CN105424670A - 一种检测溶液或细胞中10-7~105m低浓度h2o2的方法 - Google Patents
一种检测溶液或细胞中10-7~105m低浓度h2o2的方法 Download PDFInfo
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Abstract
本发明一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法,属分析化学领域。以化合物(E)-2-(2,4-二羟基苯基)乙烯基-8-羟基喹啉为检测微量H2O2的荧光探针。在乙腈/H2O溶液(v/v,3/2,pH?11)中,利用探针的特征荧光强度或吸光度随H2O2浓度变化,其它共存氧化或还原物质不干扰测定。荧光法和紫外吸收法检测H2O2的浓度线性范围分别为8.3×10-7~4.3×10-5M和8.5×10-7~5.2×10-5?M,检测限分别为2.3×10-8?M和8.9×10-7M。目视检测H2O2,日光和紫外灯下探针溶液颜色由橙红色变为无色。在活细胞内H2O2使探针荧光猝灭,用荧光成像可视检测细胞内H2O2。
Description
技术领域
本发明属分析化学领域,具体地说是一种用荧光、紫外-可见吸收检测微量H2O2及细胞成像的探针方法。
背景技术
生物相关分子、离子的识别检测在生命、健康及环境研究中尤为重要。小分子荧光探针具有结构简单、合成简便、成本低廉、水溶性好、对细胞毒性小等优点,将其应用于生物体系的分子、离子检测具有灵敏度高、选择性好、可视性强、快速、简便的特点。H2O2在体内继续分解生成超氧阴离子O2 -等多种自由基,参与生物细胞的衰老过程,在病理条件、慢性病及正常生理过程中发挥着关键作用。过氧化氢进入人体后极易转变成羟基自由基(×OH),这是目前所知活性氧中对生物毒性最强、危害最大的一种自由基,可以直接或间接氧化细胞内大分子(如核酸、蛋白),破坏细胞膜结构,加速细胞衰老与凋亡,对人体产生极大的危害。有关过氧化氢的测定研究日益受到重视,建立过氧化氢的准确测定方法,从而有效控制过氧化氢的投放量和残留量显得尤为重要。目前,过氧化氢的测定主要有电化学法、高效液相色谱法、化学发光法、荧光法、光度法等。其中分光光度法操作简单、直接,得到较广泛的应用。
荧光探针与荧光显微技术的结合,使荧光探针在生物活性物质检测和细胞成像方面得到广泛应用。对生物活性物质而言,检出的灵敏度极限是单个分子而对测试技术有更高的灵敏度要求,同时也有更苛刻的测试条件要求。荧光成像分析是目前广泛应用于活细胞分析的一种高灵敏的可视化分析技术。活细胞成像技术是生命科学技术领域中重要的研究手段,与固定细胞研究结合起来,可以解释活细胞中的多种生命现象。
发明内容
本发明目的在于建立一种荧光探针技术,用荧光和紫外-可见吸收光谱方法高灵敏、高选择性、简便、快速的检测溶液或细胞中低浓度微量H2O2光谱测定方法;用荧光显微镜可视性检测活细胞中H2O2的探针成像方法。
本发明一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法是以化合物(E)-2-(2,4-二羟基苯基)乙烯基-8-羟基喹啉为检测H2O2的荧光或比色探针试剂,简称探针,其结构如下:
检测方法为:在乙腈/H2O(v/v,3/2,pH11)介质中(1)荧光光谱法检测H2O2,以415nm为激发波长,测定探针在580nm处的荧光强度降低随H2O2浓度变化,用校正曲线法检测;(2)紫外吸收光谱法检测H2O2,测定探针在410nm处的吸光度降低随H2O2浓度变化,用校正曲线法检测;(3)利用探针的颜色变化目视检测H2O2:日光下探针溶液颜色随H2O2的加入由橙红色变为无色;在365nm紫外灯下探针溶液发射荧光颜色随H2O2的加入由橙红色变为无色;(4)在活细胞内利用H2O2使探针的荧光猝灭,用荧光成像可视检测细胞内H2O2。
所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法是在浓度为10μM探针的乙腈/H2Ov/v,3/2,pH=11溶液中,用荧光或紫外-可见吸收法检测H2O2,检测时反应时间控制在20~30min。
