<scp>l</scp>-Lactate Oxidase-Based Biosensor Enables Quasi-Calibration-Free Detection of <scp>l</scp>-Lactate in Sweat of Acidic to Neutral pH

l-lactate biosensing has attracted attention in recent years in sports, medicine, and nursing care fields as well as in food manufacturing and biotechnology industries. In particular, l-lactate in human sweat, a biological indicator that can be collected noninvasively, has driven rapid progress in the research and development of sensing technology, positioning sweat as a new target to replace blood and interstitial fluid. The key to l-lactate sensing in human sweat, which contains various biological components, is using l-lactate oxidase (LOX) as a recognition element. l-lactate can be specifically, continuously, and quantitatively measured using this enzyme electrode. However, as conventional LOX is affected by acidic pH, biosensors must be calibrated for each individual for accurate l-lactate quantification owing to individual differences in sweat pH. Furthermore, fluctuations in sweat pH during exercise lead to inaccuracies in the detected l-lactate levels. Therefore, identifying LOX active in acidic pH is crucial. Here, we report a novel LOX with acidic pH tolerance and a technology that enables constant detection of l-lactate levels in acidic to neutral pH sweat. Phylogenetic analysis of α-hydroxy acid oxidase in a public protein database, with the evaluation of heterologously expressed enzymes, revealed the existence of a novel LOX with better acidic pH tolerance compared to that observed with conventional LOX. Furthermore, applying the novel LOX to a paper electrode screen-printed with MgO-templated carbon enhanced the l-lactate response at acidic pH compared to that observed with conventional enzyme electrodes while maintaining a pH-independent response to l-lactate. Overall, biosensors utilizing this novel LOX will be quasi-calibration-free, by eliminating the need for adjusting the calibration according to changes in pH. Thus, our findings contribute to expanding the use of l-lactate biosensors targeting sweat and accelerating their societal application.