Development of a simple bioelectrode for the electrochemical detection of hydrogen peroxide using Pichia pastoris catalase immobilized on gold nanoparticle nanotubes and polythiophene hybrid

Abstract

In this paper, a simple and innovative electrochemical hydrogen peroxide biosensor has been proposed using catalase (CAT_pp) derived from Pichia pastoris as bioelectrocatalyst. The model biocomponent was immobilized on gold nanoparticle nanotubes (AuNPNTs) and polythiophene composite using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (EDC–NHS) coupling reagent. In this present work, we have successfully synthesized gold nanoparticles (AuNPs) by ultrasonic irradiation. The tubular gold nanostructures containing coalesced AuNPs were obtained by sacrificial template synthesis. The assembly of AuNPNTs onto the graphite (Gr) electrode was achieved via S–Au chemisorption. The latter was pre-coated with electropolymerized thiophene (PTh) to enable S groups to bind AuNPNTs. The combination of AuNPNTs–PTh, i.e., an inorganic–organic hybrid, provides a stable enzyme immobilization platform. The physical morphology of the fabricated biosensor Gr/PTh/AuNPNTs/EDC–NHS/CAT_pp was investigated using scanning electron microscopy and energy-dispersive microscopy. The analytical performance of the bioelectrode was examined using cyclic voltammetry, differential pulse voltammetry and chronoamperometry. Operational parameters such as working potential, pH, and thermal stability of the modified electrode were examined. The beneficial analytical characteristics of the proposed electrode were demonstrated. Our results indicate that the Gr/PTh/AuNPNTs/EDC–NHS/CAT_pp bioelectrode exhibits a wide linear range from 0.05 mM to 18.5 mM of H₂O₂, fast response time of 7 s, excellent sensitivity of 26.2 mA mM⁻¹ cm⁻², good detection limit of 0.12 μM and good Michaelis–Menten constant of 1.4 mM. In addition, the bioelectrode displayed good repeatability, high stability and acceptable reproducibility, which can be attributed to the AuNPNTs–PTh composite that provides a biocompatible micro-environment.