CN106397451A - 一种含有谷氨酸结构的罗丹明pH荧光探针及其制备方法和应用 - Google Patents
一种含有谷氨酸结构的罗丹明pH荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种含有谷氨酸结构的罗丹明pH荧光探针及其制备方法和应用，所述荧光探针为G‑pH荧光探针,G‑pH荧光探针的结构通式如(Ⅰ)所示。该G‑pH荧光探针在pH4.46‑9.0范围内呈现出较宽的pH响应，并且该探针的pKa为7.36，处于合理的生理pH区间，此外该探针还具有较好的化学、光稳定性，较好的溶解性和生物兼容性，不受金属离子的干扰。显微成像实验表明这类探针具有较好的细胞通透性，对细胞和生物体无毒副作用。
Description
技术领域
本发明涉及一种荧光探针，具体的涉及一种含谷氨酸结构的罗丹明pH荧光探针及其作为生物体内的pH荧光探针的应用。
背景技术
pH值是一个在环境监测、生物过程、生物医学中很常用的测量参数。pH在各种细胞活动中发挥着重要的作用，如细胞生长，钙调节，内吞作用，细胞粘附以及其他细胞过程等。细胞内pH值的微小变化可能引起细胞功能紊乱甚至诱发某些疾病的发生，同时某些疾病在发病前也会诱导pH出现波动，例如：结直肠癌，囊性纤维化和神经退行性等疾病均会出现pH异常。在生物体受到外来污染物的侵蚀过程中，其pH也可能出现波动。借助于荧光探针测量pH值，由于其对细胞没有损害性，而且具有高灵敏度、高选择性、便捷的可视化成像，使得荧光探针比其他技术具有着显著的优势。理想的pH荧光探针的pKa值应处于7.0±0.5范围内，并对pH值的变化高度敏感。目前基于罗丹明的pH荧光探针的pKa值几乎都不在上述范围内。为了充分利用罗丹明对光稳定的优越性能，现急需开发能够监测中性pH(7.0±0.5)变化的罗丹明荧光探针。
发明内容
本发明的目的是提供一种含有谷氨酸结构的罗丹明pH荧光探针，该罗丹明pH荧光探针可以快速响应、高灵敏度检测细胞中的pH变化。
本发明采用的技术方案是：一种含有谷氨酸结构的罗丹明pH荧光探针，所述的荧光探针是G-pH荧光探针，G-pH荧光探针的结构通式如(I)所示：
其中，
R1＝R2＝R3＝R4＝H；
或R1＝R4＝H，R2＝-CH2CH3，R3＝-CH3；
或R1＝R2＝-CH3，R3＝R4＝H；
或R1＝R2＝-CH2CH3，R3＝R4＝H；
或R1＝R4＝-(CH2)3-，R2＝R3＝-(CH2)3-。
上述含有谷氨酸结构的罗丹明pH荧光探针的pKa为7.36，处于中性pH区间(7.0±0.5)。
一种含有谷氨酸结构的罗丹明pH荧光探针的制备方法，包括如下步骤：将罗丹明类化合物与三氯氧磷加入到1，2-二氯乙烷中，加热回流反应3-5小时，冷却至室温后，加入氨基酸类化合物的乙腈溶液和三乙胺，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，干燥，经硅胶柱层析提纯，得到粉色固体；将该粉色固体溶于甲醇中，再加入氢氧化钠饱和水溶液，70-90℃加热水解，得到目标产物G-pH荧光探针。
优选的，上述的方法，所述罗丹明类化合物为罗丹明B、罗丹明6G、四甲基罗丹明TMR、罗丹明110或罗丹明101。
优选的，上述的方法，所述氨基酸类化合物为谷氨酸甲酯。
优选的，上述的方法，按摩尔比，罗丹明类化合物：三氯氧磷：氨基酸类化合物：三乙胺＝1:(3-6):1:(2-4)。
含有谷氨酸结构的罗丹明pH荧光探针的制备反应式如下，
本发明的含谷氨酸结构的罗丹明pH荧光探针可用于生理体系中pH的检测。主要用于检测活细胞体中pH的变化。所述的活细胞为Hela细胞株、MCF-7细胞株或RAW264.7细胞株。所述pH范围为4.46-9.0。
