CN106431986B - 一种检测肼的荧光探针及其应用 - Google Patents

一种检测肼的荧光探针及其应用 Download PDF

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CN106431986B
CN106431986B CN201610867281.XA CN201610867281A CN106431986B CN 106431986 B CN106431986 B CN 106431986B CN 201610867281 A CN201610867281 A CN 201610867281A CN 106431986 B CN106431986 B CN 106431986B
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林伟英
王建勇
刘展榕
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Abstract

本发明公开了一种检测肼的荧光探针，该荧光探针分子式为C16H13NO3，结构式如下所示：该荧光探针的合成只需要一步就可以完成，且后处理过程简单，实现了N2H4分子探针的选择性快速检测，并且选择性好，抗其他离子干扰能力强。基于其特异性且显著的颜色变化，该试剂可作为显示水溶液中和生物细胞内N2H4分子存在的专一性指示剂，可进行实时定性的目视比色法检测。故而，本发明是一种简单，快速，灵敏的N2H4分子特异性检测试剂，在生物分子检测领域具有广阔的应用前景。

Description

一种检测肼的荧光探针及其应用
技术领域
本发明涉及一种用于检测肼的荧光探针，具体的说涉及一种检测肼的荧光探针及其应用，属于有机小分子荧光探针领域。
背景技术
肼(又称为联氨、无水联氨)被广泛应用于医药、化工、军事和航天等领域，该物质用于制造异烟肼、照相显影药剂、喷气式发动机燃料、火箭燃料、抗氧剂、还原剂、高压锅炉给水脱氧剂等。但肼对人体的血液和神经系统均有毒害，且其毒性可长时间积累。肼极毒。小鼠口服LD50为59mg/kg，静脉注射LD50为57mg/kg。肼已成为人们公认的致癌物质，也是环境中重要的有害物质之一。
目前检测肼的常用方法有色谱法、电化学法、分光光度法、化学滴定法等。其中荧光光谱分析法有着操作简单、灵敏度高、专一性强、生物相容性好等优势。最近，已报道不少对肼进行检测的荧光探针，但这些探针往往灵敏度不高，响应时间较长等缺陷，影响在复杂的生物环境中对肼的检测。所以，本发明主要开发一种新的荧光平台的新型荧光探针，通过利用萘荧光平台，发展具有新型识别位点的双光子肼荧光探针，具有非常重要的科学意义。
发明内容
针对目前有机小分子荧光探针在N2H4的检测中所面临的问题，本发明通过分子设计，合成出一种具有选择性高的N2H4荧光探针。
本发明采用以下技术方案：
一种检测肼的荧光探针，它的分子式为：C16H13NO3，其结构式如下所示：
简记为N-N2H4-CN；
所述荧光探针的制备方法如下：
将0.5mmol化合物1与0.55mmol化合物2在氮气氛围下加至反应瓶中，然后将0.55mmol哌啶和5.0mL乙醇/乙腈按照体积比1/1制备的混合溶液一次加至上述反应器中，室温下反应5h后，点板检测反应至原料消失，抽滤得到目标探针化合物；
所述化合物1结构式为：
所述化合物2结构式为：
探针N-N2H4-CN的合成路线如下：
上述的荧光探针的应用，该荧光探针可应用于水环境和生物体系中N2H4的传感检测；所述的传感检测包含荧光检测、细胞成像以及环境中肼的检测。
本发明的优点：(1)探针的合成只需要一步就可以完成，且后处理过程简单；(2)本发明实现了N2H4分子探针的选择性快速检测，并且选择性好，抗其他离子干扰能力强。基于其特异性且显著的颜色变化，该试剂可作为显示水溶液中和生物细胞内N2H4分子存在的专一性指示剂，可进行实时定性的目视比色法检测。故而，本发明是一种简单，快速，灵敏的N2H4分子特异性检测试剂，在生物分子检测领域具有广阔的应用前景。其性能将在实施例中结合附图给予详细说明。
附图说明
图1是实施例1中探针N-N2H4-CN的1H NMR图谱；
图2是探针N-N2H4-CN吸收光谱；
图3是探针N-N2H4-CN随不同当量N2H4的加入荧光强度的变化情况；
图4是探针N-N2H4-CN随时间变化荧光强度变化情况；
图5是探针N-N2H4-CN选择性柱状图数据，1-15号离子分别加入的：probe，Na2SO3，NaNO2，NaNO3，CaCl2，Fe2SO4，KBr，NiSO4，ZnCl2，MgCl2，LiCl，KI，H2O2，Cys，N2H4；
图6是探针N-N2H4-CN应用于细胞中N2H4的荧光成像；
图7是探针N-N2H4-CN应用于环境中N2H4的荧光成像。
