Electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere label for detection of Vibrio cholerae

• Lyophilized AuNPs–PSA–avidin conjugate can be used as a universal reporter label.
• One-step sandwich hybridization technique is time-saving and highly sensitive.
• One-step sandwich hybridization technique can be used for PCR and LAMP products.
• Different stabilizers were evaluated for lyophilization of AuNPs–PSA–avidin conjugate.

Vibrio cholerae is a Gram-negative bacterium that causes cholera, a diarrheal disease. Cholera is widespread in poor, under-developed or disaster-hit countries that have poor water sanitation. Hence, a rapid detection method for V. cholerae in the field under these resource-limited settings is required. In this paper, we describe the development of an electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere (AuNPs–PSA) reporter label. The reporter label mixture was prepared by lyophilization of AuNPs–PSA–avidin conjugate with different types of stabilizers. The best stabilizer was 5% sorbitol, which was able to preserve the dried conjugate for up to 30 days. Three methods of DNA hybridization were compared and the one-step sandwich hybridization method was chosen as it was fastest and highly specific. The performance of the assay using the lyophilized reagents was comparable to the wet form for detection of 1 aM to 1 fM of linear target DNA. The assay was highly specific for V. cholerae, with a detection limit of 1 fM of PCR products. The ability of the sensor is to detect LAMP products as low as 50 ng µl−1. The novel lyophilized AuNPs–PSA–avidin reporter label with electrochemical genosensor detection could facilitate the rapid on-site detection of V. cholerae.

The rehydration of the lyophilized AuNPs–PSA–avidin conjugate and one-step DNA hybridization method followed by DPASV measurement on SPE.Figure optionsDownload as PowerPoint slide