CN113200935B - 一种用于次氯酸检测的荧光探针及其制备方法 - Google Patents
一种用于次氯酸检测的荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测次氯酸的荧光探针及其制备方法。该探针可以选择性地与次氯酸作用，释放出强荧光物质。其优点在于对次氯酸具有高的选择性和灵敏度，与次氯酸响应后伴随着明显的颜色的变化。可以在细胞和活体层次上对次氯酸进行识别。该探针可经化学合成获得，合成工艺简单，并且检测方法操作简单、灵敏度高、选择性好，使得该类探针在生物领域具有实际的应用价值。
Description
技术领域
本发明属于分析化学技术领域，具体设计一种可用于次氯酸检测的荧光探针及其制备方法。
背景技术
作为生物体内一种重要的活性氧物种，次氯酸在生物体内具有重要的生理作用，参与众多生理过程，在生物体内，次氯酸是在髓过氧化物酶的催化作用下由过氧化氢和氯离子发生反应而产生。然而，生物体内一旦产生过量的次氯酸，就会引起包括类风湿关节炎和癌症等在内的各种疾病，因此，快速、灵敏、高选择性检测生物体内次氯酸具有非常重要的意义。
荧光探针就是这样一种小分子或者蛋白质，通过与分析物作用之后使荧光信号(如荧光强度、荧光寿命、检测波长)发生变化从而达到定性定量检测的目的荧光探针具有操作简便、非侵入性、灵敏度高、选择性好和实时监控等优点，己经日益成为生命科学研究领域的重要工具。
发明内容
本发明目的在于提供一种新的可用于次氯酸检测的荧光探针，以及提供该荧光探针的制备方法和其在溶液和生物成像中的应用。
本发明涉及一种可用于次氯酸检测的荧光探针，该荧光探针的具有式I所示结构：
本发明涉及具有式I结构化合物的制备方法，如下：
上述合成路线具体步骤如下：
(1)中间体C的制备
将化合物A和B溶解在二氯甲烷中，加入碱，反应得到化合物C。所述碱选自各种有机碱和无机碱，优先选三乙胺和碳酸钠。
(2)化合物I的制备
将化合物C溶于甲醇中，加入钠，反应得到化合物I。
本发明涉及具有式I结构的荧光探针自身没有荧光，在其水溶液中加入次氯酸后能够产生强红色荧光。
本发明涉及具有式I结构的荧光探针，可做成试纸用于次氯酸的检测，还可以在溶液和生物成像层次应用。
检测溶液的制备及检测步骤如下：
(1)式I化合物溶于有机溶剂(甲醇，N，N-二甲基甲酰胺，二甲基亚讽)中，然后用缓冲溶剂稀释，用于次氯酸的检测。
(2)式I化合物溶于有机溶剂中(乙醇，二氯甲烷，乙腈)中，将干净的纸条浸泡在化合物I的溶液中一段时间，取出，晾干，用于次氯酸的检测。
(3)式I化合物溶于有机溶剂(甲醇，N，N-二甲基甲酰胺，二甲基亚讽)中，注射到动物活体模型中用于动物层次生物成像检测。
本发明的探针分子具有以下显著优点：(1)具有新颖的结构；(2)可以对次氯酸快速识别；(3)该制备方法简单，产品易于分离、纯化。
附图说明
图1为本发明中化合物C的1H NMR谱图。
图2为本发明中化合物C的13C NMR谱图。
图3为本发明式I荧光探针分子的1H NMR谱图。
图4为本发明式I荧光探针分子的13C NMR谱图。
图5为本发明式I荧光探针分子的选择性荧光谱图。荧光探针I的浓度为10μM，次氯酸浓度为20μM，其它分析物浓度为50μM。比较的是590nm处的荧光强度，激发波长为560nm。
图6为本发明式I荧光探针分子加入次氯酸后，溶液荧光强度随时间的变化。荧光探针I的浓度为10μM，次氯酸浓度为20μM。激发波长为560nm，发射波长为590nm。
图7为本发明式I荧光探针分子做成次氯酸试纸条后，滴加次氯酸后在日光和365nm紫外灯下的变化。
图8为本发明式I探针分子在小鼠炎症模型中的成像应用。
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应上述方案中化合物的号码。
实施例1
化合物C的合成：
