CN110041314B - 一种用于检测水合肼的水溶性比率型荧光探针 - Google Patents

一种用于检测水合肼的水溶性比率型荧光探针 Download PDF

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CN110041314B
CN110041314B CN201910305033.XA CN201910305033A CN110041314B CN 110041314 B CN110041314 B CN 110041314B CN 201910305033 A CN201910305033 A CN 201910305033A CN 110041314 B CN110041314 B CN 110041314B
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李俊
位灯国
崔元超
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Abstract

本发明公开了一种用于检测水合肼的水溶性比率型荧光探针，它具有以下所示的化学结构，该探针对于水合肼具有很好的荧光和紫外选择性，能够实现纯水相中检测，并且具有较好的生物学应用前景，能够实现细胞和斑马鱼体内比率型荧光成像。
Figure DDA0002029532270000011

Description

一种用于检测水合肼的水溶性比率型荧光探针
技术领域
本发明涉及一种检测水合肼的水溶性比率型荧光探针。
背景技术
水合肼是重要的化工原料，在工业中常用于农药和医药中间体的制备。然而，水合肼具有高毒性，其蒸汽可以刺激鼻和上呼吸道，引起头晕和中枢神经伤害，长时间皮肤接触水合肼会经皮肤吸收引起中毒，由水合肼引起的水污染也会进入鱼类体内并可能导致其死亡。因此，对于水合肼的检测对于环境污染监控十分重要。
传统检测水合肼的方法主要有质谱法，电化学法，电泳法等，相比以上方法，荧光探针具有灵敏度高，操作简易，可视化程度高等优势。目前虽然报道了一些用于水合肼检测的荧光探针，但仍存在一些不足之处：1.检测过程需要大量的有机溶剂参与，难以实现纯水相检测；2.荧光响应模式单一，主要是荧光增强型或者猝灭型，难以消除生物体内背景干扰；3.一些探针缺乏选择性难以区分水合肼和其它有机胺。
CN 108003173 A公开了一种荧光探针，该探针以香豆素为荧光母体，以乙酸酯作为识别基团，能特异性识别水合肼，但是该探针本身荧光发射峰在500nm处，与水合肼结合后荧光波长蓝移至425nm处，位于蓝光区，并且没有提及生物活体如斑马鱼类的成像和肼蒸汽的检测。此外，该专利中并没有提及化合物对其它有机胺类化合物的选择性。
发明内容
本发明的目的是针对上述缺陷，提供一种新的水合肼荧光探针，该探针由于引入了水溶性烷氧链，从而增强水溶性，减少了有机溶剂的参与，同时由于探针的荧光发射峰位于远红外区，从而降低了生物体内的背景干扰，可用于斑马鱼等生物体的检测。
本发明提供的荧光探针具有以下化学结构：
Figure BDA0002029532250000021
它是由取代的邻羟基苯甲醛为起始原料，经环化反应、脱羧反应、维尔斯迈尔－哈克反应、缩合反应最终得到目标产物。
试验结果表明：
(1)该探针具对水合肼具有很好的比率型荧光响应。
(2)该探针能实现细胞内和斑马鱼体内的水合肼荧光成像。
本发明的有益效果是：
(1)本发明探针对于水合肼具有很好的荧光和紫外选择性，能够实现纯水相中检测，并且具有较好的生物学应用前景，能够实现细胞和斑马鱼体内比率型荧光成像。
(2)该探针由于引入了水溶性烷氧链，从而增强水溶性，减少了检测中有机溶剂的参与。
(3)该探针具有较长的荧光发射波长，在560nm的激发光下，荧光发射波峰处于640nm处，位于远红外区；与水合肼反应后，在450nm的激发光下，荧光发射波峰处于510nm处，位于绿光区。
附图说明
图1探针与水合肼等物质作用后的紫外吸收响应曲线。
图2探针在不同水合肼浓度下的紫外响应曲线。
图3红光通道下探针对于水合肼的荧光强度变化(激发波长560nm)。
图4绿光通道下探针对于水合肼的荧光强度变化(激发波长450nm)。
图5(a)绿光通道下探针对于各种分析物的荧光响应(激发波长450nm)；(b)红光通道下探针对于各种分析物的荧光响应(激发波长560nm)，探针浓度10μM，分析物浓度100μM。
图6探针对于水合肼的荧光响应线性关系。
图7探针对于水合肼在低浓度下的荧光响应线性关系。
图8肼蒸汽检测实验，第一行：可见光下检测试纸颜色变化，第二行：365nm紫外灯下检测试纸荧光变化。
图9探针用于水合肼的细胞成像图。
图10探针用于斑马鱼体内水合肼的检测。
具体实施方式
下面通过具体实施例对本发明进行详细地说明。
实施例1探针的合成和结构表征
由取代的邻羟基苯甲醛为起始原料，经环化反应，脱羧反应，维尔斯迈尔－哈克反应，缩合反应最终得到目标产物。
探针的合成探针路线如下：
Figure BDA0002029532250000031
合成步骤：
(1)化合物1、2、3的制备参考文献(Chem.Commun.2016，52，8675-8678)。
(2)化合物4的制备：将化合物3(6mmol,1.48g)和4-吡啶乙腈(7mmol,0.83g)溶于20mL无水乙醇中，再加入10滴哌啶；反应体系在60℃下搅拌6小时后停止加热，并自然冷却至室温。析出的固体经减压过滤，干燥得到化合物4为棕色固体1.43g，产率68％。该中间体由于溶解性差，难以用核磁共振进行结构表征，因此将该化合物直接投入下一步反应中。
