Analytical Sciences

Amperometric Immunosensor for the Detection of Vibrio cholerae O1 Using Disposable Screen-printed Electrodes

Vepa K. RAO, Mukesh K. SHARMA, Ajay K. GOEL, Lokendra SINGH, and Krishnamurthi SEKHAR

Defence Research & Development Establishment, Jhansi Road, Gwalior-474002, India

A disposable amperometric immunosensor was studied for the rapid detection of Vibrio cholerae (V. cholerae), the causative agent of cholera, employing an indirect sandwich enzyme linked immunosorbent assay (ELISA) principle. Screen-printed electrodes (SPEs) were fabricated (by using commercial and homemade carbon inks), electrochemically characterized and the assay conditions were optimized for capturing antibodies and antigen. Whole cell lysate (WCL) of V. cholerae was used to raise antibodies in rabbits and mice. The antibodies raised against WCL of V. cholerae were found to be specific, and no cross reactivity was observed with other enteric bacteria. 1-Naphthyl phosphate was used as a substrate with the amperometric detection of its enzymatic hydrolysis product 1-naphthol at a potential of +400 mV vs. Ag/AgCl reference electrode. A comparison between the amperometric detection technique and the standard ELISA was made in terms of the total assay time, the amount of biological materials used and the sensitivity of detection. The minimum detection limit of the amperometric immunosensor for V. cholerae was found to be 10⁵ cells/ml in 55 min, while ELISA detected 10⁶ cells/ml in 4 h.

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