在25mL的圆底烧瓶中，将化合物B(0.12g,0.55mmol)、碳酸钠(0.12g,1.11mmol)和4-甲基吡啶(0.07g,0.55mmol)加到5mL的二氯甲烷中，再将化合物2(0.20g,0.55mmol)溶解在5mL的二氯甲烷中，滴加到上述反应液，室温条件下继续反应3—5小时。反应结束后，将反应液倒进冰水中，用二氯甲烷萃取，有机相用无水硫酸钠干燥，过滤，蒸除溶剂，硅胶柱层析分离提纯，得到淡黄色固体0.19g，收率63％。1H NMR和13C NMR谱图如图1和图2所示。1H NMR(400MHz,CDCl3):δ7.53(d,J＝8.2Hz,2H),7.42(d,J＝8.2Hz,2H),7.09(d,J＝8.2Hz,2H),6.83–6.86(m,4H),5.30(s,2H),3.47(s,3H),3.35(s,3H),2.30(s,6H)；13C NMR(100MHz,CDCl3):δ187.5,169.1,154.0,152.9,150.3,148.5,133.0,129.2,125.9,125.3,123.1,116.6,110.5,67.9,43.3,38.8,21.1。
探针分子I的合成：
在0℃条件下，将化合物C(0.10g,0.19mmol)溶于3mL的甲醇中，将钠屑(8.60mg,0.37mmol)慢慢加到该反应液，继续在该条件下搅拌反应0.5小时，反应结束后，将反应液倒到饱和碳酸氢钠水溶液中，乙酸乙酯萃取，有机相用无水硫酸钠干燥，过滤，蒸除溶剂，硅胶柱层析分离提纯，得到淡黄色固体0.07g，收率79％。1H NMR(400MHz,Acetone-d6):δ8.61(s,2H),7.46(d,J＝8.4Hz,2H),7.42(d,J＝8.8Hz,2H),7.08(d,J＝8.4Hz,2H),6.62(dd,J＝2.8,8.8Hz,2H),6.57(d,J＝2.8Hz,2H),5.29(s,2H),3.41(s,3H),3.36(s,3H)；13C NMR(100MHz,Acetone-d6):δ187.8,156.4,154.7,153.8,151.6,134.3,129.4,123.6,121.5,110.8,103.8,67.8,43.0,38.5。1H NMR和13C NMR谱图如图3和图4所示。
实施例2
探针分子I对次氯酸的选择性研究。实施例1制备的荧光探针I溶于二甲基亚砜中，配制成浓度为1mM的储备液，然后用PBS(10mM,pH＝7.4)配制浓度为10μM探针测试溶液。如图5所示，向探针I溶液中分别加入50μM其它活性分析物(半胱氨酸Cys，高半胱氨酸Hcy，还原性谷胱甘肽GSH，氯化钠NaCl，亚硝酸钠NaNO2，亚硫酸钠Na2SO3，亚硫酸氢钠NaHSO3，过氧化钾KO2，双氧水H2O2，氢氧根自由基·OH，过氧化叔丁醇TBHP，一氧化氮NO，过氧根自由基1O2，过氧亚硝酸钠NaONOO)，发现以上这些分析物的加入并不能引起探针I溶液荧光强度发生变化，但向探针I溶液中加入20μM次氯酸(HClO)时，探针I溶液荧光强度明显增强。
实施例3
探针分子I对次氯酸检测的时间研究。向10μM探针I的溶液中加入20μMHClO后，荧光强度随时间的变化。如图6所示，荧光强度能够快速达到平衡，说明该探针可用于次氯酸(HClO)的快速检测。
实施例4
探针分子I做成检测次氯酸试纸条的研究。将普通滤纸剪成大小相似的长条，浸泡在10μM探针分子I的乙醇溶液中，10分钟后取出，晾干。将不同浓度的HClO滴到试纸上，结果如图7所示，随着HClO浓度的增大，滴到试纸上后颜色红色颜色越明显，并且在365nm的紫外等下荧光也在增强。
实施例5
探针分子I在活体成像中的研究。如图8所示，使用不同体积角叉菜胶在小鼠的左侧脚踝处构建不同程度的炎症模型，再在两侧脚踝处注射100μL,1mM的探针分子I后，放在小动物活体成像系统里观察，发现炎症越严重，荧光越强。该荧光探针可用于炎症成像。
由此得出结论，本发明合成一种新型荧光探针I可用于次氯酸的检测，该探针对次氯酸具有很高的选择性，具有较好的应用前景。
Claims (6)
1.一种可用于次氯酸检测荧光探针分子，其特征在于具有如式I所示的结构式：
2.一种如权利要求1所述的一种可用于次氯酸检测荧光探针分子的制备方法，其主要合成路线如下：
。
3.根据权利要求2所述的一种可用于次氯酸检测荧光探针分子的制备方法，其特征在于：第一步，碱选自有机碱和无机碱。
4.根据权利要求1所述的一种可用于次氯酸检测荧光探针分子的用途，其特征在于：能够对水溶液中的次氯酸专一性快速检测。
5.根据权利要求1所述的一种可用于次氯酸检测荧光探针分子，其特征在于：能够做成试纸条用于次氯酸的检测。
6.一种如权利要求1所述的一种可用于次氯酸检测荧光探针分子的用途，其特征在于利用次氯酸检测荧光探针分子与次氯酸反应产生荧光将其用于生物成像。
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