He Qingrong, Wang Cheng, Jain Rohit, Byrnes James, Farquhar Erik R, Reed Elliot, Berezovsky Elizabeth, Chance Mark R, Lodowski David, An Ran
Department of Biomedical Engineering, University of Houston, United States.
Biomolecular Structure and Integration of Sensors (BioSIS) Program, Department of Nutrition, School of Medicine, Case Western Reserve University, United States.
Heliyon. 2024 Jul 9;10(14):e34301. doi: 10.1016/j.heliyon.2024.e34301. eCollection 2024 Jul 30.
Lactate levels in humans reveal intensity and duration of exertion and provide a critical readout for the severity of life-threatening illnesses such as pediatric sepsis. Using the lactate oxidase enzyme (Lox) from , we demonstrated its functionality for lactate electrochemical sensing in physiological fluids in a lab setting. The structure and dynamics of LOx were validated by crystallography, X-ray scattering, and hydroxyl radical protein footprinting. This provided a validated protein template for understanding and designing an enzyme-based electrochemical sensing elements. Using this template, LOx enzyme variants were generated and compared. Comparison of the variants demonstrates that one exhibits effective lactate sensing at significantly reduced operating voltages. Additionally, we demonstrate that the four hexahistidine-tags on each enzyme tetramer are sufficient for immobilization to create a durable, functional sensor, with no need for a covalent attachment, enabling self-immobilization and eliminating the need for additional immobilization steps. The functionality of the LOx enzyme variants was verified at physiological lactate concentrations in both human serum (0-4 mM) and artificial sweat (0-100 mM) using 3-electrode setups for analysis of the three variants in parallel. Accuracy of measurement in both artificial sweat and human serum were high. Employing a microfluidic flow cell, we successfully monitored varying lactate levels in physiological fluids continuously over a 2h period. Overall, this optimized LOx enzyme, which self-immobilizes onto gold sensing electrodes, facilitates efficient and reliable lactate detection and continuous monitoring at reduced operating voltages suitable for further development towards commercial use.
人体中的乳酸水平揭示了运动的强度和持续时间，并为诸如小儿败血症等危及生命疾病的严重程度提供了关键指标。利用来自[具体来源未提及]的乳酸氧化酶（Lox），我们在实验室环境中展示了其在生理流体中进行乳酸电化学传感的功能。通过晶体学、X射线散射和羟基自由基蛋白质足迹法验证了LOx的结构和动力学。这为理解和设计基于酶的电化学传感元件提供了一个经过验证的蛋白质模板。利用该模板，生成并比较了LOx酶变体。变体比较表明，其中一种在显著降低的工作电压下表现出有效的乳酸传感。此外，我们证明每个酶四聚体上的四个六组氨酸标签足以用于固定化，以创建一个耐用、功能性的传感器，无需共价连接，实现自固定化并消除了额外固定化步骤的需求。使用三电极设置并行分析三种变体，在人血清（0 - 4 mM）和人工汗液（0 - 100 mM）中的生理乳酸浓度下验证了LOx酶变体的功能。人工汗液和人血清中的测量准确性都很高。采用微流控流动池，我们成功地在2小时内连续监测了生理流体中变化的乳酸水平。总体而言，这种能自固定在金传感电极上的优化LOx酶，有助于在降低的工作电压下进行高效、可靠的乳酸检测和连续监测，适合进一步开发用于商业用途。
