CN105181666B - 一种荧光检测半胱氨酸的试剂和方法 - Google Patents
一种荧光检测半胱氨酸的试剂和方法 Download PDFInfo
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Abstract
本发明提供了一种荧光检测半胱氨酸的试剂和方法。本发明提供的检测半胱氨酸的试剂,它是一种色烯衍生物MOPCC(7‑methyl‑1‑oxo‑1,2,3,3a‑tetrahydrocyclopenta[b]chromene‑5‑carbaldehyde)。本发明提供的检测Cys的方法,是基于色烯衍生物MOPCC,在DMSO/HEPES(v/v,1:1,pH 7.4)溶液中定量地检测半胱氨酸的含量。该检测方法,对半胱氨酸显示了高的灵敏性和选择性,检测过程简便、灵敏、快速,检测结果准确。
Description
技术领域
本发明涉及半胱氨酸检测分析技术,具体涉及一种荧光检测半胱氨酸的试剂和方法。
背景技术
L-半胱氨酸(L-Cys)是20种天然氨基酸中唯一含有还原性基团巯基的氨基酸。作为一种人体必需的氨基酸,Cys参与细胞内谷胱甘肽、蛋白质的合成,承担着解毒、消炎和抗衰老的作用。同时,研究表明人体内非正常的Cys水平与儿童生长缓慢、肝损伤、皮肤损伤及肌肉损伤等疾病有着密切的关系。快速、准确地检测样品中的Cys含量对于疾病诊断具有极其重要的意义。目前,对Cys检测分析的方法主要有色谱法(张苏,王凤山.高效液相色谱法测定酶促反应液中的L-半胱氨酸含量[J].中国生化药物,2009,30(5):318-320)、光度法(陈亚红,李占灵,张会霞.酶抑制动力学光度法测定L-半胱氨酸[J].分析试验室,2008,27(1):38-41)、电化学方法(Vandeberg,P.J.,Johnson,D.C.Comparison of pulsedamperometric detection and integrated volatmmetrc detection for inorganicsulfur compounds in liquid chromatography[J].Anal.Chim.Acta,1994,290,317-327;Maleki,N.,Safavi,A.,Sedaghati,F.,et al.Efficient electrocatalysis of L-cysteine oxidation at carbon ionic liquid electrode[J].Anal.Biochem,2007,369:149-153)。然而,这些检测方法程序繁琐、设备昂贵,且无法用于细胞内Cys的检测。所以,发展新的过程简单、精度高、成本低、适用性广的检测Cys的方法非常必要。
本发明中,我们合成了一种色烯化合物MOPCC,基于Cys与化合物的“点击”开环反应引起的荧光变化,实现对Cys的检测。
发明内容
本发明的目的是提供一种合成简单,选择性高,适用性广的定量检测Cys的试剂,以及该试剂在Cys检测中的应用。
本发明提供的检测Cys的方法,是基于一种色烯衍生物MOPCC(7-methyl-1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromene-5-carbaldehyde),在DMSO/HEPES(v/v,1:1,pH 7.4)溶液中定量地检测Cys。该检测方法对Cys显示了优良的灵敏性和选择性,检测过程简便、灵敏、快速,检测结果准确。
MOPCC的合成:取pyridinium chlorochromate(PCC)(2mmol,0.42g)分散于20mLdichloromethane中,将5-(hydroxymethyl)-7-methyl-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one(HMMPC)(2mmol,0.46g)逐渐加入体系。室温搅拌20h后,减压蒸干溶剂,粗产品经柱色谱分离(ethyl acetate:petroleum=1:1)得到黄色纯品。
