current methods are limited by low-quality probes, instrument dependency, and time-consuming procedures. In this study, we developed a high-performance, nanobody pair-based, multichannel homogeneous platform for the simultaneous and rapid detection of three viruses─SARS-CoV-2, norovirus, and influenza A virus─in aquatic environments. To identify robust and sensitive multiepitope nanobody pairs for these viruses, we utilized pressure-assisted screening and docking techniques. For multichannel detection, we synthesized three distinct SiO2@TQD types with unique excitation wavelengths, each acting as an independent signal label. By integrating these multichannel antibody arrays and signal labels into a unified platform, we developed a homogeneous sensor capable of detecting all three viruses within 30 min. The system demonstrated detection limits of as low as 1.56 pg/mL for the SARS-CoV-2 antigen, 0.1 pg/mL for the norovirus antigen, and 0.39 pg/mL for the influenza A virus antigen, surpassing conventional antigen detection kits with a sensitivity enhancement of 160.26-6.25 × 104-fold. Notable advantages include exceptional specificity, accuracy, and stability. This work not only provides a transformative solution for monitoring waterborne pathogens but also establishes a versatile framework for developing multichannel detection platforms for other infectious agents.