Peroxidase-like Nanoparticles of Noble Metals Stimulate Increasing Sensitivity of Flavocytochrome b2-Based L-Lactate Biosensors

We report the development of amperometric biosensors (ABSs) employing flavocytochrome <i>b</i>₂ (Fc<i>b</i>₂) coupled with nanoparticles (NPs) of noble metals on graphite electrode (GE) surfaces. Each NPs/GE configuration was evaluated for its ability to decompose hydrogen peroxide (H₂O₂), mimicking peroxidase (PO) activity. The most effective nanoPO (nPO) was selected for developing ABSs targeting L-lactate. Consequently, several Fc<i>b</i>₂/nPO-based ABSs with enhanced sensitivity to L-lactate were developed, demonstrating mediated ET between Fc<i>b</i>₂ and the GE surface. The positive effect of noble metal NPs on Fc<i>b</i>₂-based sensor sensitivity may be explained by the synergy between their dual roles as both PO mimetics and electron transfer mediators. Furthermore, our findings provide preliminary data that may prompt a re-evaluation of the mechanism of L-lactate oxidation in Fc<i>b</i>₂-mediated catalysis. Previously, it was believed that L-lactate oxidation via Fc<i>b</i>₂ catalysis did not produce H₂O₂, unlike catalysis via L-lactate oxidase. Our initial research revealed that the inclusion of nPO in Fc<i>b</i>₂-based ABSs significantly increased their sensitivity. Employing other PO mimetics in ABSs for L-lactate yielded similar results, reinforcing our hypothesis that trace amounts of H₂O₂ may be generated as a transient intermediate in this reaction. The presence of nPO enhances the L-lactate oxidation rate through H₂O₂ utilization, leading to signal amplification and heightened bioelectrode sensitivity. The proposed ABSs have been successfully tested on blood serum and fermented food samples, showing their promise for L-lactate monitoring in medicine and the food industry.