MOPCC检测Cys的方法,步骤为:
(1)、配制pH=7.4、浓度为10mM的HEPES缓冲溶液,配制2mM MOPCC的DMSO溶液,配制2mM Cys的水溶液;
(2)、按体积比40:1,将DMSO/HEPES(v/v,1:1,pH 7.4)和MOPCC的DMSO溶液加到干净的荧光比色皿中,在荧光分光光度仪上检测,随着待测样Cys的加入,515nm的荧光强度逐渐增强;
(3)、取2mL的DMSO/HEPES(v/v,1:1,pH 7.4)溶液、50μLMOPCC的DMSO溶液加到另一个荧光比色皿中,分别加入体积为5、10、15、20、25、30、35、40、45μL的Cys溶液时,在荧光分光光度仪上测定515nm对应的荧光强度F为761、1128、1410、1764、2047、2311、2598、2904、3027,以Cys浓度为横坐标,以相对荧光强度F-F0为纵坐标绘制图,F0﹦328,得到Cys浓度的工作曲线;线性回归方程为:F-F0=62.12c+120.4,c的单位为μM;
(4)、取2mL的DMSO/HEPES(v/v,1:1,pH 7.4)溶液、50μLMOPCC的DMSO溶液加入荧光比色皿中,用微量进样器吸取VμL待测样品溶液,加入到此干净的荧光比色皿中,在荧光分光光度仪上检测,将测得的荧光强度代入(3)的线性回归方程,得到浓度c,待测样品C待测样=2000×c×10-6/V,单位M。
与现有技术相比,本发明具有如下有益效果:1、检测体系成本低廉,试剂由本课题组之前报道的HMMPC和PCC在室温下制得,原料便宜,反应条件简单,易于大规模生产;2、本发明的检测方法对Cys显示出高的选择性和灵敏性,其它氨基酸对Cys的测定没有干扰;3、检测过程在水溶液中进行;4、检测手段简单,只需要借助荧光分光光度仪即可实现。
附图说明:
图1实施例1MOPCC与Cys作用的荧光发射图
图2实施例2MOPCC与各种分析物的荧光柱状图
图3实施例3测定Cys的工作曲线
图4实施例4测定样品的荧光发射图
图5a MOPCC氢谱表征
图5b MOPCC碳谱表征
图5c MOPCC质谱表征
具体实施方式:
实施例1
配制pH=7.4、浓度为10mM的HEPES缓冲溶液,配制2mM MOPCC的DMSO溶液,配制2mMCys的水溶液;取2mL的DMSO/HEPES(v/v,1:1,pH 7.4)溶液、50μLMOPCC的DMSO溶液加到一个荧光比色皿中,取Cys的溶液,逐渐用微量进样器加到此比色皿中,边加样边在荧光分光光度仪上检测(365nm激发),随着Cys的加入,515nm处荧光强度逐渐增强。荧光发射图见图1。
实施例2
配制pH=7.4、浓度为10mM的HEPES缓冲溶液,配制2mM MOPCC的DMSO溶液,配制2mMCys,Ala,Arg,Hcy,GSH,Asn,Asp,Gln,Glu,Gly,His,Ile,Leu,Lys,Met,Ser,Thr,Trp,Phe,Pro,Tyr,Val的水溶液;在22个荧光比色皿中,各加入2mL的DMSO/HEPES(v/v,1:1,pH 7.4)溶液、50μLMOPCC的DMSO溶液,再分别加入1摩尔当量的Cys,以及5摩尔当量的各种分析物:Cys,Ala,Arg,Hcy,GSH,Asn,Asp,Gln,Glu,Gly,His,Ile,Leu,Lys,Met,Ser,Thr,Trp,Phe,Pro,Tyr,Val在荧光分光光度仪上检测(365nm激发),绘制不同分析物对应的515nm处荧光强度的柱状图,(见图2)。Cys使得MOPCC的荧光强度由292变到3299,其它的分析物基本没有引起MOPCC荧光强度的变化。
经实验证明,其它分析物不干扰体系对Cys的测定。
实施例3
