Real-Time Fluorescence Aptasensor Based on Programmable Switch-Coupled Exponential Amplification Transduction for Ultrasensitive and Specific Vibrio parahemolyticus Determination

The accurate and early detection of Vibrio parahemolyticus (V. parahemolyticus) is crucial for ensuring food safety and protecting consumer health. However, current detection methods face challenges in preventing the rapid transmission of V. parahemolyticus due to insufficient sensitivity and limited coverage. Herein, an aptamer-based real-time fluorescence biosensor was developed, termed CAPANA (a catalytic nucleic acid exponential cascade amplification network-powered magnetic aptasensor) for simple and ultrafast sensitive diagnosis of V. parahemolyticus. By integrating the programmable truncated aptamer and the EXPAR-TtAgo cascade reaction, the CAPANA system dexterously converts the epitope recognition events of V. parahemolyticus into fluorescence signals. The CAPANA system can not only address the low sensitivity of the single aptasensor transduction and the interference from nonessential bases in amplicons but also maintain dynamic balance between repeated regeneration of target and exponential signal amplification. Moreover, considering the potential variations in reaction conditions among multiple enzymes and the possible impacts of their interactions on reaction efficiency and specificity, engineering improvements in the CAPANA system can optimize coordination and complexity among multiple enzymes. The CAPANA system demonstrated a 60.61-fold higher detection efficacy for V. parahemolyticus than the TtAgo-based cleavage method. The detection limit achieved is 10⁰ CFU/mL, and the rapid results take only 40 min. This system was confirmed by identifying contaminated aquatic products and challenged with ELISA, qPCR, or culture methods. This work enriches the arsenal of signal transduction based on aptamer epitope recognition, highlighting its potential as a precision on-site diagnostic tool for pathogenic bacteria.