Dual-Working Electrode Electrochemical Immunosensor with Au-B-N-Codoped Graphene for Ultrasensitive Detection of Tetanus Toxin in Human Blood

Tetanus is a lethal bacterial infection; however, gold standard diagnostic methods and blood biomarkers have not yet been established. In this study, a label-free dual-working-electrode electrochemical immunosensor was developed for the detection of tetanus neurotoxin (TeNT). The sensor was designed as a four-electrode system with two vertically grown gold-boron-nitrogen codoped graphene (Au-BNG) spiral working electrodes. On the Au-BNG-based sensor, a response signal was generated based on the specific immune binding between monoclonal tetanus antibodies and TeNTs, and the dual-working electrodes synchronously tracked the response signal with a sensitivity deviation of ~0.7%. The dual-working electrode TeNT immunosensor had a detection range of 0.09 fg mL-1 to 300 ng mL-1 and an average detection limit of 0.012 fg mL-1. The metal enzyme antibody immobilization effect, adsorption and rapid desorption of the probe molecules, and high electrocatalytic activity of the signal-amplification platform composed of vertically grown Au-BNG nanosheets contributed to the high TeNT sensitivity of the sensor. The immunosensor had a recovery rate of 83%‒104% for detecting TeNT in serum and quantitatively detected TeNT levels in the sera of several patients. This sensor exhibited excellent specificity, reproducibility, and long-term stability, and demonstrated the ability to simultaneously detect two targets and continuously measure multiple times. Furthermore, the dual-working electrode immunosensor was designed as a test box equipped with a mini-incubator and real-time analysis software, with the potential to serve as a portable tool for tetanus screening

破伤风是一种致命的细菌性感染，但目前尚未确立金标准诊断方法与血液生物标志物。本研究开发了一种无标记双工作电极电化学免疫传感器（label-free dual-working-electrode electrochemical immunosensor），用于检测破伤风神经毒素（TeNT）。该传感器采用四电极体系，搭载两个垂直生长的金硼氮共掺杂石墨烯（Au-BNG）螺旋工作电极。在基于Au-BNG的传感器上，通过破伤风单克隆抗体与TeNT之间的特异性免疫结合产生响应信号，双工作电极可同步追踪响应信号，其灵敏度偏差约为0.7%。该双工作电极TeNT免疫传感器的检测范围为0.09 fg mL⁻¹至300 ng mL⁻¹，平均检测限为0.012 fg mL⁻¹。垂直生长的Au-BNG纳米片构成的信号放大平台，其具备的金属酶抗体固定化效果、探针分子的吸附与快速解吸特性，以及高电催化活性，共同赋予了该传感器对TeNT的高灵敏度。该免疫传感器用于血清中TeNT检测时的回收率为83%~104%，并可对多名患者血清中的TeNT水平进行定量检测。此外，该传感器展现出优异的特异性、重现性与长期稳定性，具备同时检测两种靶标并多次连续测量的能力。进一步地，本双工作电极免疫传感器被设计为搭载微型孵育箱与实时分析软件的检测试剂盒，有望成为用于破伤风筛查的便携式工具。