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

Abstract

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.

Keywords: DNA detection; box turtle; electrochemical DNA biosensor; nanocomposite; screen-printed carbon electrode.

Figure 1

The Cuora amboinensis specific probe (31 bases) and corresponding aligned sequences. A, T, G, C (A, adenine; T, thymine; G, guanine; C, cytosine) stand for the corresponding nucleotides. The dot (.) indicates a match with BT, and the corresponding nucleotide alphabet indicates a mismatch with BT.

Figure 2

Morphological and structural characterisation of different nanoparticles and their composites. FESEM images of (a) Gr and (b) AuNPs/Gr; EDX of images of (c) Gr and (d) AuNPs/Gr; UV–vis spectra of (e) Gr and (f) AuNPs/Gr; and FTIR spectra of (g) Gr, AuNPs/Gr, and DNA/AuNPs/Gr. AuNPs were taken (33.33%, 1:3) in the AuNPs/Gr nanocomposite.

Figure 3

Electrochemical characterisation of various modified electrodes. (a) The relationship between the peak current and square root of the scan rate within the linear range from 40 to 150 mV for SPCE and composite- (Gr/AuNPs) modified SPCE for determination and comparison of the effective surface area. (b) CV for SPCE and modified SPCE within the potential window of −0.1 to 0.6 V at a scan rate of 100 mV/s vs Ag/AgCl. (c) DPV for SPCE and modified SPCE at a potential range from −0.1 to 0.6 V, a step potential of 5 mV, and modulation amplitude of 25 mV. (d) EIS for SPCE and modified SPCE was carried out at a frequency range from 0.10 to 100 KHz and an amplitude of 10 mV_RMS. All experiments were carried out in a 2.0 mM K₄[Fe(CN)₆] solution in 0.2 M KCl.

Figure 4

Electrochemical study for the optimisation of reverse complementary DNA hybridisation parameters. (a) Optimisation of hybridisation time (min), and (b) optimisation of hybridisation temperature (°C).

Figure 5

DPV of hybridised DNA in the presence of MB for the RC, different types of mismatch and nontarget species of DNAs in a 10× PB (pH 7.4) solution. (a) DPV voltammogram curves of different DNAs. (b) Peak current histogram of the same DNAs (n = 3). ss: single stranded; BT: BT probe; ds: double stranded (to indicate hybridisation state); RC: Reverse complementary; MN: mismatch nucleotide(s) and numbers 1 or 3 correspond to the number of mismatch nucleotides. CW: cow, BF: buffalo, HS: horse, and DK: duck.

Figure 6

Comparison of the DPV responses of different concentrations of reverse complementary DNA hybridised with BT probe DNA. (a) DPV curve at various concentrations (from 10⁻⁵ to 10⁻¹⁵ M) of reverse complementary DNAs against corresponding values of reverse complementary DNA concentrations, indicated by corresponding different legends and colours. (b) Scattered plot with error bars for the peak current (µA) as a function of the corresponding log concentrations [log₁₀(M)] of DNA. All DNA values are at the molar (M) concentration. ‘E’ stands for exponential (power of 10) and (−) stands for negative power.

Figure 7

DPV curve of hybridised DNA isolated from raw meat muscle samples. BTS stands for box turtle DNA isolated from a meat sample; ds: double stranded; CWS: cow sample, BFS: buffalo sample, HRS: horse sample, and DKS: duck sample.

Figure 8

Step-by-step fabrication procedure of the electrochemical DNA biosensor for the detection of the BT cytb gene. Figure 8 was created with BioRender.com. This content is not subject to CC BY 4.0.