A new strategy based on a cascade amplification strategy biosensor for on-site eDNA detection and outbreak warning of crown-of-thorns starfish.

Population outbreaks of the crown-of-thorns starfish (COTS) seriously threaten the sustainability of coral reef ecosystems. However, traditional ecological monitoring techniques cannot provide early warning before the outbreaks, thus preventing timely intervention. Therefore, there is an urgent need for a more accurate and faster technology to predict the outbreaks of COTS. In this work, we developed an electrochemical biosensor based on a programmed catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cyclic amplification strategy for sensitive and selective detection of COTS environmental DNA (eDNA) in water bodies. This biosensor exhibited excellent electrochemical characteristics, including a low limit of detection (LOD = 18.4 fM), low limit of quantification (LOQ = 41.1 fM), and wide linear range (50 fM - 10 nM). The biosensing technology successfully allowed the detection of COTS eDNA in the aquarium environment, and the results also demonstrated a significant correlation between eDNA concentration and COTS number (r = 0.990; P < 0.001). The reliability and accuracy of the biosensor results have been further validated through comparison with digital droplet PCR (ddPCR). Moreover, the applicability and accuracy of the biosensor were reconfirmed in field tests at the COTS outbreak site in the South China Sea, which has shown potential application in dynamically monitoring the larvae before the COTS outbreak. Therefore, this efficient electrochemical biosensing technology offers a new solution for on-site monitoring and early warning of the COTS outbreak.