CN106632442A - 一种识别纯水体系中过氧化氢的荧光探针 - Google Patents

一种识别纯水体系中过氧化氢的荧光探针 Download PDF

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CN106632442A
CN106632442A CN201611168269.6A CN201611168269A CN106632442A CN 106632442 A CN106632442 A CN 106632442A CN 201611168269 A CN201611168269 A CN 201611168269A CN 106632442 A CN106632442 A CN 106632442A
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林伟英
刘勇
牛杰
孟芳芳
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Abstract

本发明提供了一种在纯水体系中识别过氧化氢的荧光探针，其化学结构式如式（I）所示。本发明提供的荧光探针通过荧光淬灭能识别过氧化氢，该探针在活细胞靶标分子的标记领域具有潜在的应用价值，（I）。

Description

一种识别纯水体系中过氧化氢的荧光探针
技术领域
本发明涉及一种识别过氧化氢（H2O2）的荧光探针，尤其涉及一种在纯水体系内专一识别H2O2的咔唑类化合物荧光探针；属于有机小分子荧光探针领域。
背景技术
过氧化氢(H2O2) 在各种生理过程中发挥重要作用，H2O2水平异常与许多类型的疾病如癌症、糖尿病、唐氏综合症和罕见病相关联。近来，H2O2已证实是学术界与人类重大疾病相关的活性小分子。基于此，在体内和体外对H2O2的检测也引起人们的高度重视。
目前，H2O2的检测手段主要分为两类：直接法和间接法。直接法是一类直接利用H2O2自身物理、化学性质对其进行分析检测的方法，包括原子吸收、发射光谱法和离子选择性电极法；间接法是一类利用H2O2的识别位点进行检测，主要是通过设计合成光物理性质较好的荧光平台，基于该平台的荧光探针进行在体外和生物体内进行检测。但是，综合分析目前报道的H2O2荧光探针主要存在以下不足：第一，在纯水体系内检测H2O2 依然很难，由于合成探针的本身具有较大的疏水性结构；第二，在纯水体系内H2O2探针的选择性不高，容易受到其他氧化应激分析物的干扰；基于此，开发新型荧光探针在纯水体系内中专一识别过氧化氢（H2O2）的荧光探针，并给出检测曲线判断H2O2水平，有着重要的实际应用价值。
发明内容
针对以上现有技术的不足，本发明提供了一种在纯水体系中检测H2O2的荧光探针并研究了其光谱性质。
本发明所述识别过氧化氢的荧光探针，其特征在于：所述荧光探针命名为CAI，其化学结构式如式（I）所示：
（）。
上述CAI制备方法是：
将化合物1与吡啶盐2以乙醇做溶剂，哌啶作催化剂，在氮气保护下回流反应。反应结束后，柱层层析得到CAI。
上述CAI的制备反应式如下：
本发明所述识别过氧化氢的荧光探针在纯水体系内检测H2O2。
上述的应用中：所述荧光探针是以荧光淬灭的方式实现对溶酶体中H2O2的识别。
相关实验证实：检测环境是水相时，本发明所述的荧光探针CAI在纯水体系内荧光增强明显，而在过氧化氢存在的条件下荧光淬灭，证明该探针可能是过氧化氢探针（图1）。在H2O2选择性试验中，当用405nm光激发，只有H2O2发生10倍的淬灭（图2）；在H2O2滴定实验中（图3），随着过氧化氢的逐渐增加，探针CAI荧光逐渐淬灭。在探针的动力学研究中，在405nm氙灯连续照射半小时，探针在过氧化氢存在的条件下，探针荧光强度趋于缓慢淬灭状态（图4）。推断，该探针在细胞内能检测到细胞器的过氧化氢活性分子。
基于上述实验结果，可以证明本发明所述的在存水体系内识别H2O2的荧光探针是一类新型的高选择性荧光探针分子，该探针的识别机理是通关荧光团中过氧化氢的识别位点硼酸酯与过氧化氢发生氧化反应导致的荧光团的淬灭效应。其识别反应产物如（II）所示：
。
本发明提供的识别纯水体系内的过氧化氢的咔唑类化合物荧光探针与其功能相近的传统的过氧化氢荧光探针相比具有显著优势，且本发明所述咔唑类化合物在体外对活性小分子高选择性检测和合成手段也具有新颖性和简便性。本发明的荧光探针在纯水体系内通过荧光增强而为检测过氧化氢荧光淬灭性提供荧光平台，该探针及其研究为生物学成像应用奠定了理论基础，预示该探针在活细胞内过氧化氢的检测的实现提供重要工具。
附图说明
图1探针本身及其在过氧化氢存在下的荧光光谱。激发波长405 nm。
图2探针对过氧化氢的选择性。激发波长为405 nm；探针母液的浓度：10-3M，选择性离子的浓度为0.2 mM。
