An electrochemical immunosensor for Vibrio cholerae toxin (VCT) has been developed using electrospun carbon nanofibers (CNFs) as the electrode platform. To fabricate the immunosensor, the anti-cholera toxin antibody (Ab) was covalently immobilized on the electrode platforms using the carbodiimide chemistry for the amide bond formation. Every step of the formation of the immunosensor and the subsequent binding of the VCT subunit antigen (Ag) was electrochemically interrogated. The immunosensor gave excellent reproducibility and sensitivities: limits of detection (ca. 1.2 × 10 −13 g mL −1 ), limits of quantification (ca. 1.3 × 10 −13 g mL −1 ), and a wide linear range for the anti-cholera detection of 8 orders of magnitude (10 −13 to 10 −5 g mL −1 ). One of the key findings was the enhanced sensitivity of the VCT detection using aged rather than the freshly prepared redox probe, described here as Redox Probe Aging-Induced Sensitivity Enhancement ("Redox-PrAISE"). The Redox-PrAISE was found more useful in the real application of these immunosensors, showing comparable or even better sensitivity for eight real cholera-infested water samples than the conventional clinical culture method. This immunosensor shows promise for the potential development of point-of-care diagnosis of VCT. Importantly, this study highlights the importance of considering the nature of the redox probe on the electrochemical sensing conditions when designing impedimetric immunosensors.

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