In this work, we investigated the effect of multi-walled carbon nanotubes (MWCNTs) and bio-graphene (bG) on the structure and activity of glucose oxidase (GOx), as well as on the performance of the respective electrochemical glucose biosensors. Various spectroscopic techniques were applied to evaluate conformational changes in GOx molecules induced by the presence of MWCNTs and bG. The results showed that MWCNTs induced changes in the flavin adenine dinucleotide (FAD) prosthetic group of GOx, and the tryptophan residues were exposed to a more hydrophobic environment. Moreover, MWCNTs caused protein unfolding and conversion of α-helix to β-sheet structure, whereas bG did not affect the secondary and tertiary structure of GOx. The effect of the structural changes was mirrored by a decrease in the activity of GOx (7%) in the presence of MWCNTs, whereas the enzyme preserved its activity in the presence of bG. The beneficial properties of bG over MWCNTs on GOx activity were further supported by electrochemical data at two glucose biosensors based on GOx entrapped in chitosan gel in the presence of bG or MWCNTs. bG-based biosensors exhibited a 1.33-fold increased sensitivity and improved reproducibility for determining glucose over the sweat-relevant concentration range of glucose.

In this work, we report the green production of few-layer bio-Graphene (bG) through liquid exfoliation of graphite in the presence of bovine serum albumin. Microscopic characterization evaluated the quality of the produced nanomaterial, showing the presence of 3–4-layer graphene. Moreover, spectroscopic techniques also confirmed the quality of the resulted bG, as well as the presence of bovine serum albumin on the graphene sheets. Next, for the first time, bG was used as support for the simultaneous covalent co-immobilization of three enzymes, namely β-glucosidase, glucose oxidase, and horseradish peroxidase. The three enzymes were efficiently co-immobilized on bG, demonstrating high immobilization yields and activity recoveries (up to 98.5 and 90%, respectively). Co-immobilization on bG led to an increase of apparent K_M values and a decrease of apparent V_max values, while the stability of the nanobiocatalysts prevailed compared to the free forms of the enzymes. Co-immobilized enzymes exhibited high reusability, preserving a significant part of their activity (up to 72%) after four successive catalytic cycles at 30 °C. Finally, the tri-enzymatic nanobiocatalytic system was applied in three-step cascade reactions, involving, as the first step, the hydrolysis of p-Nitrophenyl-β-D-Glucopyranoside and cellobiose.

Graphene has been studied thoroughly for its use in biomedical applications over the last decades. A crucial factor for a material to be used in such applications is its biocompatibility. Various factors affect the biocompatibility and toxicity of graphene structures, including lateral size, number of layers, surface functionalization, and way of production. In this work, we tested that the green production of few-layer bio-graphene (bG) enhances its biocompatibility compared to chemical-graphene (cG). When tested against three different cell lines in terms of MTT assays, both materials proved to be well-tolerated at a wide range of doses. However, high doses of cG induce long-term toxicity and have a tendency for apoptosis. Neither bG nor cG induced ROS generation or cell cycle modifications. Finally, both materials affect the expression of inflammatory proteins such as Nrf2, NF-kB and HO-1 but further research is required for a safe result. In conclusion, although there is little to choose between bG and cG, bG’s sustainable way of production makes it a much more attractive and promising candidate for biomedical applications.