Ultra‑Sensitive Electrochemical DNA Biosensor for Vibrio cholerae Detection Using Polystyrene‑Acrylic Acid Nanoparticles and Gold Nanoparticles

Abstract

We report a highly sensitive electrochemical DNA biosensor for detecting Vibrio cholerae DNA. The sensor uses polystyrene‑co‑acrylic acid (PSA) latex nanospheres loaded with gold nanoparticles (AuNPs) as a DNA carrier matrix. Differential pulse voltammetry (DPV) with an anthraquinone‑based oligonucleotide label measures the hybridisation signal. The AuNPs significantly amplify the faradaic current, yielding a reproducible, linear response from 1.0×10⁻²¹ to 1.0×10⁻⁸ M (RSD = 4.5 %, n = 5) and a limit of detection (LOD) of 1.0×10⁻²¹ M (R² = 0.99). Recovery in spiked samples ranged from 91 % to 109 % (n = 3). The sensor retains 95 % of its original response after 58 days and can be reused six times with an RSD of 5 % (n = 5).

Background

Vibrio cholerae remains a global health threat, especially in low‑resource settings. Traditional culture‑based methods are slow and laborious, while PCR requires costly equipment and skilled operators. Rapid, inexpensive, and highly sensitive diagnostics are essential for timely outbreak control. Electrochemical DNA biosensors offer high sensitivity, specificity, and potential for miniaturisation, making them ideal for point‑of‑care testing.

Methods

Chemical Reagents

Polystyrene, acrylic acid, HAuCl₄·3H₂O, trisodium citrate, and other reagents were sourced from Fluka, Sigma‑Aldrich, and Riedel‑De Haën. Target, mismatch, and reporter oligonucleotides were synthesized by Bio Service Unit (NSTDA) and Bioneer.

Apparatus

DPV measurements were performed on a screen‑printed carbon electrode (SPE) with an Ag/AgCl reference and platinum counter electrode, using an Autolab PGSTAT12 potentiostat. Electrochemical parameters: 0.02 V step, 0.5 V s⁻¹ scan, 0.05 M phosphate buffer, pH 7.

Synthesis of AuNPs and PSA Latex Particles

AuNPs were prepared by citrate reduction (Turkevich method). PSA latex spheres were synthesized by soap‑free emulsion copolymerisation of styrene and acrylic acid, yielding sub‑micron particles (~186 nm) with abundant carboxyl groups for DNA attachment.

Electrode Modification and DNA Immobilisation

PSA particles were drop‑cast onto the SPE, followed by AuNP deposition. EDC/NHS coupling in pH 5 buffer activated carboxyl groups, then a 5 µM capture probe was immobilised. Hybridisation with 1 µM target DNA and a 5 mM anthraquinone label (AQMS) proceeded in two stages: capture–target and reporter binding, each 1 h.

Optimisation

Probe loading (1–6 µM) and AQMS concentration (0.1–5 mM) were optimised; 4 µM probe and 1 mM AQMS gave maximal response. pH 7, 0.05 M phosphate buffer, and 2.0 M NaCl provided optimal ionic conditions. Hybridisation time of 60 min and probe immobilisation time of 8 h were selected.

Performance Assessment

The sensor demonstrated a linear range of 1.0×10⁻²¹ to 1.0×10⁻⁸ M with an LOD of 1.0×10⁻²¹ M. Recovery tests in spiked samples (1.0×10⁻⁴ to 1.0×10⁻⁶ µg µL⁻¹) yielded 91.4 %–108.9 % (n = 3). Shelf life studies showed 95 % activity after 58 days; the sensor could be regenerated with 0.1 M NaOH and reused six times with 5 % RSD.

Results and Discussion

SEM confirmed uniform PSA spheres (~186 nm). DPV analysis showed that AuNP loading reduced peak potential separation (ΔEₚ), indicating improved electron transfer. Hybridisation experiments revealed a marked increase in DPV peak current only when both target and reporter probes were present, confirming sandwich‑type specificity. Optimal probe loading and AQMS concentration were identified; higher probe densities led to plateauing response, while excessive AQMS caused background noise. pH 7, Na⁺ ions, and high ionic strength (2.0 M NaCl) maximised hybridisation efficiency.

The calibration curve exhibited excellent linearity (R² = 0.99). The sensor’s sub‑zeptomolar LOD surpasses previously reported PSA‑based or avidin‑biotin systems, while maintaining a rapid assay time (~60 min). Stability tests demonstrated minimal signal loss over 58 days, and regeneration protocols restored full activity for up to six cycles.

Conclusions

We have developed a robust, amplification‑free electrochemical DNA biosensor that detects V. cholerae DNA at sub‑zeptomolar concentrations with high specificity and reproducibility. Its simple fabrication, rapid assay time, and long shelf life make it suitable for field deployment in outbreak settings.