Covalently and regioselectively immobilized horseradish peroxidase on graphene oxide as a hydrogen peroxide sensing electrode with a broad single linear detection range and fast response

Covalent immobilization is a useful technique for fabricating enzyme-based sensors; however, a well-designed immobilization reaction is necessary to synthesize a high-performance sensing material. In this study, pH-dependency of epoxide ring opening reaction was utilized to induce regioselective reaction between the epoxides on graphene oxide (GO) and amines in the polypeptide chains of horseradish peroxidase (HRP) under mildly basic condition, thus effectively avoiding deformation of the heme group of HRP. The HRP/GO (HRP covalently immobilized on GO) with 3.6 wt% HRP, as a material for electrochemical sensing of hydrogen peroxide (H 2 O 2 ), exhibited a broad linear range of detection that ranges between 32.6 μM and 12 mM, which is suitable for detecting oxidative stresses caused by various health conditions (e.g. diabetes, Parkinson's, etc.) from blood and urine. 3.6 wt% HRP/GO also showed an outstanding response time of 2.52 s due to low interfacial resistance (between HRP and GO) and diffusion impedance achieved as the result of uniform and intact immobilization of HRP on GO. Real-time detection with high precision and linearity, and excellent selectivity for H 2 O 2 detection was also demonstrated. • Regioselective covalent immobilization of HRP on GO achieved via epoxy ring opening. • Highly uniform coating of HRP on GO was observed up to 3.6 wt% HRP. • 3.6 wt% HRP/GO exhibited a broad single linear range of detection for H 2 O 2 . • A fast response time of 2.52 s was observed from 3.6 wt% HRP/GO sensing electrodes. • High selectivity and ability for real-time sensing of HRP/GO were demonstrated.