Analytical Sciences

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

Yue WANG*,** and Yasushi HASEBE*,***

*Department of Materials Science and Engineering, Graduate School of Engineering, Saitama Institute of Technology, 1690 Fusaiji, Fukaya, Saitama 369-0293, Japan
**School of Chemical Engineering, University of Science and Technology Liaoning, Anshan, Liaoning 114044, China
***Department of Life Science and Green Chemistry, Saitama Institute of Technology, 1690 Fusaiji, Fukaya, Saitama 369-0293, Japan

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 (H₂O₂), 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 H₂O₂. 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 H₂O₂ 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).

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