Studies on an on-line reaction of micro lactate dehydrogenase in capillary electrophoresis using electrochemical detection

The method for an on-line reaction of micro lactate dehydrogenase in capillary electrophoresis using electrophoretically mediated micro-assay (EMMA) coupled with electrochemical detection (EC) was developed. The running electrolyte (pH 9.2) was composed of 50 mmol/L Tris-HCl, 1.0 mmol/L nicotinamide adenine dinuceotide (NAD+), 5.0 mmol/L lithium lactate and 0.2 mmol/L cetylpridinium bromide (CPB). LDH was injected into the capillary. As it migrated electrophoretically in the capillary, LDH catalyzed the reaction between lactate and NAD+. The product NADH of the enzyme-catalyzed reaction was monitored by a bundle of carbon fiber electrode (150 μm diameter) held at +0.8 V vs. SCE. The detection limits for LDH activity using constant-voltage mode and zero-voltage mode in EMMA were determined to be 1.1 × 10-9 U and 6.0 × 10-10 U, respectively. The formulae concerning the width or the height of the plateau in the electropherogram for EMMA were deduced, and some significant conclusions were drawn from these formulae. The developed method has been used for the determination of ultramicro LDH activity in serum with satisfactory results.