CN105777794B - 一种双光子氟离子荧光探针化合物的制备与应用 - Google Patents
一种双光子氟离子荧光探针化合物的制备与应用 Download PDFInfo
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Abstract
本发明涉及一种氟离子荧光探针化合物及其制备与应用，所述氟离子荧光探针化合物具有式I所示的结构。制备方法是：将2,4‑二羟基苯甲醛，环己烯酮，以咪唑为催化剂，在氮气保护下反应；将分离所得产物与叔丁基二甲基氯硅烷反应得到纯净的氟离子荧光探针化合物。该探针化合物对氟离子具有很好的选择性和灵敏性，且对细胞没有毒性。该探针分子可以用双光子激发从而避开了紫外区。因此，该探针分子不仅可以应用于水中氟离子的检测，还可应用于细胞内氟离子的检测和成像。
Description
技术领域
本发明涉及一种氟离子荧光探针化合物及其制备与应用，属于荧光探针技术领域。
技术背景
氟是一种非金属元素，也是人体必需的微量元素，少量的氟可以代替磷构筑牙齿，使牙齿更坚固，饮用水和牙膏中含有适量的氟离子可以有效的预防龋齿，人体内氟含量过多会导致氟斑牙、氟骨病，人体内氟含量过少会导致骨质疏松，龋齿等。因此，定性定量的检测氟离子具有十分重要的意义。
荧光探针检测法由于其具有灵敏度高、选择性好、响应速度快等优点，近几年来备受研究人员的关注。双光子荧光探针的激发波长位于近红外区，在生物组织中的穿透力较强，可以实现生物组织深层物质的层析成像，另外，双光子荧光还能避免单光子成像中的荧光漂白等不利干扰，在生物成像中组织中自发荧光物质的激发波长一般在350-560nm的范围内，双光子荧光能有效的避免组织自发荧光的干扰。
在现有的技术中，CN104449677A提供了一种基于苯并噻唑的氟离子荧光探针的制备方法和用途，该探针可以应用于细胞中氟离子的检测，但是其存在以下不足：1、该探针是一个猝灭型的荧光探针，不利于生物成像的观察；2、该探针的激发波长300nm，对生物体有一定的伤害作用，而且细胞内物质的自发荧光会发生干扰；3、该探针没有报道工作曲线，不能定量的测定氟离子的含量。此外，CN104845611A提供了一种基于香豆素和荧光素的氟离子荧光探针的制备方法，该荧光探针可应用于水中的氟离子检测，该探针未能应用于细胞中氟离子的检测以及细胞成像研究。
发明内容
针对现有技术的不足，本发明提供一种双光子氟离子荧光探针化合物，既可以在单光子波长下激发检测水中的氟离子，还可以在双光子波长下激发，避免生物体内自发荧光物质干扰，特异性检测生物组织中的氟离子。
本发明还提供所述氟离子荧光探针化合物的制备方法与应用。
本发明的技术方案如下：
一种双光子氟离子荧光探针化合物，具有式I所示的结构：
本发明所述的氟离子荧光探针化合物的制备方法，包括如下步骤：
（1）将2,4-二羟基苯甲醛和环己烯酮，以咪唑为催化剂，四氢呋喃和水为溶剂，在室温氮气保护下反应，得到化合物II；
（2）上步制得化合物Ⅱ溶于二氯甲烷中，加入叔丁基二甲基氯硅烷，室温下搅拌反应，得到化合物Ⅰ；
根据本发明，优选的，步骤（1）所述2,4-二羟基苯甲醛与环己烯酮的摩尔比为1:1.1；
根据本发明，优选的，步骤（2）所述化合物Ⅱ与叔丁基二甲基氯硅烷的摩尔比为1:1.1；
根据本发明，优选的，步骤（1）、（2）全程在氮气保护下进行；
根据本发明，优选的，步骤（1）和（2）反应温度为25℃。
更为详细的，所述的双光子氟离子荧光探针化合物的制备方法，步骤如下：
(a) 取2,4-二羟基苯甲醛1.106g，咪唑0.816g置于三口烧瓶中，氮气保护，取1.3mL环己烯酮加入烧瓶中；另取四氢呋喃6mL，水6mL加入烧瓶中，25℃下反应72小时，然后加入20mL1M稀盐酸，水相用乙酸乙酯萃取，蒸发溶剂，用体积比为1:6的乙酸乙酯与正己烷的混合溶剂为展开剂，柱层析分离，得0.46克化合物II，产率19%。
(b) 取0.1克化合物Ⅱ、叔丁基二甲基氯硅烷0.331克、咪唑0.163克溶于10mL二氯甲烷中，室温下氮气保护反应8小时，反应完毕后，用体积比为10:1的石油醚与乙酸乙酯混合溶剂为展开剂，柱层析分离，得0.232克化合物III，产率69%。
本发明所述的双光子氟离子荧光探针化合物的应用，可用于测试细胞和水中的氟离子含量。
进一步优选的，所述荧光探针用于在pH=7.4的乙醇和水体积比1:20的溶液中氟离子的快速检测。在5%乙醇溶液中最低可探测的氟离子浓度为7.4*10-8 mol/L。
本发明通过实验验证，所述荧光探针在pH=7.4的乙醇和水体积比1:20的溶液中，用波长422nm的光作为激发波长，无荧光发射，单加入氟离子一个小时后检测，溶液在512nm处荧光迅速增强，而其他离子加入后溶液的荧光没有明显变化，该探针对氟离子具有很高的选择性。
