An Ultrasensitive Voltammetric Genosensor for the Detection of Bacteria Vibrio cholerae in Vegetable and Environmental Water Samples

Abstract

In view of the presence of pathogenic Vibrio cholerae (V. cholerae) bacteria in environmental waters, including drinking water, which may pose a potential health risk to humans, an ultrasensitive electrochemical DNA biosensor for rapid detection of V. cholerae DNA in the environmental sample was developed. Silica nanospheres were functionalized with 3-aminopropyltriethoxysilane (APTS) for effective immobilization of the capture probe, and gold nanoparticles were used for acceleration of electron transfer to the electrode surface. The aminated capture probe was immobilized onto the Si-Au nanocomposite-modified carbon screen printed electrode (Si-Au-SPE) via an imine covalent bond with glutaraldehyde (GA), which served as the bifunctional cross-linking agent. The targeted DNA sequence of V. cholerae was monitored via a sandwich DNA hybridization strategy with a pair of DNA probes, which included the capture probe and reporter probe that flanked the complementary DNA (cDNA), and evaluated by differential pulse voltammetry (DPV) in the presence of an anthraquninone redox label. Under optimum sandwich hybridization conditions, the voltammetric genosensor could detect the targeted V. cholerae gene from 1.0 × 10^-17-1.0 × 10^-7 M cDNA with a limit of detection (LOD) of 1.25 × 10^-18 M (i.e., 1.1513 × 10^-13 µg/µL) and long-term stability of the DNA biosensor up to 55 days. The electrochemical DNA biosensor was capable of giving a reproducible DPV signal with a relative standard deviation (RSD) of <5.0% (n = 5). Satisfactory recoveries of V. cholerae cDNA concentration from different bacterial strains, river water, and cabbage samples were obtained between 96.5% and 101.6% with the proposed DNA sandwich biosensing procedure. The V. cholerae DNA concentrations determined by the sandwich-type electrochemical genosensor in the environmental samples were correlated to the number of bacterial colonies obtained from standard microbiological procedures (bacterial colony count reference method).

Keywords: DNA biosensor; Vibrio cholerae; gold nanoparticles; sandwich hybridization; silica nanospheres.

Figure 1

Schematic illustration of the electrochemical genosensor fabrication process based on Si-Au nanocomposite-modified carbon SPE for voltammetric sandwich DNA detection of toxigenic bacteria, V. cholerae DNA sequences.

Figure 2

The morphology of the silica nanospheres synthesized via the emulsification technique was captured under scanning electron microscopy.

Figure 3

Differential pulse voltammograms of the AQMS redox indicator on the gold nanoparticle−, Si-Au nanocomposite−, and DNA−modified carbon SPE. The DPV measurement was carried out in 0.05 M K-phosphate buffer (pH 7.0) at a scan rate of 0.5 Vs⁻¹ versus the Ag/AgCl reference electrode.

Figure 4

Effects of gold nanoparticles (A), silica nanospheres (B), capture probes (C), and reporter probe loadings (D) on the electrochemical sandwich-type V. cholerae DNA biosensor DPV response using a 1 mM AQMS DNA hybridization label.

Figure 5

The pH (A), buffer capacity (B), ionic strength (C), capture probe immobilization time (D), and DNA hybridization duration (E) profiles of the voltammetric genosensor based on Si-Au-SPE towards sandwich DNA detection of V. cholerae.

Figure 6

The dynamic linear response range of the V. cholerae DNA biosensor at the presence (A) and absence of a reporter probe (B) using various cDNA concentrations from 1.0 × 10⁻⁵ to 1.0 × 10⁻¹⁹ M with a 45−minute DNA hybridization time at 25 °C.

Figure 6

The dynamic linear response range of the V. cholerae DNA biosensor at the presence (A) and absence of a reporter probe (B) using various cDNA concentrations from 1.0 × 10⁻⁵ to 1.0 × 10⁻¹⁹ M with a 45−minute DNA hybridization time at 25 °C.

Figure 7

Regeneration behaviour of the V. cholerae DNA biosensor using 0.1 M NaOH regeneration solution (15 min exposure). A1 is the DPV response before regeneration and A2-A6 are current responses from rehybridization (in 1.0 × 10⁻⁶ M cDNA for 30 min).The DPV current of B1–B6 are the responses after regeneration processes without rehybridization to indicate that the process of regeneration has occurred.

Figure 8

The stability behaviour of the DNA biosensor for the determination of V. cholerae DNA over a period of 100 days.