Electroanalysis of Infection with Methyl Pyruvate.

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.