A ratiometric electrochemical DNA biosensor for the detection of crown-of-thorns starfish eDNA

Recurring infestations of crown-of-thorns starfish (COTS) inflict severe harm on coral reef ecosystems. To prevent irreversible harm to coral ecosystems, early monitoring of COTS distribution before outbreaks is crucial for timely intervention and effective management. However, traditional monitoring methods are time-consuming, labor-intensive, and lack the sensitivity required for early warning and proactive control. In this study, a ratiometric electrochemical DNA biosensor was constructed for the detection of environmental DNA (eDNA) from COTS. This biosensor combines a dual-signal detection mechanism with internal calibration provided by a reference probe, along with a target recycling amplification strategy mediated by exonuclease III (Exo III). The biosensor exhibited outstanding analytical performance, with a low detection limit (LOD)of 93 fM and a linear response range spanning from 300 fM to 10 nM. Moreover, digital droplet PCR (ddPCR) verified the biosensor's accuracy and reliability (P > 0.05). The biosensor was successfully applied to detect COTS eDNA in environmental samples collected from the Paracel Islands. Therefore, the electrochemical biosensor is promising for detecting low-density COTS individuals or larvae before outbreaks, enabling early warning and facilitating timely ecological intervention.