Hybrid carbon nanostructures as efficient electron transfer platforms for lactate biosensing

Two bioelectrocatalytic systems for lactate oxidation were developed by immobilizing lactate oxidase (LOx) onto nanostructured carbon-based platforms: 4-(pyrrole-1-yl)benzoic acid modified multi-walled carbon nanotubes (MWCNT/PyBA) and a hybrid system combining electrochemically reduced graphene oxide with MWCNT/PyBA (ERGO/MWCNT/PyBA). The modification of MWCNT with PyBA enhanced electron transfer between the enzyme's active site and the electrode surface, while the ERGO/MWCNT/PyBA hybrid, prepared via non-covalent π-π stacking interactions, provided improved conductivity and electrocatalytic performance. Both systems exhibited well-defined, reversible FMN/FMNH₂ redox peaks with formal potentials of -0.453 V and -0.446 V vs. Ag/AgCl at pH 7.0, respectively. Notably, this study presents the first determination of electron transfer rate constants (k_s) for LOx using Laviron method, yielding values of 3.9 s^-1 for MWCNT/PyBA-LOx and 9.5 s^-1 for ERGO/MWCNT/PyBA-LOx, confirming the enhanced electron transfer in the hybrid system. Apparent Michaelis-Menten constants (K_M^app) were 73.7 mM and 8.25 mM, respectively, demonstrating higher enzymatic affinity with the hybrid system. These results introduce a novel hybrid nanostructured matrix for enzyme immobilization and highlight its potential in constructing sensitive, rapid, and selective lactate biosensors applicable to real samples such as dairy products, alcoholic beverages, and clinical fluids.