所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法是探针荧光和紫外-可见吸收法检测H2O2时,其它共存氧化剂或还原剂:NO3 -,NO2 -,t-BuOOH(过氧化氢叔丁醇),ClO-,GSH(谷胱甘肽),O2 -,t-BuO×(叔丁氧基自由基),×OH(羟基自由基)在浓度与H2O2相同时,不干扰探针对H2O2的测定。
所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法是探针用荧光法检测H2O2的浓度线性范围为8.3×10-7~4.3×10-5M,检测限为2.3×10-8M;紫外-可见吸收法检测H2O2的浓度线性范围为8.5×10-7~5.2×10-5M,检测限为8.9×10-7M。
所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法是所述探针的荧光成像检测活性细胞内H2O2方法为:活性PC3细胞经复苏、传代、接种、培养、清洗后,将细胞浸入pH值为11的含10mM探针的培养液,在37℃,5%CO2以及饱和湿度为100%的培养箱中孵育1h,吸出含探针的培养液,用新鲜培养基清洗细胞,用荧光显微镜检测,呈现清晰的红色荧光细胞图像;再将上述细胞浸入含20mMH2O2的培养液中孵化10min后,吸出含H2O2的培养液,用新鲜培养基清洗细胞3次,用荧光显微镜检测,观察到微弱的红色荧光细胞图像;再经H2O2孵化30min后,用荧光显微镜检测,观察不到细胞的荧光图像。
所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法是所用的试剂为分析纯或生化试剂,所用水为二次蒸馏水或生理盐水;所用活性PC3细胞为人体前列腺癌细胞;所用的拍照设备为荧光倒置显微镜。
本发明一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法有别于其他H2O2的测定方法:(1)探针可同时用于荧光和紫外-可见吸收法检测,方法简便、快速灵敏,无需加入过多试剂;(2)荧光光谱法检测H2O2的检出限低至2.3×10-8M,且不受常见氧化物或还原物的干扰。检测方法不仅适宜在生物样品、极小体系等特殊环境中使用,而且便于荧光成像应用;(3)不仅可用于荧光和吸收光谱定量及目视法定性检测H2O2,用于荧光成像可视化检测活细胞中H2O2;(4)测量方式多样,应用前景好;(5)可用于低浓度微量H2O2的定性定量分析;(6)本发明所选用的探针试剂是用于溶液或细胞中H2O2定性定量的最新试剂。
附图说明
图1探针与氧化剂或还原剂作用的荧光光谱图:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,分别加入氧化剂或还原剂:1mM的NO3 -、NO2 -、t-BuOOH(过氧化氢叔丁醇)、ClO-;0.5mM的GSH(谷胱甘肽)、O2 -;100mM的t-BuO×(叔丁氧基自由基)、×OH(羟基自由基),反应30min后,没有观察到荧光光谱明显的变化;而加入50μM的H2O2反应30min后,探针在580nm处的荧光峰猝灭。表明在此条件下探针仅对H2O2有识别检测作用。激发波长为415nm。
图2探针与氧化剂或还原剂在不同作用时间的荧光强度变化图:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,分别加入氧化剂或还原剂:1mM的NO3 -、NO2 -、t-BuOOH(过氧化氢叔丁醇)、ClO-;0.5mM的GSH(谷胱甘肽)、O2 -;100mM的t-BuO×(叔丁氧基自由基)、×OH(羟基自由基),分别反应1min、5min、10min、30min后,没有观察到荧光光谱明显的变化;而加入50μM的H2O2反应30min后,探针在580nm处的荧光峰猝灭,表明在此条件下探针仅对H2O2有识别检测作用。激发波长为415nm。
