Graphitic Carbon Nitride: A Reusable and Stable Support for Glucose Oxidase

This study demonstrates the successful immobilization of the enzyme glucose oxidase (GOx) onto 2D thermally exfoliated graphitic carbon nitride (GCN-T) via physical adsorption, producing a stable, reusable, and highly active bioconjugate. Binding was confirmed by scanning transmission electron microscopy (STEM) coupled with energy-dispersive spectroscopy (EDS), thermogravimetric analyses (TGAs), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). At the optimal conditions, GCN-T/GOx showed an immobilization yield of 88% and an activity of 0.54 U mg^-1. Immobilized GOx retained 81% of its initial activity over 10 consecutive cycles and 50% after 10 storage/reuse cycles. While the free enzyme lost 27% of activity at 70 °C, the immobilized enzyme showed no activity loss after 2 h at the same temperature. Moreover, the 1.8-fold decrease in the Michalis-Menten kinetic constant indicates a better affinity with the substrate upon immobilization. As a proof of concept, GCN-T/GOx was used as a glucose sensor, showing a linear response from 2.5 to 25 mM, a sensitivity of 3.9 μA mM^-1 cm^-2, and a limit of detection (LOD) of 1 mM. The creation of the GCN-T/GOx bioconjugate is a breakthrough in sustainable solutions for the reusability and enhanced performance of enzymes, offering insights into the design of advanced biofunctional surfaces for nanotechnology, biosensing, biocatalysis, and beyond.