Rapid and sensitive detection of box turtles using an electrochemical DNA biosensor based on a gold/graphene nanocomposite.

The Southeast Asian box turtle, Cuora amboinensis, is an ecologically important endangered species which needs an onsite monitoring device to protect it from extinction. An electrochemical DNA biosensor was developed to detect the C. amboinensis mitochondrial cytochrome b gene based on an in silico designed probe using bioinformatics tools, and it was also validated in wet-lab experiments. As a detection platform, a screen-printed carbon electrode (SPCE) enhanced with a nanocomposite containing gold nanoparticles and graphene was used. The morphology of the nanoparticles was analysed by field-emission scanning electron microscopy and structural characteristics were analysed by using energy-dispersive X-ray, UV-vis, and Fourier-transform infrared spectroscopy. The electrochemical characteristics of the modified electrodes were studied by cyclic voltammetry, differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy. The thiol-modified synthetic DNA probe was immobilised on modified SPCEs to facilitate hybridisation with the reverse complementary DNA. The turtle DNA was distinguished based on hybridisation-induced electrochemical change in the presence of methylene blue compared to their mismatches, noncomplementary, and nontarget species DNA measured by DPV. The developed biosensor exhibited a selective response towards reverse complementary DNAs and was able to discriminate turtles from other species. The modified electrode displayed good linearity for reverse complementary DNAs in the range of 1 × 10-11-5 × 10-6 M with a limit of detection of 0.85 × 10-12 M. This indicates that the proposed biosensor has the potential to be applied for the detection of real turtle species.