Carbon-felt-based bioelectrocatalytic flow-detectors: role of ultrasound irradiation during the adsorption of horseradish peroxidase and thionine for a highly sensitive amperometric determination of H2O2.

This study reports on the first example, to our knowledge, of the usefulness of an ultrasound (US)-irradiation during an enzyme adsorption step, for enhancing the performance of a redox-enzyme-based amperometric biosensor. Horseradish peroxidase (HRP) and thionine (Th) were coadsorbed from a mixed aqueous solution of HRP and Th onto a carbon-felt (CF) under US-irradiation for 5 min with an ultransonic bath operating at 40 kHz frequency and 55 W of electric power output. The resulting HRP and Th-coadsorbed CF (HRP/Th-CF) was successfully used as a working electrode unit of a bioelectrocatalytic flow-detector for hydrogen peroxide (H(2)O(2)), which detects the cathodic peak currents based on the direct (unmediated) reduction of oxidized HRP intermediates at 0 V vs. Ag/AgCl. Compared with ordinary adsorption without US-irradiation, US-irradiation during the HRP adsorption step was effective to obtain highly sensitive peak current responses to H(2)O(2). The measurements of electrochemical impedance spectroscopy and cyclic voltammetry suggested that the adsorption of HRP and Th under the US-irradiation provides a suitable interfacial microenvironment for a favorable orientation and conformation of an enzyme with active site available for both substrates and the electrode, which results in larger bioelectrocatalytic activity. The peak currents for H(2)O(2) increased up to 3 × 10(-6) M (sensitivity, 4.72 µA/µM) with a lower detection limit of 2 × 10(-8) M (S/N = 3; current noise level, 0.03 µA).