Voltammetric detection of DNA hybridization using a non-competitive enzyme linked assay

A new and simple voltammetric assay method for DNA hybridization events using a disposable DNA sensor was developed. The DNA sensor was prepared by self-assembling horseradish peroxidase (HRP)-linked single-stranded DNA (HRP-ssDNA) onto gold nanoparticles-modified composite membrane at a carbon paste electrode (CPE). With a non-competitive format, the injected sample containing the complementary DNA sequence (cDNA) was produced transparent hybridization reaction with the immobilized HRP-ssDNA. The formed double strand DNA (dsDNA) inhibited partly the active center of HRP, and decreased the immobilized HRP to H2O2 reduction. The fabrication procedure of the DNA sensor was characterized by using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The performance and factors influencing the performance of the DNA sensor were investigated. The hybridization reaction between the probe and its complementary sequence as the target was measured by differential pulse voltammetry (DPV). Under optimal conditions, the current change obtained from the binding HRP relative to H2O2 system was proportional to the cDNA concentration in the range of 1.5 × 10−10 to 9.5 × 10−9 M with a detection limit of 5.0 × 10−11 M (at 3δ). Tests relating to the detection of cDNA demonstrated that the developed DNA sensor exhibited short assay time, high sensitivity, acceptable reproducibility and long-term stability.