A signal-amplified electrochemical DNA biosensor incorporated with a colorimetric internal control for Vibrio cholerae detection using shelf-ready reagents

• Published in: Biosensors and Bioelectronics
• Main Author: Low K.-F.; Zain Z.M.; Yean C.Y.
• Format: Article
• Language: English
• Published: Elsevier Ltd 2017
• Online Access: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84983784777&doi=10.1016%2fj.bios.2016.08.064&partnerID=40&md5=30a0279d26a9039ecdfdf0a00cc09699

A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5 ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0–100 ng/ml (R2=0.992) and log10 (1–104 CFU/ml) (R2=0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4–37 °C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens. © 2016 Elsevier B.V.