Photo-Oxidized Highly Oriented Pyrolytic Graphite as a Reactive Carbon Biointerface for Biomolecule Immobilization

Abstract Highly oriented pyrolytic graphite (HOPG) is a promising carbon material for biointerfacial applications. However, its chemically inert surface limits efficient biomolecule immobilization. In this study, we developed a carbon-based biointerface by introducing oxygen-containing functional groups onto HOPG surfaces using photoactivated chlorine dioxide radical treatment. Surface characterization using contact angle measurements, Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS) demonstrated enhanced hydrophilicity and the formation of oxygen-containing functionalities, including C–O, carbonyl, and carboxy-type species, on the oxidized HOPG surface. The effects of surface functionalization on biomolecule immobilization were also investigated. Bicinchoninic acid (BCA) assays revealed a significant increase in the amount of immobilized protein on oxidized HOPG compared to that on untreated surfaces. Additionally, fluorescence imaging using fluorescently labeled immunoglobulin G (IgG) and washing stability tests demonstrated high fluorescence intensity and enhanced retention of immobilized biomolecules after repeated washing. To evaluate the functionality of immobilized biomolecules, β-d-galactosidase (β-Gal) was immobilized on the surface, and its enzymatic activity was assessed using fluorescein di-β-d-galactopyranoside (FDG) as a substrate. The oxidized HOPG exhibited a strong fluorescence response even at low enzyme concentrations and showed improved signal retention during repeated measurements. These results demonstrate that photoactivated chlorine dioxide radical treatment effectively functionalizes HOPG surfaces with oxygen-containing groups, thereby enhancing biomolecule immobilization and supporting the retention of enzymatic activity after immobilization. This simple and effective surface modification strategy provides a promising approach for constructing stable carbon-based biointerfaces and may facilitate the development of carbon-material-based biosensing platforms.