本发明的有益效果是：本发明G-pH荧光探针的pKa为7.36，处于合理的生理pH区间，能够快速的进入活细胞体内，其对H+响应迅速，具有较高的灵敏度，并且具有良好的酸碱可逆性，能够对其中的pH进行在线实时的检测，另外，该探针还具有较好的化学稳定性，较好的溶解性和生物兼容性，不受其他常见的金属离子等物种的干扰。
附图说明
图1是实施例1制备的G-pH1对pH变化的荧光的光谱响应。
图2是实施例1制备的G-pH1对pH变化的荧光最高点的拟合结果。
图3是实施例1制备的G-pH1在pH7.4对金属干扰性的荧光响应。
图4是实施例1制备的G-pH1的可逆性测试。
图5是实施例1制备的G-pH1对活细胞的荧光显微成像；
其中，a：Hela细胞株；b：染有G-pH1的Hela细胞株。
具体实施方法
实施例1 含有谷氨酸结构的罗丹明pH荧光探针(G-pH1荧光探针)
将1摩尔的罗丹明B与5摩尔的POCl3加入到干燥的1，2-二氯乙烷中，加热回流反应3-5小时，冷却至室温后，加入1摩尔的谷氨酸甲酯的乙腈溶液和3摩尔的三乙胺，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，再用无水硫酸镁干燥，经硅胶柱层析提纯，得到粉色固体。将该粉色固体溶于甲醇中，再加入氢氧化钠饱和水溶液，70-90℃加热水解，得到目标产物G-pH1荧光探针。HRMS：587.7058。
实施例2 含有谷氨酸结构的罗丹明pH荧光探针(G-pH2荧光探针)
将1摩尔罗丹明6G与3摩尔POCl3加入到干燥的1，2-二氯乙烷中，加热回流反应3-5小时，冷却至室温后，加入1摩尔的谷氨酸甲酯的乙腈溶液和3摩尔的三乙胺，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，再用无水硫酸镁干燥，经硅胶柱层析提纯，得到粉色固体。将该粉色固体溶于甲醇中，再加入3摩尔氢氧化钠饱和水溶液，70-90℃加热水解，得到目标产物G-pH2荧光探针。HRMS：559.6527。
实施例3 含有谷氨酸结构的罗丹明pH荧光探针(G-pH3荧光探针)
将1摩尔的四甲基罗丹明TMR与5.5摩尔的POCl3加入到干燥的1，2-二氯乙烷中，加热回流反应3-5小时，冷却至室温后，加入1摩尔的谷氨酸甲酯的乙腈溶液和3摩尔的三乙胺，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，再用无水硫酸镁干燥，经硅胶柱层析提纯，得到粉色固体。将该粉色固体溶于甲醇中，再加入3摩尔氢氧化钠饱和水溶液，70-90℃加热水解，得到目标产物G-pH3荧光探针。HRMS：559.6527。
实施例4 含有谷氨酸结构的罗丹明pH荧光探针(G-pH4荧光探针)
将1摩尔的罗丹明110与6摩尔的POCl3加入到干燥的1，2-二氯乙烷中，加热回流反应3-5小时，冷却至室温后，加入1摩尔的谷氨酸甲酯的乙腈溶液和3摩尔的三乙胺，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，再用无水硫酸镁干燥，经硅胶柱层析提纯，得到粉色固体。将该粉色固体溶于甲醇中，再加入3摩尔氢氧化钠饱和水溶液，70-90℃加热水解，得到目标产物G-pH4荧光探针。HRMS：531.5995。
实施例5 含有谷氨酸结构的罗丹明pH荧光探针(G-pH5荧光探针)
将1摩尔的罗丹明101与4摩尔的POCl3加入到干燥的1，2-二氯乙烷中，加热回流反应3-5小时，冷却至室温后，加入1摩尔的谷氨酸甲酯的乙腈溶液和3摩尔的三乙胺，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，再用无水硫酸镁干燥，经硅胶柱层析提纯，得到粉色固体。将该粉色固体溶于甲醇中，再加入3摩尔氢氧化钠饱和水溶液，70-90℃加热水解，得到目标产物G-pH5荧光探针。HRMS：611.7272。
实施例6 应用试验
本应用试验采用实施例1制备的G-pH1荧光探针进行。
