The l-lactate oxidase (LOx) based lactate sensors are widely used for clinical diagnostics, sports medicine, and food quality control. However, dissolved oxygen interference and electroactive interferent effects are inherent issues of current lactate sensors. In this paper, a quasi-direct electron transfer (quasi-DET) type lactate sensor was developed using rationally engineered Aerococcus viridans LOx (AvLOx) modified with amine-reactive phenazine ethosulfate (PES). Since the modification of wild type AvLOx by PES did not result quasi-DET, engineered AvLOx with additional Lys residue was designed. The additional Lys residue was introduced by substituting residue locating on the surface of AvLOx, and within 20 Å of the isoalloxazine ring of FMN. Among several constructed mutants, Ala96Leu/Asn212Lys double mutant showed the highest dye-mediated dehydrogenase activity with negligible oxidase activity, showing quasi-DET properties after PES modification, when the enzyme was immobilized on screen printed carbon electrode. The constructed electrode did not show oxygen interference in cyclic voltammetric analysis and distinct catalytic current with 20 mM l-lactate. The sensor performance of a chronoamperometric l-lactate sensor employing PES modified Ala96Leu/Asn212Lys AvLOx, marked with linear range between 0 and 1 mM, with sensitivity of 13 μA/mM∙cm2, and a limit of detection of 25 μM for l-lactate. By applying -200 mV vs. Ag/AgCl, l-lactate could be monitored with negligible interference from 170 μM ascorbic acid, 1.3 mM acetaminophen, 1.4 mM uric acid or 20 mM glucose. These results indicated that a quasi-DET type lactate sensor was developed that did not suffer from the interference of oxygen and representative electroactive ingredient compounds.

中文翻译：

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合理设计工程绿藻球菌乳酸氧化酶进行介体修饰，以实现准直接电子转移型乳酸传感器。

基于l-乳酸氧化酶（LOx）的乳酸传感器广泛用于临床诊断，运动医学和食品质量控制。然而，溶解氧干扰和电活性干扰效应是当前乳酸传感器的固有问题。在本文中，使用胺反应性吩嗪乙硫酸盐（PES）修饰的合理设计的拟球菌绿单球菌LOx（AvLOx），开发了一种准直接电子传输（准DET）型乳酸传感器。由于PES对野生型AvLOx的修饰未得到准DET，因此设计了带有额外Lys残基的工程AvLOx。通过取代位于AvLOx表面且在FMN的异四恶嗪环20Å之内的残基引入额外的Lys残基。在几个构造的突变体中，当酶固定在丝网印刷的碳电极上时，Ala96Leu / Asn212Lys双突变体显示出最高的染料介导的脱氢酶活性，氧化酶活性可忽略不计，在PES修饰后显示出准DET特性。所构建的电极在循环伏安分析中没有显示出氧干扰，并且在20 mM的l-乳酸盐中没有明显的催化电流。采用PES改良的Ala96Leu / Asn212Lys AvLOx的计时电流型乳酸盐传感器的传感器性能，线性范围介于0和1 mM之间，灵敏度为13μA/ mM∙cm2，对L-乳酸盐的检测极限为25μM 。通过施加相对于Ag / AgCl的-200 mV，可以用来自170μM抗坏血酸，1.3 mM对乙酰氨基酚，1.4 mM尿酸或20 mM葡萄糖的微不足道的干扰监测l-乳酸。