Electroanalysis of Infection with Methyl Pyruvate

Area of Science:

• Electrochemistry
• Biochemistry
• Analytical Chemistry

Background:

• Leukocyte esterase (LE) is an infection enzyme relevant in human biofluids.
• Existing methods for LE detection, like ELISA, can be time-consuming and laborious.

Purpose of the Study:

• To develop a novel electroanalytical method for quantifying leukocyte esterase (LE) in human biofluids.
• To couple the LE-catalyzed hydrolysis of methyl pyruvate (MP) with an electrochemical detection system.
• To establish kinetic and analytical parameters for the developed method.

Main Methods:

• Coupling LE + MP reaction with alcohol oxidase to produce hydrogen peroxide.
• Electrochemical reduction of hydrogen peroxide at a nitrogen-doped carbon nanotube electrode.
• Kinetic analysis using turnover rate (kcat) and specificity constant (kcat/Km).
• Immuno-electroanalysis using immunosorption with internally calibrated amperometry.

Main Results:

• The method demonstrated fast enzyme kinetics with kcat = 15 s−1 and kcat/Km = 2.3 × 10^6 M−1 s−1.
• Analytical assays were rapid (5 min) with LE quantification in the clinically relevant range (22–300 μg L−1).
• Immuno-electroanalysis detected picomole quantities of LE, showing matrix independence and high spike-and-recovery rates (99–104%).
• Reduced incubation time from 4 h (ELISA) to 30 min (immuno-electroanalysis).

Conclusions:

• The developed electroanalytical method provides a sensitive, rapid, and less laborious approach for LE detection in biofluids.
• The method exhibits excellent kinetic properties and analytical performance, suitable for clinical relevance.
• The proposed immunosorption and amperometry combination is versatile for determining other enzymes forming active immune complexes.