[1]荧光光谱的测定。配制浓度为2×10-5mol/L的含有30％乙醇的G-pH1探针水溶液，该探针溶液分别用氢氧化钠和盐酸调节pH，分别制备pH范围在4.0-9.5不同pH值的溶液，测试荧光光谱，结果如图1所示，图1中每条线分别代表不同pH值的测试结果，最下端的线代表最高pH值为13.56，往上pH值依次降低最上端为pH值最低为4.57。结果表明，随着pH值的降低，荧光强度不断增强。探针在pH处于9.0～4.46范围内较为敏感，其荧光强度增强约500倍。
[2]图2是G-pH1对pH变化的荧光最高点的拟合结果，对图1中pH最高点值进行拟合，得知pKa为7.36。
[3]金属离子干扰性测定。分别配制浓度为2×10-5mol/L的含有30％乙醇的探针溶液，其pH分别为7.4。pH为7.4的探针溶液中分别加入金属盐的量为探针摩尔量的100倍，进行荧光光谱测定。结果如图3所示。结果显示，金属离子对探针的吸收与荧光光谱没有影响，即此探针在生物体内使用过程中不会受到来自金属离子的干扰。
[4]可逆性测试。光谱测定探针G-pH1在pH在5和10左右荧光变化的可逆性。用NaOH，HCl调节pH在5和10左右，进行荧光光谱测定。结果如图4所示，由图4可知，该探针响应迅速，荧光强度变化稳定而可逆。
[5]荧光显微成像。向含有活细胞为Hela细胞株的培养皿中，加入浓度为5×10-5M的G-pH1的二甲基亚砜溶液，与细胞培养液混合均匀，染色5min后，用pH＝7.4的磷酸盐缓冲溶液进行清洗三次，最后将该培养皿置于共聚焦显微镜下进行观察。实验结果发现，染有G-pH1的Hela细胞株(图中b)中呈现出明显的荧光，如图5所示，实验结果表明，G-pH1具有较好的细胞膜透过性，能够定位于细胞中。结果显示探针具有很好的透膜性，可以进入细胞并在细胞中指示pH的变化。
Claims (10)
1.一种含有谷氨酸结构的罗丹明pH荧光探针，其特征在于，所述含有谷氨酸结构的罗丹明pH荧光探针为G-pH荧光探针，其结构通式如(Ⅰ)所示：
其中，
R1＝R2＝R3＝R4＝H；
或R1＝R4＝H，R2＝-CH2CH3，R3＝-CH3；
或R1＝R2＝-CH3，R3＝R4＝H；
或R1＝R2＝-CH2CH3，R3＝R4＝H；
或R1＝R4＝-(CH2)3-，R2＝R3＝-(CH2)3-。
2.根据权利要求1所述的一种含有谷氨酸结构的罗丹明pH荧光探针，其特征在于：所述的G-pH荧光探针的pH区间为7.0+0.5。
3.根据权利要求2所述的一种含有谷氨酸结构的罗丹明pH荧光探针，其特征在于：所述的G-pH荧光探针的pKa为7.36。
4.权利要求1-3任一所述的含有谷氨酸结构的罗丹明pH荧光探针的制备方法，其特征在于，包括如下步骤：将罗丹明类化合物与三氯氧磷加入到1，2-二氯乙烷中，加热回流反应3-5小时，冷却至室温后，加入氨基酸类化合物的乙腈溶液和三乙胺，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，干燥，经硅胶柱层析提纯，得到粉色固体；将该粉色固体溶于甲醇中，再加入氢氧化钠饱和水溶液，70-90℃加热水解，得到目标产物G-pH荧光探针。
5.根据权利要求4所述的方法，其特征在于，所述罗丹明类化合物为罗丹明B、罗丹明6G、四甲基罗丹明TMR、罗丹明110或罗丹明101。
6.根据权利要求4所述的方法，其特征在于，所述氨基酸类化合物为谷氨酸甲酯。
7.根据权利要求4所述的方法，其特征在于，按摩尔比，罗丹明类化合物：三氯氧磷：氨基酸类化合物：三乙胺＝1:(3-6):1:(2-4)。
8.权利要求1、2或3所述的含有谷氨酸结构的罗丹明pH荧光探针在检测生理体系pH中的应用。
9.根据权利要求8所述的应用，其特征在于，荧光探针在检测活细胞体内pH中的应用。
10.根据权利要求9所述的应用，其特征在于，所述活细胞为Hela细胞株、MCF-7细胞株或RAW264.7细胞株。
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