Rational design of direct electron transfer type l-lactate dehydrogenase for the development of multiplexed biosensor

The development of wearable multiplexed biosensors has been focused on systems to measure sweat l-lactate and other metabolites, where the employment of the direct electron transfer (DET) principle is expected. In this paper, a fusion enzyme between an engineered l-lactate oxidase derived from Aerococcus viridans, AvLOx A96L/N212K mutant, which is minimized its oxidase activity and b-type cytochrome protein was constructed to realize multiplexed DET-type lactate and glucose sensors. The sensor with a fusion enzyme showed DET to a gold electrode, with a limited operational range less than 0.5 mM. A mutation was introduced into the fusion enzyme to increase K_m value and eliminate its substrate inhibition to construct "b2LOxS". Together with the employment of an outer membrane, the detection range of the sensor with b2LOxS was expanded up to 10 mM. A simultaneous lactate and glucose monitoring system was constructed using a flexible thin-film multiplexed electrodes with b2LOxS and a DET-type glucose dehydrogenase, and evaluated their performance in the artificial sweat. The sensors achieved simultaneous detection of lactate and glucose without cross-talking error, with the detected linear ranges of 0.5-20 mM for lactate and 0.1-5 mM for glucose, sensitivities of 4.1 nA/mM∙mm² for lactate and 56 nA/mM∙mm² for glucose, and limit of detections of 0.41 mM for lactate and 0.057 mM for glucose. The impact of the presence of electrochemical interferants (ascorbic acid, acetaminophen and uric acid), was revealed to be negligible. This is the first report of the DET-type enzyme based lactate and glucose dual sensing systems.