基于NAD^+/NADH比率荧光探针的乳酸检测法

A Method of Lactate Assay Based on NAD^+/NADH Fluorescent Sensor

结合氧化型烟酰胺腺嘌呤二核苷酸/还原型烟酰胺腺嘌呤二核苷酸(NAD+/NADH)比率荧光探针SoNar与酶法分析技术,建立了一种灵敏的乳酸检测方法,并进一步对影响检测性能的几个因素,如pH、温度、底物浓度、SoNar浓度等进行了优化.结果显示,该方法的检测限(LOD)为2.60μmol/L,定量限(LOQ)为8.67μmol/L,不同时间重复测定的标准偏差(RSD)为2.37%,乳酸质量浓度0~1000μmol/L范围内的Z因子值为0.82,表明该方法具备很好的稳定性和重复性.与目前常用的基于MTT-PMS的酶偶联分析法相比,本文所建立的乳酸检测法灵敏度更高,特异性更好,且生物兼容性更强.

Lactate is the final metabolite of glycolysis, which is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. As an important marker in glycolysis pathway, lactate is closely related to the occurrence of cancer, diabetes, inflammation and neurodegeneration. The typical methods of chromatography and electrochemistry for lactate determination are time-consuming and complicated to operate, so the determination of lactate is usually based on the enzymatic reaction between lactate and cofactor oxidized nicotinamide adenine dinucleotide (NAD+). However, due to the weak fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and interference from other autofluorescent biological molecules, the sensitivity of this method is low. Therefore, it ’s necessary to develop a new method with improved sensitivity for lactate determination. In this paper, lactate dehydrogenase combined with genetically encoded NAD^+/NADH sensor SoNar for determination of lactate is reported. Lactate dehydrogenase catalyzes the interconversion of lactate and pyruvate coupled with interconversion of NAD^+ and NADH. Changes in NAD^+, NADH, and their ratio can be sensitively and immediately recognized by SoNar. The fluorescence signal of SoNar was monitored by a microplate reader. Fluorometric detection was carried out with excitation wavelength at (420 ± 10)nm or (485 ± 10)nm and emission wavelength of (528 ± 20)nm. The detection conditions, including pH, temperature, substrate and SoNar concentration, were optimized. The lower detection limit (LOD) of this method was 2.60 μmol/L and the limit of quantitation (LOQ) was 8.67 μmol/L. The relative standard deviation (RSD) was 2.37% and the Z-factor was 0.82 in the range between 0 and 1 000 μmol/L, indicating the good stability and repeatability of this method. In addition, compared with method based on MTT-PMS, the developed novel method showed improved sensitivity, specificity and biocompatibility;and the efficiency of this method was immune to the presence of nicotinamide adenine dinucleotide phosphate (NADPH), thereby providing a useful tool for lactate metabolism studies.