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

• A novel dual colorimetric/electrochemical DNA biosensor was developed.
• It enables simultaneous detection of target microbial DNA and an internal control (IC).
• All of the assay reagents are shelf-stable and ready-to-use (shelf-ready).
• Electrochemical detection of target microbial DNA was validated by an instant coloration in IC.
• Evaluation with clinical isolate-spiked fecal specimens showed excellent diagnostic sensitivity and specificity.

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.

Figure optionsDownload as PowerPoint slide