DNAzyme-Aptamer Electrochemical Biosensor for Pb(II) and Hg(II) Determination: Robust Validation in Food Matrices and Water Samples

Abstract

A dual-recognition electrochemical biosensing platform was developed for ultrasensitive and selective determination of trace toxic metal ions in complex matrices. The sensor integrates a Pb(II)-dependent DNAzyme and an Hg(II)-specific aptamer on a graphene oxide-gold nanoparticle modified gold electrode, enabling target-induced and distinguishable impedance responses. Structural and surface analyses confirmed the uniform decoration of gold nanoparticles (18.5 ± 2.1 nm) on graphene oxide sheets and the successful immobilization of thiolated nucleic acid probes through Au-S bonding. Under optimized conditions (pH 7.4, 60 min incubation), Pb(II) triggered DNAzyme-catalyzed substrate cleavage, resulting in a decrease in charge-transfer resistance, whereas Hg(II) induced aptamer folding via T-Hg-T coordination, producing an opposite impedance increase. The biosensor exhibited linear responses from 1.0 pM to 10 nM for Pb(II) and from 10 pM to 100 nM for Hg(II), with limits of detection of 0.32 pM and 2.8 pM, respectively. High selectivity was achieved against common interfering metal ions at 100-fold excess. The platform demonstrated excellent reproducibility, with inter-assay relative standard deviations below 5%, and retained more than 92% of its initial response after 28 days of storage. Practical applicability was validated through analysis of spiked tap water, river water, seawater, milk, fish, and vegetable samples, yielding recoveries of 91.2-108.0% and strong agreement with inductively coupled plasma mass spectrometry results (P > 0.05). These results demonstrate a robust and versatile sensing strategy suitable for reliable monitoring of trace metal contamination in food and environmental samples.