CN108165267B - 一种开关型pH荧光探针及其制备方法和应用 - Google Patents
一种开关型pH荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于荧光探针制备技术领域,提供了一种开关型pH荧光探针及其制备方法和应用,以酸性品红为碳源,加入无水乙醇,均匀混合得到澄清的无色溶液;加入不锈钢反应釜中并密封,置于高温烘箱中反应,得到深棕色醇溶液;深棕色醇溶液通过旋转蒸发去除乙醇,得到黑色粘稠物,二次水溶解得到棕黑色水溶液;透析去除杂质,即得到纯净的氮硫共掺杂碳量子点水溶液;冷冻干燥后得到目标碳量子点,即开关型pH荧光探针。本发明工艺简单,原料来源广泛且价格便宜,制备条件要求低,所得探针量子产率较高。制得的pH荧光探针可在活细胞荧光成像中应用,也可用作活细胞pH实时监测。
Description
技术领域
本发明属于荧光探针制备技术领域,具体涉及一种开关型pH荧光探针及其制备方法和应用,属于碳量子点pH荧光探针。
背景技术
人体细胞内pH与细胞、酶和组织的许多重要生理过程如细胞增殖和凋亡、药物耐药性、离子运输、内吞作用和肌肉收缩等活动密切相关。不同的原核生物以及真核生物的不同亚细胞器内的pH值从碱性到强酸性不等。pH异常会导致细胞功能紊乱,严重时会引发诸如炎症、癌症和阿尔茨海默症等。研究表明:细胞酸化与癌症的发生发展有密切关系。因此,对细胞内pH进行灵敏、准确的实时监测,可以为分子水平上研究细胞的生理和毒理过程提供重要的信息。
实时监测细胞内pH的动态变化对于理解细胞内许多生理功能的规则机制有着重要的作用和意义。相较于微电极、NMR和吸收光谱等pH测量方法,荧光光谱检测技术对于在时空分布上检测pH变化具有独特的优势。此外,荧光技术具有操作简单、响应快速、高信噪比、高灵敏度以及大多数情况下对细胞无损伤等优点。荧光纳米材料、荧光染料和荧光试剂作为分子探针在生命科学领域备受瞩目。
碳量子点自2004年被发现以来,已经在生物成像与传感、光催化、化学和生物探针及光电器件等领域有较广泛的应用。碳量子点与传统的有机染料和半导体量子点相比,不仅具有较好的水溶性、稳定性、低毒性、抗光漂白性、生物相容性等特性,而且具有合成成本低、工艺简单、无毒环保等优点。以碳量子点为基础,构建pH荧光探针,用于细胞内pH实时监测,具有广阔的应用前景。
发明内容
本发明的目的在于提供一种开关型pH荧光探针,并建立一种操作简单、设备简易、原料低廉和绿色环保的制备方法,以及将所述的开关型pH荧光探针用于细胞内pH实时监测。
本发明由如下技术方案实现的:一种开关型pH荧光探针的制备方法,包括如下步骤:
1)、将酸性品红和无水乙醇置于玻璃容器中,搅拌混合均匀,得到澄清的无色溶液,酸性品红和无水乙醇的比例为:0.05-1.0:4-100g/ml;
2)、将玻璃容器中的无色溶液转移到聚四氟乙烯内胆中,将内胆密闭好后,置于不锈钢反应釜中,将反应釜置于高温烘箱中反应,得到深棕色醇溶液;
3)、将深棕色醇溶液置于旋转蒸发仪中,蒸发去除深棕色醇溶液中的乙醇,得到黑色粘稠物,利用二次水溶解粘稠物,得到棕黑色水溶液;
4)、将棕黑色水溶液通过1000Da的透析袋,在玻璃容器中透析处理至少3天,即得到纯净的氮硫共掺杂碳量子点水溶液;
5)、将获得的氮硫共掺杂碳量子点水溶液冷冻干燥后得到目标碳量子点,即开关型pH荧光探针。
