Wu Jia, He Baoshan, Du Gengan, Zhang Yurong, Bai Chunqi, Wang Longdi
School of Food Science and Technology, Henan University of Technology, Zhengzhou, Henan, 450001, PR China.
School of Food Science and Technology, Henan University of Technology, Zhengzhou, Henan, 450001, PR China.
Anal Chim Acta. 2025 Oct 22;1372:344460. doi: 10.1016/j.aca.2025.344460. Epub 2025 Jul 23.
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.
脱氧雪腐镰刀菌烯醇（DON）是谷物中普遍存在的单端孢霉烯族真菌毒素，对人类健康和农业可持续发展构成严重威胁。传统检测方法在现场应用的灵敏度和操作复杂性方面存在局限性。在此，我们开发了一种集成双信号放大策略的电化学适配体传感器：Nb.BbvCI切口内切酶驱动的DNA步行器和催化发夹组装（CHA）。这项工作开创了合理设计酶可识别的发夹DNA结构（HP1/t-DNA）并结合分级纳米杂化界面的先河。通过将酶介导的靶标循环与基于CHA的级联放大相结合，所提出的系统解决了霉菌毒素电化学传感中的关键灵敏度瓶颈，同时保持了操作的简便性。该传感器结构采用聚乙烯亚胺还原氧化石墨烯/金铂核壳纳米棒（PEI-rGO/Pt@Au NRs）对金电极（AuE）进行功能化。将具有高比表面积和孔隙率的PtPdAu与分子亚甲基蓝（MB）结合引入电极作为信号探针。每个靶标引发的Nb.BbvCI激活的DNA步行器切割事件释放CHA触发链（S），放大信号。这种双放大策略实现了对DON的超灵敏检测，检测限（LOD）为9.35×10⁻⁸mg/mL（S/N = 3），超过了大多数已报道的适配体传感器。该传感器在1×10⁻⁷至5×10⁻⁶mg/mL范围内呈现宽线性范围（R² = 0.997）。在最佳条件下评估了稳定性、特异性和重现性，对于1 ng/mL、100 pg/mL和10 pg/mL的响应电流值分别保持在99.39 - 99.46%、96.55 - 98.77%和95.44 - 98.15%。在加标玉米样品中的实际验证回收率为97.15 - 107.8%，证实了其现场适用性。这项工作建立了将酶促DNA纳米技术转化为实用霉菌毒素监测工具的范例。酶驱动的分子机器与介孔纳米酶工程的协同整合克服了电化学生物传感中传统的灵敏度/便携性权衡。该平台在灵敏度上优于先前的DON适配体传感器，并在复杂基质中验证了性能，为食品安全监管提供了强大的解决方案，特别是在资源有限的环境中。模块化设计原则扩展到其他需要超灵敏监测的有害小分子污染物。
