Dual-signal amplified aptasensor based on PtPdAu mesoporous nanozymes with DNA walker-CHA for ultrasensitive DON detection.

Deoxynivalenol (DON), a prevalent trichothecene mycotoxin in cereals, poses severe threats to human health and agricultural sustainability. Conventional detection methods face limitations in sensitivity and operational complexity for on-site applications. Herein, we develop an electrochemical aptasensor integrating dual-signal amplification strategies: Nb.BbvCI nicking endonuclease-driven DNA walker and catalytic hairpin assembly (CHA). This work pioneers the rational design of enzyme-recognizable hairpin DNA structures (HP1/t-DNA) coupled with hierarchical nanohybrid interfaces. By combining enzyme-mediated target recycling with CHA-based cascade amplification, the proposed system addresses critical sensitivity bottlenecks in mycotoxin electrochemical sensing while maintaining operational simplicity. The sensor architecture incorporated polyethyleneimine-reduced graphene oxide/gold-platinum core-shell nanorods (PEI-rGO/Pt@Au NRs) to functionalize gold electrodes (AuE). PtPdAu with high specific surface area and porosity combined with molecule methylene blue (MB) was introduced into electrodes as the signal probe. Nb.BbvCI-activated DNA walker cleavage events per target released CHA-triggering strands (S), amplifying the signal. This dual-amplification strategy enabled ultrasensitive DON detection with a limit of detection (LOD) of 9.35 × 10 mg/mL (S/N = 3), surpassing most reported aptasensors. The sensor exhibited a wide linear range from 1 × 10 to 5 × 10 mg/mL (R = 0.997). Stability, specificity, and reproducibility were assessed under optimal conditions, with response current values maintained at 99.39-99.46 %, 96.55-98.77 %, and 95.44-98.15 % for 1 ng/mL, 100 pg/mL, and 10 pg/mL, respectively. Practical validation in spiked corn samples achieved recoveries of 97.15-107.8 %, confirming its field applicability. This work establishes a paradigm for translating enzymatic DNA nanotechnology into practical mycotoxin monitoring tools. The synergistic integration of enzyme-powered molecular machines with mesoporous nanozyme engineering overcomes traditional sensitivity/portability trade-offs in electrochemical biosensing. With sensitivity improvement over previous DON aptasensors and validated performance in complex matrices, this platform provides a robust solution for food safety regulation, particularly in resource-limited settings. The modular design principle extends to other hazardous small-molecule contaminants requiring ultrasensitive surveillance.