荧光探针包被法测定血浆硫化氢浓度

摘要

目的:在荧光探针的基础上建立一种简易的方法来检测血液中的硫化氢(hydrogen sulfide,H2S).方法:将荧光探针包被到96孔板上,冷藏待用.用饱和硫酸铵沉淀血浆或血清中的蛋白,离心后所得的上清液分别加到含包被探针和不含包被探针的微孔中,37℃避光孵育2h,然后使用分光光度计(波长λEx/λEm340/445 nm)读取微孔的荧光值,计算包被有探针和相应无探针微孔的荧光差值,根据标准曲线浓度计算出血液中H2S的浓度.结果:灵敏度和特异性测试表明,此方法检测灵敏度下限可以达到0.3μmol/L,血液中其他成分甚至其他含硫活性成分和含硫氨基酸等对此方法影响甚微.应用此方法检测188名健康成年志愿者的血清H2S浓度[(12.1±3.5)μmol/L,95％ CI:4.6 ～ 19.8 μmol/L],所得结果呈正态分布(单样本K-S检验,P＞0.1).对30名高血压患者和22名相匹配的健康志愿者血清H2S浓度检测表明,前者H2S浓度低于后者[(3.52±1.49) μmol/L vs.(10.23 ±2.76) μmol/L],差异具有统计学意义(配对样本t检验,t=9.937,P＜0.001).检测雄性Wistar大鼠血清[(19.66 ±2.32) μmol/L]和血浆[(18.67 ±2.07) μmol/L]以及动脉血[(19.34 ±0.51) μmol/L]和静脉血[(18.99 ±0.50) μmol/L]中的H2S浓度,结果表明雄性Wistar大鼠血清和血浆以及动脉血和静脉血两者差异都没有统计学意义(重复测量的方差分析,P=0.38).样品的重复性检测稳定性较好(两因素方差分析,P＞0.05).结论:此方法对检测血液中的H2S具有简单、高灵敏度、特异性和可重复性等优点,出结果快,可适用于大样本的高通量检测,满足实验室基础研究及临床一次性大样本H2S浓度检测的需求.%Objective:The hydrogen sulfide (H2 S) role in pathogenesis of various diseases were wildly addressed in recent decade.The circulatory (plasma or serum) and biological fluid H2S measurement is still an enormous issues due to the technical limitation.This paper aimed to develop a novel measurement method based on fluorescence probe.Methods:Firstly,20 μL ethanol was used to dissolve 100 pmol fluorescence probe,then added in a 96-well plate.An equal volume of ethanol was also added to the blank well of the plate.The plate was placed in a dark room for about 1 h until the fluorescence probe was evenly coated in the 96-well microplate and dried.The plate was frozen at-20 ℃ for later use.Secondly,the plasma or serum sample was added with saturated ammonium sulfate buffer (pH 7.8) and then centrifuged to remove the proteins.The equal volume supernatant liquid was added to the probecoated well and the probe-uncoated well.The plate was incubated in a dark environment at 37 ℃ for 2 h.Finally,after incubation,the fluorescence density was acquired at λEx/λEm 340/445 nm in a microplate reader.The differences of the fluorescence density values between the probe-coated well and probeuncoated well were counted and H2S concentration of plasma/serum was calculated by standard curve with NaHiS.Results:The method had high sensitivity (from 0.3 to 100 μmol/L) and specificity for measuring H2S as compared with other biologically relevant reactive sulfur species and sulfur-containing amino acid.Serum H2S concentrations were assayed in 188 health volunteers using this method [(12.1 ±3.5) μmol/L,95％ CI:4.6-19.8 μmol/L],and the frequency distribution showed a normal tendency(one-sample Kolmogorov-Smirnov test,P ＞ 0.1).The serum H2S concentrations in 30 hypertension patients were decreased compared with 22 age-and gender-matched health individuals (paired-samples t test,t =9.937,P ＜ 0.001).There were no differences of H2S concentration in serum [(19.66 ±2.32) μmol/L] or plasma [(18.67 ±2.07) μmol/L],between the samples acquired from artery [(19.34 ±0.51) μmol/L] or vein [(18.99 ±0.50) μ mol/L] of male Wistar rats (repeated measurement of ANOVA,P =0.38).One week frozen samples did not affect the detection.The values of the repeated measurement did not differ (two-way ANOVA,P ＞ 0.05).Conclusion:The present method is easily performed with high sensitivity,specificity and repeatability for circulatory H2S.It is also quick and may apply for large samples.