将本发明的荧光探针化合物加入含有5%乙醇的水溶液中，配成荧光探针化合物浓度为μM的溶液，用HEPES缓冲溶液调节pH=7.4，加入不同浓度的氟离子，用波长422nm的光激发，在波长511nm处的荧光依次增强。得出工作曲线如图2所示，通过测定待测样品的荧光强度，就可以定量的计算出氟离子的浓度。
本发明所述的氟离子荧光探针化合物不仅可以用于水中氟离子的检测，还可以应用于组织细胞内的氟离子检测和成像。
将MCF-7细胞在含有10%牛胎儿血清的1mL细胞培养基培养12小时，然后用100μM的氟离子处理10分钟，在用10μM的本发明的荧光探针处理30分钟。细胞用激发波长为534nm的光源激发，在共聚焦显微镜下成像。
与现有的检测技术相比，本发明的荧光探针设计新颖，选择性好，且具有良好的水溶性，而不具有生物毒性，在制备方面，均使用常用试剂，合成步骤简单。本发明的优良效果如下：
1、本发明的荧光探针化合物具有较好的水溶性、生物相容性以及较好的膜穿透性，可用于细胞内氟离子的探测及成像。
2、本发明的荧光探针化合物由于能用于水体中的定量氟离子检测且检测限较低；
3、本发明的荧光探针化合物具有良好的灵敏度和选择性，测试样品前处理简单；
4、本发明的荧光探针化合物为固体粉末，便于储存使用，并且合成方法简单、收率高、成本低、具有良好的推广前景；
5、与现有的技术CN104449677A相比，本发明的荧光探针的激发波长更长，在细胞成像研究时所受干扰更少，而且对组织细胞几乎无损伤。
附图说明
图1为本发明的荧光探针与不同浓度的氟离子作用后的荧光强度工作曲线图。
图2为本发明的荧光探针的核磁图。
图3为本发明的荧光探针培养，再加入氟离子进行培养的MCF-7细胞图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不限于此。实施例中的各种原料均来自于市场购买。
实施例1、探针分子化合物I的合成
（a）取2,4-二羟基苯甲醛1.106g，咪唑0.816g置于三口烧瓶中，氮气保护，取1.3mL环己烯酮加入烧瓶中；另取四氢呋喃6mL，水6mL加入烧瓶中，25℃下反应72h，然后加入20mL1M稀盐酸，水相用乙酸乙酯萃取，蒸发溶剂，用体积比为1:6的乙酸乙酯与正己烷的混合容易为展开剂，柱层析分离，得到0.46g化合物II，产率19%；
（b）取0.1g化合物II、叔丁基二甲基氯硅烷0.331g、咪唑0.163g溶于10mL二氯甲烷中，室温下氮气氛围下反应8h，蒸出溶剂，用体积比为10:1的石油醚与乙酸乙酯混合溶剂展开剂，柱层析分离，得到0.232g化合物 I，产率69%。如图1，核磁氢谱：1H NMR (400 MHz,CDCl3) δ ppm : 10.18 (s, 1H), 8.74 (d, J=4.83, 1H), 8.65 (dd, J=7.12, 1.95,1H), 8.56 (d, J=7.95, 1H), 7.97-8.02 (m, 2H), 7.90 (td, J=7.65, 1.64, 1H),7.40 (ddd, J=7.57, 4.87, 1.10, 1H)。
实施例2、荧光实验
取实施例1制备的荧光探针化合物，溶解到含有5%乙醇的水溶液中，用HEPES缓冲溶液调节pH=7.4；得荧光探针溶液，备用；
1、取荧光探针溶液，分11组，每组10mL，其中1组不加阴离子，10组分别加入含有CO3 2‒、SO4 2‒、NO3 ‒、Cl‒、I‒、Br‒、CH3COO‒、N3 ‒、PO4 3‒、F‒溶液，使得每组溶液中含有探针化合物的浓度为10μΜ，阴离子浓度为300μM，使得阴离子与探针化合物的摩尔比为30:1；采用激发波长为422nm，荧光光度计测试其荧光强度，本发明探针溶液本身没有荧光，一旦加入氟离子，溶液在511nm荧光迅速增强，而其他离子加入溶液后,荧光强度没有变化，对氟离子具有很高的选择性；
2、取荧光探针溶液，分15组，每组10mL，分别加入不同浓度的氟离子溶液，调节到溶液中含有探针化合物的浓度为10μM，氟离子的浓度分别为探针化合物浓度的0、1、2、3、4、5、6、7、8、9、10、20、30、40、50倍。采用激发波长为422nm，荧光光度计测试其荧光强度，如图2所示，结果显示：溶液在511nm荧光迅速增强，其荧光强度与浓度成线性关系。根据测试计算，本探针化合物的最低检测限为7.4*10-8 mol/L。
实施例3、细胞成像实验
将MCF-7细胞在含有10%牛胎儿血清的1mL细胞培养基培养12小时，然后用100μM的氟离子处理10分钟，在用10μM的本发明的荧光探针处理30分钟。细胞用激发波长为534nm的光源激发，在共聚焦显微镜下成像。如图3为本发明的荧光探针培养，再加入氟离子进行培养的MCF-7细胞图。
Claims (1)
1.一种式I所示的氟离子荧光探针化合物
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