Peroxidase and methylene blue-incorporated double stranded DNA-polyamine complex membrane for electrochemical sensing of hydrogen peroxide

Horseradish peroxidase (HRP) and methylene blue (MB) were incorporated in polyion complex membrane composed of double stranded DNA (dsDNA) and poly(allylamine) (PAA) prepared on the surface of an Au diskelectrode to fabricate highly sensitive and selective reagentless second-generation H2O2 biosensor. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of DNA/PAA membrane indicated incorporated HRP was aggregated in the DNA/PAA membrane. The embedded-MB in the DNA/PAA membrane exhibited excellent electrochemical redox property with an electron transfer rate constant of 2.692 s(-1), and shuttled electron effectively from the base Au electrode to catalytic center of the HRP. Under the applied potential of -0.20 V (versus Ag/AgCl) and pH 7.0, the resulting electrode (HRP/DNA-MB/PAA/AuE) exhibited rapid (<10s) and sensitive response to H2O2. The calibration curve of H2O2, plotting steady-state cathodic current versus H2O2 concentration, was linear up to 0.1 mM with a detection limit of 0.3 muM H2O2 (S/N = 3). The H2O2 response was scarcely interfered by ascorbic acid, L-Cysteine and uric acid which potentially reduce oxidized intermediate of the HRP and interfere with the response of peroxidase-based electrodes. (C) 2004 Elsevier B.V. All rights reserved.