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

Abstract

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.

Keywords: Chitosan; Clostridium difficile toxin B; Electrochemical immunosensor; Multiwalled carbon nanotubes; Prussian blue.