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制，实施例中化合物的号码对于上述方案中化合物的号码。
实施例1
探针化合物N-N2H4-CN的合成：
将化合物1(0.5mmol，100.0mg)与化合物2(0.55mmol,62.2mg)在氮气氛围下加至反应瓶中，然后将哌啶(0.55mmol,46.8mg)和乙醇/乙腈(v/v＝1/1，5.0mL)一次加至上述反应器中，室温下反应5h后，点板检测反应至原料消失，抽滤得到探针N-N2H4-CN。1HNMR(400MHz,DMSO-d6):10.47(s,1H),8.46(d,J＝15.2Hz,2H),8.15(dd,J1＝1.6Hz,J2＝8.8Hz,1H),7.88(dd,J1＝10Hz,J2＝20,2H),7.21(s,1H),7.20(t,J＝10,1H),4.33(dd,J1＝7.2,J2＝14.4,2H),1.32(t,J＝16,3H)。
实施例2
探针化合物N-N2H4-CN的吸收光谱
取实施例1制备的探针N-N2H4-CN溶于二甲基亚砜(DMSO)中，制成1mmol/L储备液。从储备液中取两份30μL，加入两个5mL的离心管中，用PBS缓冲溶液(0.1mol/L，pH＝7.5)与DMSO体积比为1:1的溶液稀释至3mL，一份加入100equiv的N2H4标准溶液，另一份不加，反应2h进行吸收测试。如图2所示。由图2可见，加入N2H4后探针的最大吸收在340nm处。
实施例3
探针化合物N-N2H4-CN随N2H4加入当量的增加荧光谱图的变化
取实施例1制备的探针N-N2H4-CN溶于二甲基亚砜(DMSO)中，制成1mmol/L储备液。从储备液中取出30μL加入到5mL的离心管当中，加入不同当量(0-100equiv)的N2H4标准溶液，用PBS缓冲溶液(0.1mol/L，pH＝7.5)与DMSO体积比为1:1的溶液稀释至3mL，测量其荧光性质。荧光光谱如图3所示。由图3可见，随着N2H4加入当量的增加荧光逐渐增强，当加入50当量的N2H4时，体系的荧光达到饱和。
实施例4
探针N-N2H4-CN随时间变化的荧光谱图的变化
从实施例1中荧光探针储备液中取出30μL加入到5mL的离心管当中，加入N2H4(20equiv)标准溶液，用DMSO/PBS(1:1,v/v)稀释至3mL(10μM)，测量其荧光性质。荧光光谱如图4所示。由图4可见，加入探针N-N2H4-CN后，直接加入N2H4，随时间的增加荧光强度迅速达到最大，实现了N2H4的检测。
实施例5
荧光探针N-N2H4-CN对不同离子的选择性
从实施例1中荧光探针储备液中取出30μL加入到5mL的离心管当中，分别加入等摩尔量的竞争分子标准溶液，其中一个加入等摩尔量的N2H4标准溶液，120min后检测溶液的荧光发射光谱变化，结果见图5。由图5可以发现，其他干扰离子对化合物N-N2H4-CN的荧光几乎没有影响，而N2H4溶液的加入使化合物N-N2H4-CN的荧光显著增强。
实施例6
N-N2H4-CN荧光探针对细胞中N2H4荧光成像
我们将本发明探针应用于HeLa细胞中N2H4的检测进行荧光成像应用(图6)。具体操作步骤如下：a)将20μM探针DMSO溶液加入到育有HeLa细胞的培养液中在二氧化碳培养箱中培养0.5h，明场成像，可以看到细胞大致的轮廓；b)将a)用蓝光进行激发观得到成像图；c)将a)和b)图成像叠加；d)将20μM探针DMF溶液加入到育有HeLa细胞的培养液中在二氧化碳培养箱中培养0.5h，加入N2H4后，明场成像，可以看到细胞大致的轮廓；e)将d)用蓝光进行激发观得到成像图；f)将d)和e)图成像叠加。说明此荧光探针实现细胞中N2H4的检测。
实施例7
本发明的探针N-N2H4-CN作为气态肼试纸条测试：
用1mmol/mL的N-N2H4-CN溶液浸泡硅胶板，烘干制成条状，然后放置于不同浓度肼的容量瓶的瓶口，10min后置于紫外灯下拍照。随肼浓度增大，紫外灯下显示硅胶板，详见图7。

Claims (1)

1.一种 1.6’- 羟基萘基 -2- 氰基丙烯酸乙酯荧光探针化合物在检测肼中的应用，其特征在于，该荧光探针可应用于水环境和生物体系中N2H4的传感检测；所述的传感检测包含荧光检测、细胞成像以及环境中肼的检测；所述荧光探针化合物分子式为：C16H13NO3，其结构式如下所示：
结构式.jpg；
所述荧光探针化合物的制备方法如下：
将0.5 mmol化合物1与0.55mmol化合物2在氮气氛围下加至反应瓶中，然后将0.55mmol哌啶和5.0 mL乙醇/乙腈按照体积比1/1制备的混合溶液一次加至上述反应器中，室温下反应5h后，点板检测反应至原料消失，抽滤得到目标探针化合物；
所述化合物1结构式为：化合物1.jpg；
所述化合物2结构式为：化合物2.jpg。
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