步骤2)中所述的反应温度为250℃,反应时间为9-14h。步骤2)中所述的聚四氟乙烯内胆的体积为25ml,50ml,100ml,150ml,200ml或250ml。步骤3)中控制旋转蒸发仪温度为65-75℃。
本发明还提供了利用上述开关型pH荧光探针的制备方法制备的开关型pH荧光探针。所述开关型pH荧光探针发出黄色荧光,荧光量子效率为9.8-14.6%。本发明另外还提供了所述开关型pH荧光探针在活细胞内实时监测pH的应用,其检测线性范围为5.0-7.4。
所制备的开关型pH荧光探针具有较低的细胞毒性,良好的细胞相容性,作为荧光显像材料,可在活细胞荧光成像中应用。所制备的开关型pH荧光探针在活细胞中实时监测pH的应用,pH检测线性范围为5.0-7.4。
与现有技术相比,本发明具有以下优点:本发明开关型pH荧光探针的制备方法简单,原料来源广泛,价格便宜,具有很大的实用价值;所制备的开关型pH荧光探针在水溶液中具有良好的溶解度和分散性;开关型pH荧光探针的量子效率比较高,以荧光素(量子产率79%)为参照物,所得荧光探针的量子效率在9.8-14.6%之间;所制备的开关型pH荧光探针在紫外光照下发出黄色荧光,可有效克服细胞自发荧光的干扰;开关型pH荧光探针具有好的选择性,对H+的响应不受常见金属离子和生命体中一些氨基酸、葡萄糖等物质的干扰;具有很好的细胞穿透性,利用激光共聚焦成像技术可进行细胞内pH实时监测和细胞标记。
附图说明
图1为实施例1制备的开关型pH荧光探针的紫外吸收光谱、荧光激发光谱和发射光谱图,嵌入图为该探针在可见光和紫外光激发下的摄像图;其中玻璃容器盛有荧光探针水溶液,放置于紫外透射台上,经365nm激发光源激发后发出黄色荧光;图2为实施例1制备的开关型pH荧光探针在不同激发波长下的发射光谱图,该探针具有激发波长依赖性,当激发波长从320nm变化到540nm时,发射波长从532nm红移到585nm。图3为实施例1制备的开关型pH荧光探针与对照酸性品红的红外光谱图;图4为实施例1制备的开关型pH荧光探针的X射线光电子能谱总图,说明该探针主要由C、H、O、N、S五种元素组成。图5为实施例1制备的开关型pH荧光探针的透射电镜图(A)和粒径分布图(B);图6为在pH 4.4-8.0范围内,实施例1制备的开关型pH荧光探针荧光光谱图;图7为实施例1制备的开关型pH荧光探针在pH 4.4-8.0范围内S型拟合图(A)和探针在pH 5.0-7.4范围内线性关系图(B);图8为实施例1制备的开关型pH荧光探针利用MTT法进行的SiHa细胞毒性测试结果图;图9为实施例1制备的开关型pH荧光探针用于SiHa细胞内pH实时监测细胞成像图,从上到下依次为:明场图,暗场(激发为515nm)细胞图(黄色),明场和黄色暗场叠加图。
具体实施方式
下面结合实施例对本发明做详细说明,实施例给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1:一种开关型pH荧光探针的制备方法,包括如下步骤:
1)、称量0.1g酸性品红置于玻璃容器中,加入10ml无水乙醇,充分搅拌均匀,得到澄清的无色溶液;
2)、将玻璃容器中的无色澄清溶液转移到25ml聚四氟乙烯内胆中,将内胆密闭好后,置于不锈钢反应釜中,密封装好后将反应釜置于高温烘箱中,于250oC下反应12小时,得到深棕色醇溶液;
3)、将深棕色醇溶液置于旋转蒸发仪中,控制旋转蒸发仪温度为65℃,旋转蒸发去除深棕色醇溶液中的乙醇,得到黑色粘稠物,利用二次水溶解粘稠物,得到棕黑色水溶液;
