The discovery of infection enzyme leukocyte esterase (LE) hydrolyzing a mitochondrial substrate methyl pyruvate (MP) was explored in the development of electroanalytical methods for LE in human biofluids. The LE + MP reaction was coupled with alcohol oxidase to produce hydrogen peroxide, which was then reduced at a nitrogen-doped carbon nanotube electrode at -0.20 V, yielding current proportional to the LE content in a sample. The kinetic assays revealed a fast turnover (kcat = 15 s-1) and high specificity constant (kcatKm-1 = 2.3 × 106 M-1 s-1) for the LE-triggered hydrolysis of MP. The analytical assays were short (5 min) and the quantified LE was in the clinically relevant range of 22-300 μg L-1 (R2, 0.985). The immuno-electroanalysis could detect the picomole quantity of LE and yielded linear calibration plots up to 150 μg L-1 of LE with the same slope regardless of the sample matrix (urine, saliva, and phosphate buffer). The spike-and-recovery experiments displayed an LE recovery of 99-104%. The amperometric immunoassay of LE was less laborious than traditional enzyme-linked immunosorbent assay (ELISA) for LE and reduced the required sample incubation time from 4 h (sandwich ELISA) to 30 min (immuno-electroanalysis). The proposed combination of immunosorption with internally calibrated amperometry can also be used for a selective determination of other enzymes, which form enzymatically active immune complexes.

中文翻译：

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丙酮酸甲酯感染的电分析。

在人类生物流体中LE的电分析方法的开发中，探索了水解线粒体底物丙酮酸甲酯（MP）的感染酶白细胞酯酶（LE）的发现。LE + MP反应与醇氧化酶偶联生成过氧化氢，然后在-0.20 V的氮掺杂碳纳米管电极上将其还原，从而产生与样品中LE含量成比例的电流。动力学测定揭示了LE触发的MP水解的快速转换（kcat = 15 s-1）和高特异性常数（kcatKm-1 = 2.3×106 M-1 s-1）。分析测定时间短（5分钟），定量的LE在临床相关范围内22-300μgL-1（R2，0.985）。免疫电分析可以检测LE的皮孔量，并生成线性校准图，其线性高至LE的L-1线性度相同，而与样品基质（尿液，唾液和磷酸盐缓冲液）无关。加标回收实验显示LE回收率为99-104％。LE的安培免疫测定比LE的传统酶联免疫吸附测定（ELISA）省力，并将所需的样品孵育时间从4 h（夹心ELISA）减少到30 min（免疫电分析）。提议的免疫吸附与内部校准的电流分析法的组合也可用于选择性确定形成酶活性免疫复合物的其他酶。加标回收实验显示LE回收率为99-104％。LE的安培免疫测定比LE的传统酶联免疫吸附测定（ELISA）省力，并将所需的样品孵育时间从4 h（夹心ELISA）减少到30 min（免疫电分析）。提议的免疫吸附与内部校准的电流分析法的组合也可用于选择性确定形成酶活性免疫复合物的其他酶。加标回收实验显示LE回收率为99-104％。LE的安培免疫测定比LE的传统酶联免疫吸附测定（ELISA）省力，并将所需的样品孵育时间从4 h（夹心ELISA）减少到30 min（免疫电分析）。所提出的免疫吸附与内部校准的电流分析法的组合也可用于选择性测定形成酶活性免疫复合物的其他酶。