An Oxygen-Insensitive Electrochemical Biosensor for <scp>l</scp>-Lactate Using a Novel Flavin Mononucleotide-Dependent Lactate Dehydrogenase

Accurate and rapid quantification of l-lactate is essential in clinical diagnostics, bioprocess monitoring, and food quality control. Electrochemical biosensors are well suited for sensing due to their scalability, rapid response, and high sensitivity. Among these, mediated electron transfer-based enzymatic sensors offer distinct advantages by employing redox mediators to facilitate efficient electron exchange between enzymes and electrode surfaces, thereby overcoming the limitations of direct electron transfer. Despite these advantages, deployment of traditional lactate detecting enzyme systems, such as oxygen-dependent lactate oxidase (LOx) and NAD+-dependent lactate dehydrogenases (LDHs) is hindered by oxygen interference, cofactor instability, and complex assay conditions. To address these challenges, we report the development of an oxygen-insensitive l-lactate biosensor incorporating a novel flavin mononucleotide (FMN)-dependent LDH (LactaZyme Type LDHLt) immobilized within an osmium redox polymer matrix as mediator. This biosensor is unaffected by the presence of oxygen, with a signal loss of 2.1 ± 2.9 % for 5 mM l-lactate when changing from nitrogen sparging to oxygen sparging, whereas LOx-modified electrodes show a loss of 25 ± 13 % under the same conditions. The LDHLt biosensor also exhibits an enhanced electrocatalytic performance over the LOx biosensor, with a sensitivity of 0.16 ± 0.01 mA cm–2 mM–1 and a maximum current density of 2.15 ± 0.07 mA cm–2. Notably, it also maintains reliable operation across a broad pH and temperature range. These results establish the LDHLt-based biosensor as a robust and practical platform for l-lactate detection in complex sample matrices.