4)、将棕黑色水溶液通过1000Da的透析袋,在玻璃容器中透析处理至少3天,即得到纯净的氮硫共掺杂碳量子点水溶液;
5)、将上述氮硫共掺杂碳量子点水溶液冷冻干燥后得到碳量子点,即开关型pH荧光探针,其相对荧光量子产率(以荧光素为标准)为14.6%。性质表征和应用见图1-9。
实施例2:一种开关型pH荧光探针的制备方法,称量1.0g酸性品红置于玻璃容器中,加入95ml无水乙醇,充分搅拌,得到澄清的无色溶液;无色溶液转移到200ml聚四氟乙烯内胆中,于250oC下反应14小时,得到深棕色醇溶液;将深棕色醇溶液置于旋转蒸发仪中,控制旋转蒸发仪温度为75℃,旋转蒸发去除深棕色醇溶液中的乙醇,得到黑色粘稠物,其余制备方法同实施例1所述方法。获得的开关型pH荧光探针,其相对荧光量子产率(以荧光素为标准)为11.8%。
实施例3:一种开关型pH荧光探针的制备方法,称量0.05g酸性品红置于玻璃容器中,加入4ml无水乙醇,充分搅拌,得到澄清的无色溶液;将无色澄清溶液转移到50ml聚四氟乙烯内胆中,于250oC下反应9小时,得到深棕色醇溶液;将深棕色醇溶液置于旋转蒸发仪中,控制旋转蒸发仪温度为70℃,旋转蒸发去除深棕色醇溶液中的乙醇,得到黑色粘稠物,其余制备方法同实施例1所述方法。获得的开关型pH荧光探针,其相对荧光量子产率(以荧光素为标准)为10.2%。
实施例4:一种开关型pH荧光探针的制备方法,称量0.5g酸性品红置于玻璃容器中,加入55ml无水乙醇,充分搅拌,得到澄清的无色溶液;将无色澄清溶液转移到100ml聚四氟乙烯内胆中,于250oC下反应10小时,得到深棕色醇溶液;将深棕色醇溶液置于旋转蒸发仪中,控制旋转蒸发仪温度为73℃,旋转蒸发去除深棕色醇溶液中的乙醇,得到黑色粘稠物,其余制备方法同实施例1所述方法。获得的开关型pH荧光探针,其相对荧光量子产率(以荧光素为标准)为12.1%。
实施例5:一种开关型pH荧光探针的制备方法,步骤1,称量0.3g酸性品红置于玻璃容器中,加入30ml无水乙醇,充分搅拌,得到澄清的无色溶液;将无色澄清溶液转移到150ml聚四氟乙烯内胆中,于250oC下反应13小时,得到深棕色醇溶液;将深棕色醇溶液置于旋转蒸发仪中,控制旋转蒸发仪温度为68℃,旋转蒸发去除深棕色醇溶液中的乙醇,得到黑色粘稠物,其余制备方法同实施例1所述方法。获得的开关型pH荧光探针,其相对荧光量子产率(以荧光素为标准)为11.5%。
实验例1:石英比色皿盛有实施例1所制备的“开关型”pH荧光探针水溶液,放置于紫外透射台上,经365nm激发光源激发后发出明亮的黄色荧光,结果见图1,荧光量子产率约14.6%。实施例1制备的“开关型”pH荧光探针在不同激发波长下的发射光谱图见图2,由图2可知:该探针具有激发波长依赖性,当激发波长从320nm变化到540nm时,发射波长从532nm红移到585nm。
实施例1制备的“开关型”pH荧光探针与对照酸性品红的红外光谱图见图3,图中横坐标为检测波长,纵坐标为透过率,该图说明酸性品红经过水热反应后完全碳化为氮硫共掺杂碳量子点,且表面富含OH/NH/C=N/CON-H/C-S/C-O-C等官能团,该探针的X射线光电子能谱总图见图4,由图3图4可知该探针主要由C、H、O、N、S五种元素组成。
实施例1制备的开关型pH荧光探针的透射电镜图(A)和粒径分布图(B)见图5,由图5可知:该探针的粒径范围为2.0-4.1nm,平均粒径为2.99 ± 0.2 nm(见图5)。
