Dual Adjacent Capture Probe-Induced Hybridization Chain Reaction in a Cyclic Array Reactor for Rapid and Sensitive...

Norovirus (NoV) is a leading global cause of foodborne illness, necessitating the development of rapid and sensitive detection methods. However, conventional proximity ligation assays (PLA) often suffer from limited sensitivity and poor compatibility with array-based analysis. Here, we present a novel detection strategy that integrates a proximity-ligation hybridization chain reaction with catalytic hairpin assembly (NPLA-HCR/CHA) and a multidirectional cyclic fluorescence detection system (MDCFS). In this design, protein targets are first converted into amplifiable DNA barcodes via NPLA-HCR/CHA, enabling exponential signal amplification. The MDCFS device, employing electronic fluorinated liquid spacers, enables cyclic array-based readout with enhanced sensitivity. Specific lectin- and antibody-based probes were designed to ensure selective recognition of NoV capsid protein VP1. Signal amplification is achieved through proximity-activated hybridization chain reaction and catalytic hairpin assembly. For practical samples, this method demonstrated a detection limit of 0.15 pg·mL^-1 (approximately 8.51 × 10³ copies·mL^-1) and a linear range of 0.50-50 pg·mL^-1 (approximately 2.84 × 10⁴ to 2.84 × 10⁶ copies·mL^-1), exhibiting high selectivity and accuracy comparable to those of real-time polymerase chain reaction (RT-PCR). The combination of NPLA-HCR/CHA with MDCFS thus provides a powerful platform for ultrasensitive, multiplexed, and array-compatible detection of NoV, offering significant potential for applications in public health monitoring and food safety assurance.