图3探针与氧化剂或还原剂作用的紫外-可见吸光谱图:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,分别加入氧化剂或还原剂:1mM的NO3 -、NO2 -、t-BuOOH、ClO-;0.5mM的GSH、O2 -;100mM的t-BuO×、×OH,反应30min后,没有观察到紫外吸收光谱明显的变化;而加入50μM的H2O2反应30min后,探针在410nm处的吸收峰降低。表明在此条件下探针仅对H2O2有识别检测作用。
图4探针与氧化剂或还原剂的紫外-可见吸收强度变化图:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,分别加入氧化剂或还原剂:1mM的NO3 -、NO2 -、t-BuOOH、ClO-;0.5mM的GSH、O2 -;100mM的t-BuO×、×OH,分别反应1min、5min、10min、30min后,没有观察到紫外吸收光谱明显的变化;而加入50μM的H2O2反应30min后,探针在410nm处的吸收峰降低,表明在此条件下探针仅对H2O2有识别检测作用。
图5探针荧光光谱检测H2O2的反应时间曲线:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,加入50μM的H2O2到探针溶液中,测定不同反应时间下探针溶液的荧光光谱及探针在580nm处的荧光强度随时间的变化,随时间的增长,在580nm处的荧光强度逐渐降低至猝灭,反应时间在20~30min内荧光强度的降低保持稳定。插图为荧光强度值随反应时间的变化,测试的激发波长为415nm。
图6探针紫外-可见吸收光谱检测H2O2的反应时间曲线:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,加入50μM的H2O2到探针溶液中,测定不同反应时间下探针溶液的紫外-可见吸收光谱及探针在410nm处的吸光度的变化,随时间的增大,在410nm处吸光度逐渐降低,反应时间在20~30min内吸光度的降低保持稳定,插图为吸光度值随反应时间的变化。
图7不同浓度的H2O2对探针的荧光光谱滴定图:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,分别加入不同浓度H2O2到探针溶液中,反应20min后,随H2O2的加入,测得荧光光谱滴定曲线,探针在580nm处的荧光强度随H2O2浓度的增加逐渐降低至猝灭,测试的激发波长为415nm。
图8不同浓度的H2O2对探针的紫外-可见吸收光谱滴定图:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,分别加入不同浓度H2O2到探针溶液中,反应20min,随H2O2的加入,测得紫外-可见吸收光谱滴定曲线,探针在410nm处的吸光度随H2O2浓度的增加逐渐降低。
图9共存氧化剂或还原剂对探针荧光法检测H2O2的影响:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,加入50μM的H2O2反应20min后,探针的荧光强度在580nm逐渐降低至猝灭;再分别向探针-H2O2混合溶液中加入其他不同氧化剂或还原剂:1mM的NO3 -、NO2 -、O2 -、t-BuOOH、ClO-、GSH;100mM的t-BuO×、×OH后测其荧光强度变化,黑色条表示在探针中加入不同氧化剂或还原剂在波长580nm处的荧光强度,白色条表示在探针-H2O2混合溶液再分别加入上述其他共存氧化剂或还原剂后在波长580nm处的荧光强度变化,表明探针检测H2O2的荧光强度不受上述其他氧化剂或还原剂共存的影响,测试的激发波长为415nm。
图10共存氧化剂或还原剂对探针紫外-可见吸收法检测H2O2的影响:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)中,加入50μM的H2O2反应20min后,探针在410nm处的吸光度逐渐降低至猝灭;再分别向探针-H2O2混合溶液中加入其他不同氧化剂或还原剂:1mM的NO3 -、NO2 -、O2 -、t-BuOOH、ClO-、GSH;100mM的t-BuO×、×OH后测其吸光度变化,黑色条表示在探针中加入不同氧化剂或还原剂在波长410nm处的吸光度,白色条表示在探针-H2O2混合溶液再分别加入上述其他共存氧化剂或还原剂后在波长410nm处的吸光度变化,表明探针检测H2O2的吸光度不受上述其他氧化剂或还原剂共存的影响。
图11探针检测H2O2的荧光光谱法校正曲线:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中分别加入不同浓度H2O2,反应20min后测定荧光光谱,纵坐标为580nm处荧光强度值,横坐标为H2O2的浓度,H2O2浓度在0~45μM范围内与探针的荧光强度成线性关系,激发波长为415nm。
图12探针检测H2O2的紫外-可见吸收光谱法校正曲线:
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中分别加入不同浓度H2O2,反应20min后测定吸收光谱,纵坐标为410nm处吸光度,横坐标为H2O2的浓度,H2O2浓度在0~50μM范围内与探针的吸光度成线性关系。
图13探针检测H2O2的溶液颜色变化:
左图为可见光下观察,10μM探针的颜色为橙红色,当加入50μMH2O2反应20min后,探针检测H2O2溶液颜色由橙红色变为无色。右图为365nm紫外灯下观察,探针的颜色为橙红色,当加入H2O2反应20min后,探针检测H2O2溶液颜色由橙红色变为无色。
图14探针的荧光成像方法对活性细胞内H2O2检测的荧光显微镜拍摄照片:
a:在pH=11的HEPES-NaOH介质中,10μM探针对PC3细胞染色的暗场荧光显微照片,用荧光显微镜的红色通道(激发波长为510~550nm)检测,观测到细胞的明亮红色荧光图像;
b:再将经过pH=11的10mM探针的培养液孵化过的细胞浸入含50mM的H2O2的培养液中,孵化10min后,用荧光显微镜的红色通道检测,观测到细胞的微弱红色荧光图像;
c:b述细胞继续孵化30min后,用荧光显微镜的红色通道检测,观测不到细胞的荧光图像;
d:在pH=11的HEPES-NaOH介质中,10μM探针对PC3细胞染色后加H2O2孵化20min后的明场显微照片。
具体实施方式
实施例一:本发明中各溶液、试剂的配制方法:
(1)探针溶液的配制方法:称取探针28mg(分子式:C17H13NO3分子量:279.09),用乙腈溶解,配制成100mL溶液,浓度为1mM;
(2)HEPES缓冲溶液:用浓度为50mM的4-羟乙基哌嗪乙磺酸(HEPES)和50mM的NaOH配制,调pH至所需;
(3)pH为11的探针溶液的配制方法:称取探针2.8mg(分子式:C17H13NO3分子量:279.09),用乙腈/水(v/v,1/20)溶解,用HEPES-NaOH(50mM)调节pH11,配制成100mL溶液,浓度为100mM;
(4)氧化剂或还原剂溶液:取分析纯的各种氧化剂或还原剂,用二次蒸馏水溶解,并配制成浓度为50mM或100mM的水溶液;叔丁氧基自由基(t-BuO×)的配制是用二次蒸馏水溶解100mM七水合硫酸亚铁(FeSO4×7H2O)和10mM过氧化氢叔丁醇(t-BuOOH),羟基自由基(×OH)的配制是用二次蒸馏水溶解100mM七水合硫酸亚铁(FeSO4×7H2O)和10mM双氧水(H2O2);
(5)75%的乙醇溶液:无水乙醇75mL加蒸馏水至100mL,混匀,室温保存备用;
(6)磷酸盐缓冲溶液(D-hanks平衡盐溶液):0.4gKCl、0.06gKH2PO4、8.0gNaCl、1.0g葡萄糖、0.35gNaHCO3、0.152gNa2HPO4·12H2O、10万IU双抗,调整pH为7.2~7.4,去离子水定容至1000mL,针式滤器(0.22um进口微孔滤膜)过滤除菌,分装备用;
(7)1万单位(IU)/mL双抗溶液:将青霉素钠(80万单位)溶于40mLD-hanks溶液中,配成终浓度2万单位/mL;将硫酸链霉素(160万单位)溶于80mLD-hanks溶液中,配成终浓度2万单位/mL。分别取等体积的青霉素钠溶液和硫酸链霉素溶液混合,得到青霉素钠和硫酸链霉素的终浓度均为1万单位/mL的溶液;针式滤器(0.22um进口微孔滤膜)过滤除菌,分装1mL/支,-20℃保存备用;
(8)0.25%胰蛋白酶:称取0.25g胰蛋白酶,溶解于100mL的D-hanks液中,针式滤器(0.22um进口微孔滤膜)过滤除菌,分装1mL/支,-20℃保存备用;
(9)0.02%乙二胺四乙酸(EDTA):将0.02gEDTA,溶解于100mL的D-hanks液中,针式滤器(0.22mm进口微孔滤膜)过滤除菌,分装1mL/支,-20℃保存备用;
(10)培养液:用无菌移液管量取10mL已灭活的胎牛血清、90mL培养基(改良型RPMI-1640)以及1mL双抗液混合于100mL的无菌培养瓶中,2~8℃保存备用。
所用试剂为分析纯或生物试剂,试验用水为二次蒸馏水。
本发明所用荧光分光光度计型号为CaryEclipse荧光分光光度计,美国VARIAN公司生产;ThermoFisher8000储水型CO2细胞培养箱;IX-71型荧光倒置相差显微镜,日本Olympus公司;AR1530/C电子天平;25cm2细胞培养瓶,美国Corning公司立式压力蒸汽灭菌器(LS-B75);DHG-9230A电热恒温鼓风干燥箱,上海精宏实验设备有限公司。
实施例二:探针化合物的制备。
以8-羟基喹哪啶,2,4-二羟基苯甲醛为原料,分别以乙酸酐,吡啶/水为溶剂,首先合成中间体,再由中间体在吡啶/水的混合溶剂中水解得,合成路线如下:
三口烧瓶中,在溶有8-羟基喹哪啶的乙酸酐溶液中,加入2,4-二羟基苯甲醛,按摩尔比8-羟基喹哪啶:2,4-二羟基苯甲醛等于1:2,氮气保护下,回流,反应结束,浓缩除去溶剂乙酸酐,经硅胶柱层析洗脱,得到中间体。反应温度:139℃(回流),反应时间:5h,反应溶剂:乙酸酐,洗脱剂:体积比氯仿:乙酸乙酯(3:1)。
N2保护下,三口烧瓶中加入中间体,吡啶为溶剂,加热回流反应,冷却,加入水使吡啶和水的体积比为3:1,继续回流,反应结束,加入水萃取,干燥,过滤,硅胶柱层析分离和洗脱,得探针化合物。反应温度:100℃,反应时间:12h,反应溶剂:吡啶:水(3:1),洗脱剂:体积比氯仿:甲醇(9:1)。
实施例三:
(1)荧光光谱法对H2O2检测
在10mL容量瓶中加入探针(1mM,100mL),pH值11的HEPES-NaOH缓冲溶液(50mM,1mL),H2O2(5mM,0~100mL),用乙腈/H2O稀释至刻度,使测试溶液的溶剂比为乙腈/H2O(v/v,3/2),
摇匀,进行荧光光谱测定;
设置荧光激发波长为415nm,在1cm的比色皿中加入约3ml探针的乙腈/H2O溶液(10μM,v/v,3/2,pH11)进行荧光光谱扫描,探针在580nm波长处有荧光发射。分别加入氧化剂或还原剂:1mM的NO3 -、NO2 -、t-BuOOH、ClO-;0.5mM的GSH、O2 -;100mM的t-BuO×、×OH,反应30min后的荧光光谱,没有观察到荧光光谱明显的变化,而加入50μM的H2O2反应30min后,探针在580nm处的荧光峰猝灭(如附图1),表明在此条件下探针仅对H2O2有识别检测作用;上述条件下,分别测定反应时间为1min、5min、10min、30min的580nm处的荧光强度,只有H2O2在反应时间分别为5min、10min、30min下能使探针在580nm处的荧光强度明显降低(如附图2);
在浓度为10μM探针的乙腈/H2O(v/v,3/2,pH11)溶液中,加入50μM的H2O2到探针溶液中,测定不同反应时间下在580nm处的荧光强度变化。随时间的增长,在15~30min范围内,探针的荧光强度的降低保持稳定。检测时选择反应时间为20min。测试的激发波长为415nm(如附图5)。
以415nm为荧光激发波长,在浓度为10mM探针的乙腈/H2O(v/v,3/2,pH11)溶液中逐渐滴加不同浓度的H2O2,反应20min,得到荧光光谱滴定曲线(如附图7)。随H2O2浓度增加探针在580nm处的荧光强度逐渐降低至猝灭。以荧光强度对H2O2浓度作图得到校正曲线(如附图11)。由校正曲线的斜率和测定5次空白值的标准偏差,测定并计算得到探针荧光法检测H2O2的浓度线性范围和检测限列于表1。
探针检测H2O2在580nm处的荧光强度在其他氧化剂或还原剂分别作为共存物质存在于探针-H2O2混合溶液中,当1mM的NO3 -、NO2 -、O2 -、t-BuOOH、ClO-、GSH;100mM的t-BuO×、×OH共存时,测得探针检测H2O2的荧光强度保持不变,共存物质对检测H2O2的荧光强度不干扰(如附图9)。
(2)紫外-可见吸收光谱法对H2O2检测
在10mL容量瓶中加入探针(1mM,100mL),pH值11.4的HEPES-NaOH缓冲溶液(50mM,1mL),H2O2(5mM,0~100mL),用乙腈/H2O稀释至刻度,使测试溶液的溶剂比为乙腈/H2O(v/v,3/2),摇匀,进行紫外光谱测定。
在1cm的比色皿中加入约3ml10μM探针的乙腈/H2O溶液(v/v,3/2,pH11)进行紫外吸收光谱扫描,探针在410nm波长处有紫外吸收。分别加入氧化剂或还原剂:1mM的NO3 -、NO2 -、t-BuOOH、ClO-;0.5mM的GSH、O2 -;100mM的t-BuO×、×OH,分别30min后的紫外-可见吸收光谱,没有观察到光谱明显的变化;而加入50μM的H2O2反应30min后,探针在410nm处的吸收峰降低(如附图3),表明在此条件下探针仅对H2O2有识别检测作用;上述条件下,分别测定反应时间为1min、5min、10min、30min的410nm处的吸光度,只有H2O2在反应时间分别为5min、10min、30min下能使探针在410nm处的吸光度明显降低(如附图4)。
在浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液中,加入50μM的H2O2到探针溶液中,测定不同反应时间下在410nm处的吸光度变化。随时间的增长,在15~30min范围内,探针的吸光度的降低保持稳定(如附图6)。检测时选择反应时间为20min。
在浓度为10mM探针的乙腈/H2O(v/v,3/2,pH11)溶液中,逐渐滴加不同浓度的H2O2,反应20min,得到紫外-可见吸收光谱滴定曲线(如附图8)。随H2O2浓度增加探针在410nm处的吸光度逐渐降低。以吸光度对H2O2浓度作图得到校正曲线(如附图12)。由校正曲线的斜率和测定5次空白值的标准偏差,测定并计算得到探针紫外吸收法检测H2O2的浓度线性范围和检测限列于表2。
探针检测H2O2在410nm处的吸光度在其他氧化剂或还原剂分别作为共存氧化剂或还原剂存在于探针-H2O2混合溶液中,当1mM的NO3 -、NO2 -、O2 -、t-BuOOH、ClO-、GSH;100mM的t-BuO×、×OH共存时,测得探针检测H2O2的吸光度保持不变,共存物质对检测H2O2的吸光度不干扰(如附图10)。
(3)溶液中H2O2检测
在可见光下,浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液的颜色为橙红色,当加入50μM的H2O2反应20min后,探针检测H2O2溶液颜色由橙红色变为无色(如附图13左图)。在365nm紫外灯下,浓度为10μM的探针的乙腈/H2O(v/v,3/2,pH11)溶液的颜色为橙红色,当加入50μM的H2O2反应20min后,探针检测H2O2溶液颜色由橙红色变为无色(如附图13右图)。
实施例四:探针荧光成像检测活性细胞内H2O2。
活性PC3细胞经复苏接种于含10%胎牛血清以及含1%双抗的培养基(改良型RPMI-1640)中,在温度为37℃,5%CO2以及饱和湿度为100%的培养箱中培养,每隔2-3天传代1次,选择生长状态良好的细胞接种于12孔板中培养,密度为2×104个/ml,次日用新鲜培养基清洗细胞两次。将细胞浸入pH值为11的含10mM探针的培养液(10mM探针培养液配制:900mL的培养液+100mL用生理盐水配制的100mMpH值为11的探针储备液)中孵化1h,吸出含探针的培养液,用新鲜培养基清洗细胞3次。置于荧光显微镜下进行暗场拍照,用荧光显微镜的红色通道检测,观测到细胞的红色荧光图像(附图14a)。
将经pH为11的含10mM探针的培养液孵化过的细胞,再经H2O2(20mMH2O2培养液配制:900mL的培养液+100mL用生理盐水配制的200mMH2O2储备液)孵化10min后,吸出含H2O2的培养液,用新鲜培养基清洗细胞3次。用荧光显微镜的红色通道检测,观测到细胞微弱的红色荧光图像(附图14b);孵化30min后,用荧光显微镜的红色通道检测,观测不到细胞的荧光图像(附图14c);用荧光显微镜观察明场下的细胞照片(附图14d)。
实施例五:
探针荧光光谱校准曲线法测定合成样品中微量H2O2。
1.取数个10.0ml容量瓶,每个容量瓶中依次加入浓度为0.1mM探针的乙腈溶液1ml。
2.依次在各容量瓶中加入不同浓度的H2O2样品溶液,用乙腈/HEPES-NaOH(pH=11)定容到10ml,摇匀、反应20min。
3.在荧光分光光度计上以415nm为激发波长,测定波长580nm处的荧光强度,平行测定3次。
4.用本发明中探针检测H2O2的荧光光谱校正曲线(附图11),求得样品溶液中H2O2的浓度。结果如表3所示。
探针紫外-可见光谱标准曲线法测定合成样品中微量H2O2。
1.取数个10.0ml容量瓶,每个容量瓶中依次加入浓度为0.1mM探针的乙腈溶液1ml。
2.依次在各容量瓶中加入不同浓度的H2O2样品溶液,用乙腈/HEPES-NaOH(pH=11)定容到10ml,摇匀、反应20min。
3.在紫外-可见分光光度计上,测定波长410nm处的吸收值,平行测定3次。
4.用本发明中探针检测H2O2的紫外-可见吸收光谱校正曲线(附图12),求得样品溶液中H2O2的浓度。结果如表4所示。
Claims (6)
1.一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法,其特征是以化合物(E)-2-(2,4-二羟基苯基)乙烯基-8-羟基喹啉为检测H2O2的荧光或比色探针试剂,简称探针,其结构如下:
检测方法为:在乙腈/H2Ov/v,3/2,pH11介质中(1)荧光光谱法检测H2O2,以415nm为激发波长,测定探针在580nm处的荧光强度降低随H2O2浓度变化,用校正曲线法检测;(2)紫外吸收光谱法检测H2O2,测定探针在410nm处的吸光度降低随H2O2浓度变化,用校正曲线法检测;(3)利用探针的颜色变化目视检测H2O2:日光下探针溶液颜色随H2O2的加入由橙红色变为无色;在365nm紫外灯下探针溶液发射荧光颜色随H2O2的加入由橙红色变为无色;(4)在活细胞内利用H2O2使探针的荧光猝灭,用荧光成像可视检测细胞内H2O2。
2.根据权利要求1所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法,其特征是在浓度为10μM探针的乙腈/H2Ov/v,3/2,pH=11溶液中,用荧光或紫外-可见吸收法检测H2O2,检测时反应时间控制在20~30min。
3.根据权利要求1所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法,一种检测溶液和细胞中浓度低于10-5MH2O2的方法,其特征是探针荧光和紫外-可见吸收法检测H2O2时,其它共存氧化剂或还原剂:NO3 -,NO2 -,t-BuOOH(过氧化氢叔丁醇),ClO-,GSH(谷胱甘肽),O2 -,t-BuO×(叔丁氧基自由基),×OH(羟基自由基)在浓度与H2O2相同时,不干扰探针对H2O2的测定。
4.按照权利要求1所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法,其特征是探针用荧光法检测H2O2的浓度线性范围为8.3×10-7~4.3×10-5M,检测限为2.3×10-8M;紫外-可见吸收法检测H2O2的浓度线性范围为8.5×10-7~5.2×10-5M,检测限为8.9×10-7M。
5.按照权利要求1所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法,其特征是所述探针的荧光成像检测活性细胞内H2O2方法为:活性PC3细胞经复苏、传代、接种、培养、清洗后,将细胞浸入pH值为11的含10mM探针的培养液,在37℃,5%CO2以及饱和湿度为100%的培养箱中孵育1h,吸出含探针的培养液,用新鲜培养基清洗细胞,用荧光显微镜检测,呈现清晰的红色荧光细胞图像;再将上述细胞浸入含20mMH2O2的培养液中孵化10min后,吸出含H2O2的培养液,用新鲜培养基清洗细胞3次,用荧光显微镜检测,观察到微弱的红色荧光细胞图像;再经H2O2孵化30min后,用荧光显微镜检测,观察不到细胞的荧光图像。
6.根据权利要求1-5之一所述的一种检测溶液或细胞中10-7~10-5M低浓度H2O2的方法,其特征是所用的试剂为分析纯或生化试剂,所用水为二次蒸馏水或生理盐水;所用活性PC3细胞为人体前列腺癌细胞;所用的拍照设备为荧光倒置显微镜。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1844124A (zh) * | 2006-04-11 | 2006-10-11 | 山东师范大学 | 一种检测过氧化氢的荧光探针及其合成方法和用途 |
US20080160627A1 (en) * | 2003-11-19 | 2008-07-03 | Invitrogen Corporation | Environmental Sensitive Fluorogenic Compounds and Their Application for Singlet Oxygen and Protein Detection |
CN103641779A (zh) * | 2013-12-24 | 2014-03-19 | 贵州大学 | 一种喹哪啶衍生物b荧光、比色试剂及制备方法和应用 |
CN105038762A (zh) * | 2015-06-04 | 2015-11-11 | 济南大学 | 一种检测过氧化氢的比率型荧光探针及其应用 |
-
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- 2016-01-04 CN CN201610001038.XA patent/CN105424670B/zh not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080160627A1 (en) * | 2003-11-19 | 2008-07-03 | Invitrogen Corporation | Environmental Sensitive Fluorogenic Compounds and Their Application for Singlet Oxygen and Protein Detection |
CN1844124A (zh) * | 2006-04-11 | 2006-10-11 | 山东师范大学 | 一种检测过氧化氢的荧光探针及其合成方法和用途 |
CN103641779A (zh) * | 2013-12-24 | 2014-03-19 | 贵州大学 | 一种喹哪啶衍生物b荧光、比色试剂及制备方法和应用 |
CN105038762A (zh) * | 2015-06-04 | 2015-11-11 | 济南大学 | 一种检测过氧化氢的比率型荧光探针及其应用 |
Non-Patent Citations (3)
Title |
---|
JIAN XU 等: ""Mitochondria-Targeted Fluorescent Probe for Imaging Hydrogen Peroxide in Living Cells"", 《ANALYTICAL CHEMISTRY》 * |
YUAN-HUI ZHAO ET AL.: "A reversible and visible colorimetric/fluorescent chemosensor for Al3+ and F−ions with a Large Stoke’s shift", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
YUAN-YU QIAN ET AL.: "Quinoline-based fluorescent probe for ratiometric detection of hydrogen peroxide in aqueous solution", 《DYES AND PIGMENTS》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113155794A (zh) * | 2021-04-01 | 2021-07-23 | 江南大学 | 一种定量检测单个原生质体和液泡内Cd2+的方法 |
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