Efficient removal of biological contaminants from microbially reduced graphene oxide via a TRIzol-based protocol

Graphene-based nanomaterials, particularly graphene oxide (GO) and reduced graphene oxide (rGO), have emerged as promising platforms for biomedical applications due to their biocompatibility, high surface area, and versatile functionalization. Among methods to obtain rGO from GO the bacteria-mediated reduction occurs by the enzymatic transfer of electrons from bacterial respiratory chains to GO, leading to the partial depletion of the oxygen-based functionalities and restoration of the graphene lattice. However, rGO obtained via microbial reduction often retains bacterial contaminants, which can strongly adsorb to the rGO surface and interfere with in vitro cellular assays and optoelectronic applications. Furthermore, the large-scale production of high-quality rGO requires the effective elimination of these biological impurities. Here, we report a new protocol for the efficient purification of microbially produced rGO using a purification method based on TRIzol, a phenol-guanidinium reagent widely used for nucleic acid and protein extraction. Our protocol effectively disrupts cellular components and dissociates macromolecular complexes, enabling the removal of surface-bound contaminants. Purified rGO was characterized by physicochemical methods and evaluated in vitro. The protocol removes bacterial contaminants without detectable alteration of the rGO structural features probed by Raman spectroscopy, XPS and FE-SEM, and the purified material showed no detectable cytotoxicity toward BJ5ta fibroblasts at 100 µg mL^-1 after 24 and 48 h. This approach provides a practical strategy to improve reproducibility in studies involving microbially reduced graphene-based nanomaterials. The practical constraints of the method, namely reagent hazards, possible phenol carryover and reagent cost, are critically discussed, positioning it as a laboratory-scale purification step for applications in which material purity is critical.