Point-of-Care Detection of Tetanus Toxin Based on a Four-Electrode Dual-Signal Immunosensor with Non-Regenerative and Synchronous Tracking of Co-biomarker

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.