基于电子顺磁共振技术的谷胱甘肽检测方法  

Glutathione Detection Method Based on Electron Paramagnetic Resonance Spectroscopy

作　　者：王志文 邝健 刘傲锟 魏若彤 于璐 田长麟 WANG Zhi-Wen;KUANG Jian;LIU Ao-Kun;WEI Ruo-Tong;YU Lu;TIAN Chang-Lin(Department of Chemical Physics,University of Science and Technology of China,Hefei 230026,China;High Magnetic Field Laboratory,Hefei Institutes of Physical Science,Chinese Academy of Sciences,Hefei 230031,China;Department of Life Sciences and Medicine,University of Science and Technology of China,Hefei 230026,China)

机构地区：[1]中国科学技术大学化学物理系,合肥230026 [2]中国科学院合肥物质科学研究院强磁场科学中心,合肥230031 [3]中国科学技术大学生命科学与医学部,合肥230026

出　　处：《生物化学与生物物理进展》2024年第11期3034-3045,共12页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(21825703,21927814);国家重点研发计划(2019YFA0405600,2019YFA0706900,2021YFA1200104,2022YFC3400500);中国科学院战略性先导科技专项(B类)(XDB0540200,XDB37040201);安徽省科技重大专项计划(202303a07020004);中国科学院青年创新促进会(2022455)资助项目。

摘　　要：目的谷胱甘肽(GSH)是细胞中巯基(―SH)含量最丰富的非蛋白质化合物,在提供还原当量、直接中和有毒反应物质以及维持细胞氧化还原平衡等方面发挥着至关重要的作用,因此,准确检测组织中的GSH含量具有重要意义。鉴于电子顺磁共振(EPR)技术在GSH检测方面的应用报道相对较少,本文提出了一种基于EPR技术的GSH检测方法。方法首先,将ABTS(2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸)溶液与K_(2)S_(2)O_(8)溶液混合并在避光条件下反应12~16 h制备ABTS∙^(+)溶液,借助UV-Vis对ABTS∙^(+)溶液进行浓度测定。然后,基于ABTS∙^(+)的EPR信号变化来检测GSH浓度,在此基础上探究最适的反应时间和温度,建立ABTS∙^(+)的EPR信号强度和GSH浓度之间的标准方程。最后,采用计算得到的标准曲线对C57BL/6J小鼠全血的GSH浓度进行定量分析,并与文献结果进行比较,验证方法的准确性。结果实验结果显示,本方法对GSH的检测范围(50 nmol/L~15μmol/L)分布超过两个数量级,检测限(LOD)低至0.50 nmol/L,检测到的小鼠全血中GSH含量为(10660±706)nmol/g(每克血红蛋白所含GSH纳摩尔数),与文献报道结果((11200±237)nmol/g)接近。此外我们还使用类似的方法进一步发展了对GSSG的检测方法。结论本文提出了一种基于EPR技术,使用ABTS∙^(+)快速检测GSH的方法。得益于EPR技术的独特优势,相比于传统的比色法,本方法具有更高的检测灵敏度,更宽的线性范围。我们进一步拓展了其在血液样品中的检测应用,可以快速准确地检测全血中GSH的含量,为衡量组织中氧化还原平衡状态提供数据支持,具有重要意义。Objective Glutathione(γ-glutamyl-L-cysteinylglycine,GSH)is the most abundant non-protein compound containing sulfhydryl(―SH)groups in cells.It serves as a source of reducing equivalents,effectively neutralizing harmful reactive substances,and playing a crucial role in maintaining cellular redox balance.Therefore,sensitive detection and accurate measurement of GSH levels in tissues are of great importance.In this work,we presents a novel method for GSH detection utilizing electron paramagnetic resonance(EPR)spectroscopy.Methods Initially,ABTS(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate acid))solution was mixed with K_(2)S_(2)O_(8) solution and reacted in the dark for 12 to 16 h to prepare ABTS∙^(+)solution,which was then quantified using UV-Vis spectroscopy.Subsequently,the concentration of glutathione(GSH)was determined based on the changes in the EPR signal of ABTS∙^(+).On this basis,the optimal reaction time and temperature were explored to establish a standard equation correlating the EPR signal intensity of ABTS∙^(+)with GSH concentration.Finally,the derived standard curve was employed to quantitatively analyze the GSH concentration in whole blood from C57BL/6J mice,and the results were compared with those reported in the literature to verify the accuracy of the method.Results The experimental results demonstrate that this method has a linear detection range from 50 nmol/L to 15μmol/L for GSH,spanning two orders of magnitude,with a limit of detection(LOD)at 0.50 nmol/L.The measured GSH content in mouse whole blood is(10660±706)nmol/g Hb,which agrees with the value of(11200±237)nmol/g Hb as previously reported.Furthermore,a similar method was developed for detection of glutathione disulfide(GSSG)at higher reaction temperature.Conclusion This article presents a novel assay for the rapid detection of GSH using the intensity of EPR signal from ABTS∙^(+)as indicator.This method demonstrates enhanced detection sensitivity and a broader linear range compared to conventional colorimetric methods.F

关 键 词：谷胱甘肽检测 电子顺磁共振 ABTS 

分 类 号：Q5-33[生物学—生物化学] O657.61[理学—分析化学]