其中激发波长为405 nm；探针母液的浓度：10-3M，选择性离子的浓度为0.2 mM。
图3 H2O2的滴定图。
横坐标：荧光强度，纵坐标：波长。
H2O2的滴定实验；其中激发波长为405 nm；探针母液的浓度:10-3M，取5μL探针母液稀释3ml进行，加入不同量的H2O2滴定。
图4：探针加入H2O2后的动力学变化图
横坐标：相对荧光强度，纵坐标：时间。
其中激发波长为405 nm；探针的浓度:10-3M；时间1800s。
具体实施方式
实施例1
CAI的合成：
将0.35g(1mmol)化合物1，0.25g(1.1mmol) 化合物2，溶于20mL乙醇中, 滴入两滴哌啶做催化剂，在氮气保护下回流反应。反应结束。用流动相CH2Cl2/CH3OH=70:1过硅胶柱得到CAI。
1H NMR (400 MHz, CDCl3) δ 8.93 (d, J = 8.0 Hz, 2H), 8.62 (s, 1H), 8.27(d, J = 4.0 Hz, 1H), 8.06-8.05 (d, J = 4.0 Hz, 2H), 7.99-7.96 (m, 1H), 7.92(s, 1H), 7.81 – 7.79 (m,1H), 7.40-7.42 (dd, J = 8.0 Hz, J1 = 8.0 Hz, J1 = 8.0Hz, 2H), 7.18-7.14 (d, J = 16.0 Hz, 1H), 4.77-4.82 (q, J = 20.0 Hz, 2H), 4.51(q, J = 16.0 Hz, 2H), 1.72-1.69 (t, J = 12.0 Hz, 6H), 1.42 (s, 12H).
实施例2
CAI在纯水体系内对过氧化氢的响应：
预先准备1份10 mL的10-3 M本发明的探针N, N-二甲基甲酰胺溶液，然后分别取5μL加入两个相同的5mL容量瓶中，其中一个瓶中加入过氧化氢当量为探针当量的22倍，用PBS的溶液稀释到3mL，然后进行荧光检测（λEx= 405nm），结果见图1。
结论： 表明该探针在在纯水体系内响应。
实施例3
CAI对过氧化氢的选择性(见图2)：
其中：激发波长为405 nm；探针母液的浓度：10-3M，选择性离子的浓度为0.2mM。取5μL探针母液稀释3ml进行，后取3mL的上述稀释液，加入各种离子进行光谱测试,其中各种离子浓度均为0.2mM。探针的测试环境均为PBS缓冲溶液。
结论：测试结果表明，探针CAI对过氧化氢具有高选择性。加入过氧化氢后，探针响应倍数为10倍左右。
实施例 4
过氧化氢的滴定：
配制10 mL浓度为0.2mM H2O2的水溶液及本发明的探针母液（浓度为10-3M）作为备用。
将过氧化氢浓度从0当量滴定到22当量并进行荧光检测（λEx= 405nm, λEm=575nm），计算各体系中荧光强度，建立荧光强度与过氧化氢浓度标准曲线 (见图3)。
结论：随着过氧化氢的加入，探针的荧光强度趋于下降至平衡的趋势。
实施例5
过氧化氢的动力学实验：
为了研究探针的动力学，配置探针的浓度为10-3M，在405nm氙灯连续照射半小时，检测探针在过氧化氢存在的条件下，荧光强度趋于缓慢淬灭状态。建立荧光强度与时间的标准曲线(见图4)。
结论：随着时间的延长，探针的荧光强度趋于下降至平衡的趋势，该探针在细胞内能检测到细胞器的过氧化氢活性分子。

Claims (2)

1.一种识别纯水体系中过氧化氢的荧光探针，其特征在于：所述荧光探针命名为CAI，其化学结构式如式（I）所示：
（I）。
2.根据权利要求1所述的荧光探针，其特征在于，采用如下制备方法制备而成：将1mmol化合物1与1.1mmol化合物2，溶于20mL乙醇中，滴入两滴哌啶做催化剂，在氮气保护下回流反应，用CH2Cl2/CH3OH=70:1作流动相，过硅胶柱得CAI，反应式如下：
。
CN201611168269.6A 2016-12-16 2016-12-16 一种识别纯水体系中过氧化氢的荧光探针 Pending CN106632442A (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
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CN104949946A (zh) * 2015-06-08 2015-09-30 济南大学 一种荧光探针在过氧化氢分子检测中的应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104949946A (zh) * 2015-06-08 2015-09-30 济南大学 一种荧光探针在过氧化氢分子检测中的应用

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