Immobilization of horseradish peroxidase to a nano-Au monolayer modified chitosan-entrapped carbon paste electrode for the detection of hydrogen peroxide

A procedure for fabricating an enzyme electrode has been described based on the effective immobilization of horseradish peroxidase (HRP) to a nano-scaled particulate gold (nano-Au) monolayer modified chitosan-entrapped carbon paste electrode (CCPE). The high affinity of chitosan entrapped in CCPE for nano-Au associated with its amino groups has been utilized to realize the use of nano-Au as an intermediator to retain high bioactivity of the enzyme. Hydrogen peroxide (H₂O₂) was determined in the presence of hydroquinone as a mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano-Au displayed excellent electrocatalytical activity to the reduction of H₂O₂. The effects of experimental variables such as the operating potential of the working electrode, mediator concentration and pH of measuring solution were investigated for optimum analytical performance by using an amperometric method. The enzyme electrode provided a linear response to hydrogen peroxide over a concentration range of 1.22×10⁻⁵-2.43×10⁻³ mol l⁻¹ with a sensitivity of 0.013 A l mol⁻¹ cm⁻² and a detection limit of 6.3 μmol l⁻¹ based on signal per noise =3. The apparent Michaelis-Menten constant (K_m^app) for the sensor was found to be 0.36 mmol l⁻¹. The lifetime, fabrication reproducibility and measurement repeatability were evaluated with satisfactory results. The analysis results of real sample by this sensor were in satisfactory agreement with those of the potassium permanganate titration method.