配制pH=7.4、浓度为10mM的HEPES缓冲溶液,配制2mM MOPCC的DMSO溶液,配制2mMCys的水溶液;取2mL的DMSO/HEPES(v/v,1:1,pH 7.4)溶液、50μLMOPCC的DMSO溶液加到一个荧光比色皿中,分别在加入5、10、15、20、25、30、35、40、45μL的Cys溶液时,在荧光分光光度仪上测定515nm对应的荧光强度F为761、1128、1410、1764、2047、2311、2598、2904、3027(365nm激发),以Cys浓度为横坐标,以相对荧光强度F-F0为纵坐标绘制图,F0﹦328,得到Cys浓度的工作曲线(见图3);线性回归方程为:F-F0=62.12c+120.4,c的单位为μM;
实施例4
配制pH=7.4、浓度为10mM的HEPES缓冲溶液,配制2mM MOPCC的DMSO溶液,配制2mMCys的水溶液;取2mL的DMSO/HEPES(v/v,1:1,pH 7.4)溶液、50μLMOPCC的DMSO溶液加到一个荧光比色皿中,取Cys的溶液28.5μL,用微量进样器加到此比色皿中,同时在荧光光谱仪上测定515nm的对应的荧光强度F为2243(365nm激发),相对荧光强度F﹣F0﹦1915,通过实施例4的线性回归方程,求得c=28.89×10-6mol/L,偏差为1.4%。见图4。
实施例5
取pyridinium chlorochromate(PCC)(2mmol,0.42g)分散于20mLdichloromethane中,将5-(hydroxymethyl)-7-methyl-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one(HMMPC)(2mmol,0.46g)逐渐加入体系。室温搅拌20h后,减压蒸干溶剂,粗产品经柱色谱分离(ethyl acetate:petroleum=1:1)得到黄色纯品。
1H NMR(DMSO-d6,300MHz):δ(ppm):2.18(m,J=21,2H),2.35(s,3H),2.75(q,J=18.9,2H),5.49(d,J=16.2,1H),7.35(s,1H),7.57(s,1H),7.64(s,1H),10.37(s,1H)(图5a).13C NMR(DMSO-d6,75MHz)δ19.2,26.9,36.1,75.4,122.6,122.9,124.7,128.9,130.6,132.6,136.4,154.7,187.8,200.3(图5b).ESI-MS m/z:[probe+H]+Calcd forC14H13O3289.08;Found 288.92(图5c)。
Claims (2)
1.一种检测半胱氨酸的试剂:特征在于它是一种色烯衍生物MOPCC,其结构式为:
2.一种检测半胱氨酸的方法:其特征在于,步骤为:
(1)、配制pH=7.4、浓度为10mM的HEPES缓冲溶液,配制2mM如权利要求1所述的MOPCC的DMSO溶液,配制2mM Cys的水溶液;
(2)、按体积比40:1,将体积比1:1、pH 7.4的DMSO/HEPES溶液和MOPCC的DMSO溶液加到干净的荧光比色皿中,在荧光分光光度仪上检测,随着待测样Cys的加入,515nm的荧光强度逐渐增强;
(3)、取2mL的体积比1:1、pH 7.4的DMSO/HEPES溶液、50μLMOPCC的DMSO溶液加到另一个荧光比色皿中,分别加入体积为5、10、15、20、25、30、35、40、45μL的Cys溶液时,在荧光分光光度仪上测定515nm对应的荧光强度F为761、1128、1410、1764、2047、2311、2598、2904、3027,以Cys浓度为横坐标,以相对荧光强度F-F0为纵坐标绘制图,F0﹦328,得到Cys浓度的工作曲线;线性回归方程为:F-F0=62.12c+120.4,c的单位为μM;
(4)、取2mL的体积比1:1、pH 7.4的DMSO/HEPES溶液、50μLMOPCC的DMSO溶液加入荧光比色皿中,用微量进样器吸取VμL待测样品溶液,加入到此干净的荧光比色皿中,在荧光分光光度仪上检测,将测得的荧光强度代入步骤(3)的线性回归方程,得到浓度c,待测样品半胱氨酸的浓度C待测样=2000×c×10-6/V,单位M。
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