Ike Kosuke, Muto Kousuke, Hioki Takahiro, Loew Noya, Shitanda Isao, Takesue Masafumi, Okuda Mitsuyoshi
Laboratory of Biological & Material Science Research, Kao Corporation, 1334 minato, 640-8580 Wakayama, Japan.
Laboratory of Performance Chemicals Research, Kao Corporation, 1334 minato, 640-8580 Wakayama, Japan.
ACS Sens. 2025 Jun 27;10(6):4707-4716. doi: 10.1021/acssensors.5c01238. Epub 2025 Jun 6.
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.
近年来，L-乳酸生物传感在体育、医学、护理领域以及食品制造和生物技术产业中受到了关注。特别是人体汗液中的L-乳酸，作为一种可以无创收集的生物指标，推动了传感技术研发的快速进展，将汗液定位为替代血液和组织液的新目标。在含有各种生物成分的人体汗液中进行L-乳酸传感的关键是使用L-乳酸氧化酶（LOX）作为识别元件。使用这种酶电极可以特异性、连续且定量地测量L-乳酸。然而，由于传统的LOX受酸性pH值影响，由于汗液pH值的个体差异，生物传感器必须针对每个个体进行校准以实现准确的L-乳酸定量。此外，运动期间汗液pH值的波动会导致检测到的L-乳酸水平不准确。因此，鉴定在酸性pH值下具有活性的LOX至关重要。在此，我们报告了一种具有酸性pH耐受性的新型LOX以及一种能够在酸性至中性pH值的汗液中持续检测L-乳酸水平的技术。通过对公共蛋白质数据库中的α-羟基酸氧化酶进行系统发育分析，并对异源表达的酶进行评估，发现了一种新型LOX，其酸性pH耐受性比传统LOX更好。此外，将新型LOX应用于用MgO模板碳丝网印刷的纸电极上，与传统酶电极相比，在酸性pH值下增强了L-乳酸响应，同时保持了对L-乳酸的pH无关响应。总体而言，利用这种新型LOX的生物传感器将无需校准，因为无需根据pH值变化调整校准。因此，我们的发现有助于扩大针对汗液的L-乳酸生物传感器的应用，并加速其社会应用。
