Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase Immobilized in a DNA/Chitosan-Fe₃O₄ Magnetic Nanoparticle Bio-Complex Film.

A DNA/chitosan-Fe₃O₄ magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP) on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe₃O₄ nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM) was used to study the surface features of DNA/chitosan/Fe₃O₄/HRP layer. The results of electrochemical impedance spectroscopy (EIS) show that Fe₃O₄ and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET) and bioelectrocatalysis of HRP in the DNA/chitosan/Fe₃O₄ film were investigated by cyclic voltammetry (CV) and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s ^-1 . The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe ^{(III )} ) and HRP(Fe ^(II) ). The obtained electrode also displayed an electrocatalytic reduction behavior towards H₂O₂. The resulting DNA/chitosan/Fe₃O₄/HRP/glassy carbon electrode (GCE) shows a high sensitivity (20.8 A·cm ^-2 ·M ^-1 ) toward H₂O₂. A linear response to H₂O₂ measurement was obtained over the range from 2 µM to 100 µM ( R ² = 0.99) and an amperometric detection limit of 1 µM (S/N = 3). The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability.