CN109180716A - 一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 - Google Patents
一种多信号比率型区分检测h2o2和h2s的荧光探针的设计、合成及应用 Download PDFInfo
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Abstract
本发明涉及的是化学分析检测技术领域，具体涉及一种多多信号比率型区分检测H2O2和H2S的荧光探针的制备方法以及该荧光探针在体外和活细胞内检测H2O2和H2S方面的应用。该探针化合物的结构式如式(I)所示。我们将两种识别基团通过染料的桥连构建到一个分子上，比率型荧光信号通过两个荧光信号的比值作为输出信号，探针分子本身的自校准能够克服诸如探针浓度、仪器灵敏度和环境因素带来的影响，提高荧光检测的准确性。通过探针与H2S和H2O2反应后获得不同组合的荧光信号来实现对H2S和H2O2的区分检测。此外该系列化合物有望在生物医药，光电以及环境保护领域有着较好的应用前景。
Description
技术领域
本发明涉及的是化学分析检测技术领域，具体涉及一种多信号比率型区分检测H2O2和H2S的荧光探针的制备方法以及该荧光探针在体外和活细胞内检测H2O2和H2S方面的应用。
背景技术
H2O2是其他活性氧的前体分子，在宿主防御，免疫应答和细胞信号传导中起着重要的作用。作为氧化应激的标志物，越来越多的研究表明，体内H2O2浓度的增加会诱导细胞蛋白的氧化损伤，导致神经退行性疾病，心血管疾病，癌症和阿尔兹海默症等。H2S作为体内的一种信号传递分子，在神经调节，细胞凋亡，抗炎症，抗氧化和抑制胰岛素信号传导等生理过程中起着广泛而重要的作用。研究发现，H2S通过提高细胞内谷胱甘肽的水平、调节抗氧化蛋白的高表达等途径，来保护细胞免受氧化应激的损伤。H2S本身具有的还原性也会直接清除H2O2等活性氧物质。H2S和H2O2在生理过程中有如此重要的作用，同时彼此互相影响，开发一种有效的检测方法是至关重要的，特别是对细胞内H2S和H2O2的浓度的监测，对生物学研究和临床诊断至关重要。
目前对H2S和H2O2的检测方法主要有比色法、电化学分析法、气相色谱法、滴定法、分光光度法和荧光法等。在这些方法中，荧光探针成像技术由于操作简单，灵敏度高，选择性好，实时成像等优点，特别是对组织的非破坏性而受到特别的关注。近年来开发了大量的荧光探针来检测H2S和H2O2，但他们都是独立的对H2S或H2O2进行选择性检测，还没有一个探针分子可以同时区分检测H2S和H2O2。
发明内容
本发明目的之一是提供一种合成路线简单、反应条件温和、成本较低的荧光探针合成方法；目的之二是提供一种灵敏度高、选择性好，抗干扰能力强，斯托克斯位移大，发射波长在近红外，能够对体外或者活细胞内进行监测或者细胞成像的荧光探针。比率型荧光信号通过两个荧光信号的比值作为输出信号，探针分子本身的自校准能够克服诸如探针浓度、仪器灵敏度和环境因素带来的影响，提高荧光检测的准确性。通过探针与H2S和H2O2反应后获得不同组合的荧光信号来实现对H2S和H2O2的区分检测。
本发明解决问题采取的技术方案为，一种多信号比率型区分检测H2O2和H2S的荧光探针，其分子结构式如下：合成路线如下：
具体合成方法如下：将化合物(7-羟基-2-氧代-2H-色烯-4-基)甲基(E)-3-(7-((4- 叠氮苯基)氧基)-1，4-二乙基-1，2，3，4-四氢喹喔啉-6-基)-2-氰基丙烯酸酯和4-溴甲基苯硼酸频哪醇酯溶于丙酮，置于圆底烧瓶中，加入碳酸钾60℃搅拌回流反应。TLC监测反应完成后，停止反应，减压蒸馏除去溶剂，剩余物用柱层析分离纯化，二氯甲烷/乙酸乙酯(v:v ＝20:1,含1％三乙胺)作为洗脱剂。干燥得到红色固体
本发明的荧光探针测试方法如下，将探针分子溶于二甲基亚砜(DMSO)中，配制成1.0×10-3mol/L的溶液，室温下进行测试。并且对低浓度的H2S和H2O2可以进行定量检测，具体实施方法在实施实例中详细介绍。
本发明的荧光探针的作用机理如下，在探针分子中，探针本身发蓝色荧光，且有两个反应位点。当探针与H2S反应时，位点1的叠氮基被还原，随后醚键被切断，经过快速的分子内成环反应得到发蓝色荧光的HCB和发红色荧光的TQC。当探针TCAB与H2O2反应时，位点2的醚键被切断，释放出发蓝绿色荧光的化合物TCA，探针的荧光发生了从蓝光到蓝绿色的比值变化。通过探针TCAB与H2S和H2O2反应得到不同的荧光信号：H2S(蓝光+红光)， H2O2(蓝光→蓝绿光)，可以实现对H2S和H2O2的区分检测。随后，我们进一步研究了探针与两种响应物叠加响应的情况。当向探针TCAB与H2S响应后的溶液中继续加入H2O2时，前一步的产物HCB上的苯硼酸酯会继续被H2O2切断，从而释放出蓝绿色的荧光染料HC，同时之前得到的红光染料TQC不受影响。同样的，向探针TCAB与H2O2响应后的溶液中继续加入H2S时，TCA上的叠氮基会继续被H2S还原，醚键断裂，再经过快速的分子内成环反应得到发蓝绿色荧光的染料HC和发红色荧光的染料TQC。由此可见，探针TCAB可以实现对H2S和H2O2的连续检测。
探针分子的响应过程：
本发明的荧光探针探针TCAB与H2S和H2O2的荧光响应测试在HEPES缓冲液(20 mM,1.0mM CTAB,pH＝7.4)中进行。探针本身在413nm处发蓝色荧光。当探针TCAB的溶液中加入H2O2时，413nm处的荧光下降，同时在486nm处有新的蓝绿色荧光生成(激发波长为325nm)。荧光滴定实验结果显示，随着H2O2浓度的增大，486nm处的荧光强度(F486nm) 与413nm处的荧光强度(F413nm)的比值显著增大，当H2O2浓度达到400μM时，F486nm/F413nm的值达到最大。并且F486nm/F413nm的值与H2O2的浓度在20-100μM范围内有很好的线性关系，线性相关系数为0.9968。另外，当探针TCAB与H2S响应时，反应液在413nm处的蓝色荧光增强(激发波长为325nm)，同时在627nm处有新的红色荧光生成(激发波长为475 nm)。随着H2S浓度的增加，反应体系在413nm和627nm处的荧光强度逐渐增强，在350μM 的H2S加入时，荧光强度达到最大值。并且探针TCAB在413nm和627nm处的荧光强度与 H2S在浓度范围为0-150μM时具有很好的线性关系，线性相关系数分别为0.9905和0.9901。根据信噪比S/N＝3，计算出探针TCAB对H2S和H2O2的检测限分别为0.058和0.044μM。随后，我们还测定了探针TCAB与H2S和H2O2响应前后的紫外光谱变化情况
本发明所述的探针分子合成路线简单，成本较低，对H2S和H2O2的选择性好、抗干扰能力强，斯托克位移大，该荧光探针在生物化学，环境科学等领域具有实际的应用价值。
附图说明
图1为本发明荧光探针TCAB(10.0μM)在HEPES缓冲液(20mM,1.0mM CTAB, pH＝7.4)中与H2O2(400μM)和H2S(350μM)响应前后的紫外吸收光谱。
图2(A-B)探针TCAB(10.0μM)与H2O2完全反应后的溶液随H2S浓度增加的荧光光谱变化。(C-D)探针TCAB(10.0μM)与H2S完全反应后的溶液随H2O2浓度增加的荧光光谱变化。激发波长：第一列325nm；第二列475nm。激发和发射狭缝宽度为5nm/5nm。
图3为本发明的荧光探针TCAB(10.0μM)在HEPES缓冲液(20mM,1.0mM CTAB, pH＝7.4)中与H2O2(400μM)和H2S(350μM)响应时，F486nm/F413nm的比值以及413nm和627 nm处的荧光强度随时间的变化。激发波长：(A)325nm,(B)325nm,(C)475nm。
图4为本发明的荧光探针TCAB(10.0μM)在HEPES缓冲液(20mM,1.0mM CTAB, pH＝7.4)中与相关分析物响应后，F486nm/F413nm的比值以及413nm和627nm处的荧光强度变化。分析物包括：(1)ROO.,(2)NO.,(3)ClO-,(4)O2 -,(5)TBHP,(6)TBO.,(7)ONOO-,(8)1O2,(9) OH-,(10)SO4 2-,(11)NO3 -,(12)SCN-,(13)Cl-,(14)CO3 2-,(15)SO3 2-,(16)N3 -,(17)AcO-,(18) Na+,(19)Mg2+,(20)Ca2+,(21)K+,(22)Fe3+,(23)Cys,(24)GSH,(25)H2O2,(26)H2S。浓度： (1)-(25)400μM,(26)350μM，激发波长：(A)325nm,(B)325nm,(C)475nm。
图5为高效液相色谱：(a)探针TCAB(50.0μM)；(b,c,d)探针TCAB(50.0μM)分别与10.0,20.0和40.0equiv.的H2O2在HEPES缓冲液(20mM,1.0mM CTAB,pH＝7.4)中响应 120分钟；(e)化合物TCA(50.0μM).流动相：H2O/CH3CN(v/v,2/8)；流速：0.5mL/min；温度： 25℃；检测波长：350nm。
图6高效液相色谱：(a)探针TCAB(50.0μM)；(b,c,d)探针TCAB(50.0μM)分别与10.0,20.0和40.0equiv.的H2S在HEPES缓冲液(20mM,1.0mM CTAB,pH＝7.4)中响应80 分钟；(e)化合物TQC(50.0μM)；(f)化合物HCB(50.0μM).流动相：H2O/CH3CN(v/v,2/8)；流速：0.5mL/min；温度：25℃；检测波长：350nm。
图7为本发明的荧光探针TCAB(10.0μM)在不同pH环境下与H2S和H2O2的响应情况。荧光强度：(a)F486nm/F413nm,(b)413nm,(c)627nm；激发波长：(a)325nm,(b)325nm,(c)475nm。
图8不同浓度的探针TCAB对HeLa细胞毒性的分析。
图9探针TCAB在HeLa细胞内对外源性H2O2和H2S的共聚焦荧光成像。(A)细胞先与500μM H2O2孵育30分钟，再与10μM探针TCAB孵育30分钟。(B)细胞先与500μM H2S孵育30分钟，再与10μM探针TCAB孵育30分钟。(C)细胞先与10μM探针TCAB 孵育30分钟，再与500μMH2O2孵育30分钟，最后与500μM H2S孵育30分钟。(D)细胞与10μM探针TCAB孵育30分钟。第一列：蓝光通道，激发波长405nm，收集范围420-450 nm；第二列：蓝绿光通道，激发波长405nm，收集范围460-510nm；第三列：红光通道，激发波长488nm，收集范围600-650nm；第四列：合并蓝光，蓝绿光和红光通道。
具体实施实例
本发明的探针分子结构式如下：
本发明的探针合成路线如下所示；
实施例1：探针的合成；
英文名称：
(2-oxo-7-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-2H-chromen-4-yl)methyl (E)-3-(7-((4-azidobenzyl)oxy)-1,4-diethyl-1,2,3,4-tetrahydroquinoxalin-6-yl)-2-cyanoacrylate
中文名称：(2-氧代-7-((4-(4，4，5，5-四甲基-1，3，2-二氧杂环戊烷-2-基)苄基)氧基)-2H-色烯-4-基)甲基(E)-3-(7-((4-叠氮苯甲酰)氧基)-1，4-二乙基-1，2，3， 4-四氢喹喔啉-6-基)-2-氰基丙烯酸酯
结构式：
合成方法：将化合物TCA(60mg,0.1mmol)和4-溴甲基苯硼酸频哪醇酯(60mg,0.2mmol)溶于10mL丙酮，置于25mL圆底烧瓶中，加入碳酸钾(28mg,0.2mmol)，60℃搅拌回流反应3h。TLC监测反应完成后，停止反应，减压蒸馏除去溶剂，剩余物用柱层析分离纯化，二氯甲烷/乙酸乙酯(v:v＝20:1,含1％三乙胺)作为洗脱剂。干燥得到红色固体35mg，产率为43％。表征数据：HRMS(ESI)m/z:[M]calcd for C46H47BN6O8,822.3548；found,822.3605. 1HNMR(400MHz,CDCl3)δ8.69(s,1H),7.76(s,1H),7.49(d,J＝8.8Hz,1H),7.45–7.39(m, 4H),7.37(d,J＝7.8Hz,2H),7.03(d,J＝8.4Hz,2H),6.94(dd,J＝8.8,2.4Hz,1H),6.89(d,J＝2.3Hz,1H),6.42(s,1H),5.99(s,1H),5.41(s,2H),5.12(d,J＝23.9Hz,4H),3.56(s,2H),3.36(d, J＝6.2Hz,4H),3.21(s,2H),1.35(t,12H),1.26(q,J＝3.2Hz,6H).13C NMR(100MHz,CDCl3)δ 164.6,161.8,160.8,156.7,155.5,149.2,147.8,144.4,144.0,140.0,138.8,135.1,133.3,128.7, 126.6,126.1,124.7,119.4,118.4,113.2,111.0,110.4,109.9,102.4,94.2,83.9,70.8,70.4,65.3, 61.9,53.4,48.0,45.4,29.7,24.9,10.9,9.8.
实施例2：细胞培养和荧光成像；
HeLa细胞(人宫颈癌细胞)来源于湖南大学化学生物传感与计量学国家重点实验室。 HeLa细胞培养在含10％的胎牛血清和1％的盘尼西林的DMEM培养液中，在37℃和5％二氧化碳条件下培养24h。然后接种到激光共聚焦培养皿中，继续孵育12h，待细胞贴壁后可进行荧光成像实验。
外源性H2S和H2O2的检测。实验组一和二：首先移除培养基，PBS缓冲液漂洗细胞三次，向培养皿中加入500μM的Na2S或H2O2，细胞在37℃和5％二氧化碳条件下培养 30分钟，然后用PBS缓冲液漂洗细胞三次，将探针TCAB(10μM)加入到培养皿中，与细胞孵育30分钟，PBS漂洗后进行荧光成像；实验组三：移除培养基，PBS缓冲液漂洗细胞三次，将探针TCAB(10μM)加入到培养皿中，与细胞孵育30分钟，PBS缓冲液漂洗细胞三次，然后向培养皿中加入500μM的Na2S，与细胞孵育30分钟，用PBS缓冲液漂洗细胞三次，然后向培养皿中加入500μM的H2O2，继续与细胞孵育30分钟，PBS漂洗后进行荧光成像。对照组：移除培养基，PBS缓冲液漂洗细胞三次，将探针TCAB(10μM)加入到培养皿中，在 37℃和5％二氧化碳条件下与细胞孵育30分钟，PBS漂洗后进行荧光成像。
内源性H2S和H2O2的检测。实验组一：移除培养基，PBS缓冲液漂洗细胞三次，向培养皿中加入佛波酯(PMA,1μg/mL)，细胞孵育30分钟后用PBS缓冲液漂洗三次，将探针 TCAB(10μM)加入到培养皿中，与细胞孵育30分钟，PBS漂洗后进行荧光成像；实验组二：移除培养基，PBS缓冲液漂洗细胞三次，向培养皿中加入硝普钠(SNP,100μM)，细胞孵育 30分钟后用PBS缓冲液漂洗三次，将探针TCAB(10μM)加入到培养皿中，与细胞孵育30 分钟，PBS漂洗后进行荧光成像；对照组：移除培养基，PBS缓冲液漂洗细胞三次，将探针 TCAB(10μM)加入到培养皿中，在37℃和5％二氧化碳条件下与细胞孵育30分钟，PBS 漂洗后进行荧光成像。
Claims (1)
1.一种多信号比率型区分检测H2O2和H2S的荧光探针，其结构如式(I)所示；
其特征在于该荧光探针用于环境或生物样品中的H2O2和H2S的荧光检测和分析。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109970777A (zh) * | 2019-04-23 | 2019-07-05 | 湘潭大学 | 一种具有检测过氧化氢功能的荧光探针及其制备方法和应用 |
| CN110818734A (zh) * | 2019-10-23 | 2020-02-21 | 中南大学 | 一种具有双比值识别过氧化氢和次氯酸的荧光探针 |
| CN113896739A (zh) * | 2021-08-26 | 2022-01-07 | 福建师范大学 | 一种反应性罗丹明b衍生物荧光探针及其制备方法和应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603603A (zh) * | 2017-11-02 | 2018-01-19 | 中南大学 | 一种识别过氧化氢的荧光探针 |
| CN107840829A (zh) * | 2017-11-02 | 2018-03-27 | 中南大学 | 一种特异性识别硫化氢的荧光探针 |
| CN108358906A (zh) * | 2018-03-14 | 2018-08-03 | 中南大学 | 一种特异性区分不同硫醇的荧光探针 |
-
2018
- 2018-09-19 CN CN201811090636.4A patent/CN109180716B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603603A (zh) * | 2017-11-02 | 2018-01-19 | 中南大学 | 一种识别过氧化氢的荧光探针 |
| CN107840829A (zh) * | 2017-11-02 | 2018-03-27 | 中南大学 | 一种特异性识别硫化氢的荧光探针 |
| CN108358906A (zh) * | 2018-03-14 | 2018-08-03 | 中南大学 | 一种特异性区分不同硫醇的荧光探针 |
Non-Patent Citations (1)
| Title |
|---|
| LONG YI等: "A Dual-Response Fluorescent Probe Reveals the H2O2-Induced H2S Biogenesis through a Cystathionine β-Synthase Pathway", 《CHEM. EUR.J.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109970777A (zh) * | 2019-04-23 | 2019-07-05 | 湘潭大学 | 一种具有检测过氧化氢功能的荧光探针及其制备方法和应用 |
| CN110818734A (zh) * | 2019-10-23 | 2020-02-21 | 中南大学 | 一种具有双比值识别过氧化氢和次氯酸的荧光探针 |
| CN113896739A (zh) * | 2021-08-26 | 2022-01-07 | 福建师范大学 | 一种反应性罗丹明b衍生物荧光探针及其制备方法和应用 |
| CN113896739B (zh) * | 2021-08-26 | 2023-09-22 | 福建师范大学 | 一种反应性罗丹明b衍生物荧光探针及其制备方法和应用 |
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