3D Nanoporous MXene-Graphene Oxide Sponge: Mitigating Stability Degradation of NADH-Dependent Dehydrogenase Biosensors

Electrochemical biosensors based on nicotinamide adenine dinucleotide (NADH)-dependent dehydrogenases hold great potential in analytical applications. However, previous biosensors can lead to enzyme detachment and inactivation, thus limiting their applications. This study presents a novel sensing platform based on a three-dimensional MXene-graphene oxide sponge (3D MG). The nanoporous sponge was synthesized by polyethylene glycol (PEG) assisted cross-linking of MXene and small-sized graphene oxide (GO) nanosheets, thereby forming a high-strength porous conductive network and addressing the instability of enzyme-based biosensors. This sponge significantly enhances enzyme immobilization (reducing leaching by 62.27% compared to MXene films) and maintains enzymatic activity under extreme conditions. The 3D MG sensing platform was further fabricated into two biosensors by immobilizing NADH-dependent dehydrogenases (with glutamate dehydrogenase and formate dehydrogenase employed as model enzymes, respectively), using the (2,2′-bipyridyl) (pentamethylcyclopentadienyl)-rhodium complex ([Cp*Rh(bpy)Cl]+) as a mediator that enables the regeneration of bioactive NADH. The two biosensors exhibited low detection limits (0.332 μM for NH4+ and 0.188 mM for HCO3–), excellent reproducibility (relative standard deviation < 4%), and long-term stability (94.82% and 92.63% signal retention after 60 days). The fabrication of 3D MG with a robust nanoporous structure and an enzyme-friendly environment provides a strategy for designing long-life multifunctional enzyme-based biosensors.