微量乳酸脱氢酶毛细管电泳在线反应电化学检测方法的研究

以乳酸脱氢酶催化乳酸与NAD⁺反应生成丙酮酸与NADH和安培法检测NADH为基础,利用电泳中介微分析(EMMA)技术,研究了超微量乳酸脱氢酶的毛细管电泳在线反应的电化学检测方法,并从理论上对EMMA电泳图中的平台宽度和高度作了初步探讨.结果表明,在EMMA的恒高压和零高压两种模式下,使用直径为150μm和束状碳纤维电极,在+0.8V检测电位下,对LDH活性检测灵敏度分别为1.1nU和0.6nU;所导出的平台高度、宽度与实验条件的关系式对提高毛细管电泳分离效率和检测灵敏度有一定指导意义.

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.