Catalytic Activity of Horseradish Peroxidase Immobilized on Pristine and Two-Photon Oxidized Graphene

Biosensors based on graphene and bio-graphene interfaces have gained momentum in recent years due to graphene's outstanding electronic and mechanical properties. By introducing the patterning of a single-layer graphene surface by two-photon oxidation (2PO), the surface hydrophobicity/hydrophilicity and doping can be varied at the nanoscale while preserving the carbon network, thus opening possibilities to design new devices. In this study, the effect of 2PO on the catalytic activity of the noncovalently immobilized enzyme horseradish peroxidase (HRP) on single-layer graphene-coated Si/SiO2 chips is presented. To monitor the activity continuously, a simple well-plate setup is introduced. Upon controllable 1-2-layer immobilization, the catalytic activity decreases to a maximum value of 7.5% of the free enzyme. Interestingly, the activity decreases with increasing 2PO area on the samples. Hence, the HRP catalytic activity on the graphene surface is locally controlled. This approach can enable the development of graphene-bio interfaces with locally varying enzyme activity. The study investigates the catalytic performance of horseradish peroxidase immobilized on pristine and laser-oxidized graphene surfaces in a 1-2-layer range. UV-vis spectroscopy is employed to track substrate oxidation. The results show a higher catalytic reaction rate on the pristine surface compared to the laser-oxidized one, revealing surface-related effects on the enzyme.image