实验例2:将实施例1所制备的开关型pH荧光探针在pH 4.4-8.0范围内检测荧光光谱,谱图见图6,说明随着pH变化,探针荧光强度发生显著变化。该探针在pH4.4-8.0范围内pH值与荧光强度线性关系图与探针在pH 5.0-7.4范围内pH值与荧光强度线的线性关系图,见图7,可以看出该探针在pH4.4-8.0范围内pH值与荧光强度为S型拟合关系,而在pH 5.0-7.4范围内为线性关系,经计算得到pK a 为6.06。
实验例3:实施例1制备的开关型pH荧光探针利用MTT法进行的SiHa细胞毒性进行测试,结果见图8,由图8可得:该开关型pH荧光探针具有较低的细胞毒性和良好的细胞相容性。
该荧光探针水溶液(0.5mg/mL)用于SiHa细胞内pH变化实时监测细胞,如图9所示,细胞形态良好,可见荧光探针没有细胞毒性,可用于活细胞标记和pH实时监测。当pH由7.4调节到5.0时,SiHa细胞荧光显著增强,且SiHa细胞可发出探针的特征黄色荧光,可有效避免细胞自发荧光的干扰,可见该开关型pH荧光探针有望用于细胞内pH变化实时监测,为癌症早期诊断提供新思路。
以上所述,仅是本发明的部分较好实施例而已,并非对本发明做任何形式上的限制。
Claims (9)
1.一种开关型pH荧光探针的制备方法,其特征在于:包括如下步骤:
1)、将酸性品红和无水乙醇置于玻璃容器中,搅拌混合均匀,得到澄清的无色溶液,酸性品红和无水乙醇的比例为:0.05-1.0:4-100g/ml;
2)、将玻璃容器中的无色溶液转移到聚四氟乙烯内胆中,将内胆密闭好后,置于不锈钢反应釜中,将反应釜置于高温烘箱中反应,得到深棕色醇溶液;
3)、将深棕色醇溶液置于旋转蒸发仪中,蒸发去除深棕色醇溶液中的乙醇,得到黑色粘稠物,利用二次水溶解粘稠物,得到棕黑色水溶液;
4)、将棕黑色水溶液通过1000Da的透析袋,在玻璃容器中透析处理至少3天,即得到纯净的氮硫共掺杂碳量子点水溶液;
5)、将获得的氮硫共掺杂碳量子点水溶液冷冻干燥后得到目标碳量子点,即开关型pH荧光探针。
2.根据权利要求1所述的一种开关型pH荧光探针的制备方法,其特征在于:步骤2)中所述的反应温度为250℃,反应时间为9-14h。
3.根据权利要求1所述的一种开关型pH荧光探针的制备方法,其特征在于:步骤2)中所述的聚四氟乙烯内胆的体积为25ml,50ml,100ml,150ml,200ml或250ml。
4.根据权利要求1所述的一种开关型pH荧光探针的制备方法,其特征在于:步骤3)中控制旋转蒸发仪温度为65-75℃。
5.利用权利要求1至4任一项所述的一种开关型pH荧光探针的制备方法制备的开关型pH荧光探针。
6.根据权利要求5所述的利用开关型pH荧光探针的制备方法制备的开关型pH荧光探针,其特征在于:所述开关型pH荧光探针发出黄色荧光,荧光量子效率为9.8-14.6%。
7.根据权利要求5所述的利用开关型pH荧光探针的制备方法制备的开关型pH荧光探针,其特征在于:所述开关型pH荧光探针在活细胞荧光成像中的应用。
8.根据权利要求5所述的利用开关型pH荧光探针的制备方法制备的开关型pH荧光探针,其特征在于:所述开关型pH荧光探针在活细胞内实时监测pH的应用。
9.根据权利要求8所述的利用开关型pH荧光探针的制备方法制备的开关型pH荧光探针,其特征在于:所述开关型pH荧光探针在活细胞中实时监测pH,pH检测线性范围为5.0-7.4。
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