Simple approach for ultrasensitive electrochemical immunoassay of Clostridium difficile toxin B detection

• We designed a novel sandwich-type electrochemical immunosensor for Clostridium difficile toxin B antigen detection.
• The immunosensor combined a simple immunosensor array and an effectively designed trace tag.
• This proposed method shows better accuracy, stability, reproducibility, and sensitivity.
• The LOD for Tcd B was remarkably lower compared with other current techniques.
• The proposed system can provide a further platform for other immunoassays.

Clostridium difficile toxin B (Tcd B), as one of the primary contributing factors to the pathogenesis of C. difficile-associated diseases, has raised serious public concerns due to its virulence, spore-forming ability and persistence with major types of infectious diarrhea diseases, and been used as a potential biomarker in clinical diagnoses. Thus, a simple method for the determination of Tcd B was developed based on a sandwich-type electrochemical immunosensor. Greatly enhanced sensitivity was achieved based on fabricating the immunosensor by layer-by-layer coating carbon nanotubes (MWCNTs), Prussian blue (PB), Chitosan (CS), Glutaraldehyde (GA) composite on the working electrode as well as using graphene oxide (GO) as a nanocarrier in a multienzyme amplification strategy. In comparison with conventional methods, the proposed immunoassay exhibited high sensitivity and selectivity for the detection of Tcd B, providing a better linear response range from 0.003 to 320 ng/mL and a lower limit of detection (LOD) of 0.7 pg/mL (S/N=3) under optimal experimental conditions. The immunosensor exhibited convenience, low cost, rapidity, good specificity, acceptable stability and reproducibility. Moreover, satisfactory results were obtained for the determination of Tcd B in real human stool samples, indicating that the developed immunoassay has the potential to find application in clinical detection of Tcd B and other tumor markers as an alternative approach.