(3)目标化合物-肼探针的制备：将化合物4(1.2mmol,0.42g)和二乙二醇-2-溴乙基甲醚(3.6mmol,0.82g)溶于20mL无水乙腈中回流12小时。薄层色谱监测反应结束后，将溶剂乙腈蒸干，得到的残渣经柱层析得到产品为暗红色固体157mg,产率23％.
通过核磁共振氢谱(图3)，碳谱(图4)和高分辨质谱(图5)确认该探针的结构。
1H NMR(600MHz,DMSO-d6)δ:8.98(d,J＝6.6Hz,2H),8.86(s,1H),8.41(s,1H),8.32(d,J＝7.2Hz,2H),7.67(d,J＝9.0Hz,1H),6.88(d,J＝7.2Hz,1H),6.69(s,1H),4.78(t,J＝4.2Hz,2H),3.91(t,J＝4.8Hz,2H),3.58-3.55(m,6H),3.48-3.44(m,4H),3.41-3.39(m,2H),3.24(s,3H),1.18(t,J＝6.6Hz,6H).
13C NMR(150MHz,DMSO-d6)δ:160.52,158.11,154.21,150.22,145.68,145.32,144.81,133.08,122.73,116.96,111.57,111.19,108.86,101.98,97.25,71.75,70.13,70.01,69.99,69.10,59.88,58.58,45.27,12.94.
HRMS[M+H+]calc:492.2493,found 492.2489。
最终确认探针化学结构式如下：
Figure BDA0002029532250000041
实施例2探针对水合肼紫外吸收的影响
1.如图1所示，在10mM，pH为7.4的PBS缓冲溶液中，探针(10μM)本身紫外吸收在560nm处，加入100μM(10当量)水合肼后，位于560nm处的紫外吸收峰明显降低，新的吸收峰在位于450nm处生成。其它类型胺类(包括甲胺，氨水，吗啡啉，N,N-二异丙基乙基胺和苯胺)以及金属离子(锌离子，汞离子和镉离子)不会产生紫外响应。
2.如图2所示，随着水合肼浓度增加(0-100μM)，560nm处的紫外吸收峰逐渐降低，450nm处的紫外吸收峰逐渐增强。
3.如图3所示，该探针对水合肼在560nm的激发波长下，探针的荧光发射波长在640nm处，随着水合肼的浓度增加(0-100μM)，其在640nm出的荧光逐渐减弱。
4.如图4所示，在450nm激发波长下，位于510nm处的荧光发射峰逐渐增强。
5.如图5所示，探针对于水合肼具有很好的选择性荧光响应，其它类型胺类(包括甲胺，氨水，吗啡啉，N,N-二异丙基乙基胺和苯胺)以及金属离子(锌离子，汞离子和镉离子)不会产生的荧光响应。
6.线性范围和检测限
此外，该探针在0-35μM范围内对于水合肼具有较广的荧光比率(I510/I640)线型响应范围(图6)(R2＝0.99)。
检测限(LOD)的测定方法：根据参考文献Chron.Young Sci.2011,2,21-25，使用一下公式求得，其中S/N为仪器信噪比，数值为3。δ为扫描空白样品12次求得的标准差，数值为0.001562，k为低浓度下线型曲线的斜率0.0122(图7)，将数值代入以上公式求得检测限为0.38μM。
Figure BDA0002029532250000051
综上，该探针可以用于对水合肼的比率型荧光响应检测。同时，该探针对于其它类型胺类(包括甲胺，氨水，吗啡啉，N,N-二异丙基乙基胺和苯胺)以及金属离子(锌离子，汞离子和镉离子)不会产生的荧光响应，因此该探针能够很好地区分水合肼和其它类型的有机胺。
实施例3探针对肼蒸汽的检测
将20μL的探针(1mM)溶液浸于圆形滤纸中，放入通风厨中自然晾干后至于10mL样品瓶盖中，样品瓶内分别盛有空气，水，0.1％，1％和5％浓度的水合肼溶液1mL，将盖子盖上后静置20min。如图8所示，与空气和水蒸汽接触的试纸没有颜色的变化和荧光响应，与肼蒸汽接触后，随着肼蒸汽溶度的增加，试纸颜色逐渐由暗红色变为黄色，在365nm的手提式紫外灯下，荧光逐渐变为绿光。
实施例4探针用于细胞及斑马鱼成像实验
为进一步验证该探针的生物学应用功能，我们将10μM探针注入到A549细胞内，孵育30min后将培养基置于荧光共聚焦显微镜下拍照，可以看见细胞在红色荧光信号通道(λem＝570-625nm，λex＝559nm)呈现强烈的红色荧光；而绿光通道(λem＝500-545nm，λex＝488nm)没有明显的荧光。加入500μM的水合肼后，随着时间的增加，红光通道内的荧光逐渐减弱，绿光通道的荧光逐渐增强(图9)。由此可见，该探针是可以用作检测细胞内水合肼的比率型成像的。
同样，该探针也能用于斑马鱼体内的水合肼荧光比率型成像(图10)。
综上，该探针对于水合肼具有很好的荧光和紫外选择性，能够实现纯水相中检测，并且具有较好的生物学应用前景，能够实现细胞和斑马鱼体内比率型荧光成像。

Claims (2)

1.一种用于检测水合肼的水溶性比率型荧光探针，其特征在于具有以下化学结构：
Figure FDA0002975692840000011
2.权利要求1所述的探针在检测斑马鱼体内的水合肼成像和肼蒸汽试纸中的应用。
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