酶荧光毛细管分析法测定微量血清中乳酸脱氢酶活性

基于丙酮酸/还原型辅酶I/乳酸脱氢酶(LDH)/乳酸/氧化型辅酶I荧光猝灭体系和荧光毛细管分析技术,建立了可用于微量样品中LDH酶活性测定的方法。优化的测定条件为:激发及发射波长分别为350和460nm;测定温度为25℃;酸度为pH 6.5;NADH浓度为300μmol/L;丙酮酸浓度为1.2mmol/L。本方法的测定范围为50～1500IU/L,检出限为30IU/L,相对标准偏差2.1%～2.2%(n=10),回收率在96.4%～105%范围内。本方法操作简单,每次测定仅需样品2.0μL、试剂18.0μL,分析速度约为30样/h,利用本方法测定了微量血清中LDH的活性。

An Novel Enzyme Fluorescence Capillary Analysis for Determination of Lactate Dehydrogenase Activity in Micro-volume Serums

Based on a fluorescence quenching reaction system of pyruvic acid / reduced form of nicotinamide-adenine dinucleotide(NADH)/ lactate dehydrogenase(LDH)/ lactic acid / oxidized form of nicotinamide-adenine dinucleotide and a technology of the fluorescence capillary analysis,we proposed a new method for determining the LDH′s activity in micro-volume samples.First,the optimum conditions for the method were as follows : the excitation and the emission wavelength were 350nm and 460 nm respectively;the temperature was 25 ℃;the acidity was pH 6.5;the concentration of NADH was 300 μmol / L;the concentration of pyruvate was 1.2 mmol / L.Afterwards,the measurement range(50-1500IU / L),the detection limit(30IU / L),the relative standard deviation(2.1%-2.2%,n=10) and the recovery(96.4%-105%) of the method were attained.This method is easy to operate,the dosages of the sample and reagents are only 2.0 μL and 18.0 μL,res pectively,and its analytical speed is 30samples per hour.The LDH′s activity in the serums was successfully determined by utilizing this method.