CN110330965B - 一种含脲结构的六元螺环罗丹明pH荧光指示剂及其应用 - Google Patents
一种含脲结构的六元螺环罗丹明pH荧光指示剂及其应用 Download PDFInfo
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- RLOWWWKZYUNIDI-UHFFFAOYSA-N phosphinic chloride Chemical compound ClP=O RLOWWWKZYUNIDI-UHFFFAOYSA-N 0.000 description 4
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Abstract
本发明涉及一种含脲结构的六元螺环罗丹明pH荧光指示剂及其应用。所述荧光指示剂具有通式(I)的结构。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，冷却至室温后，去除溶剂；然后加入叠氮化钠的丙酮水溶液，室温下搅拌，所得反应液用二氯甲烷萃取，干燥，减压蒸除溶剂，将所得反应液用干燥的乙腈溶解后移入反应釜中，加入过量氨水，100℃油浴反应10小时；所得产物用二氯甲烷萃取，干燥后，经硅胶柱层析提纯，得目标产物Rh‑NH。本发明的指示剂具有较好的pH可逆性。使用本发明的指示剂对大型蚤进行染色后，发现指示剂Rh‑NH1能够定位于大型蚤的肠道内，并指示其显酸性。
Description
技术领域
本发明涉及一种含脲结构的六元螺环罗丹明pH荧光指示剂。
背景技术
任何化学反应或生物活动都需要精确地控制相关参数，pH值就是关键的指示参数之一。在正常生理条件下，细胞外液的H+浓度约为40nmol/L(pH＝7.40)，正常变动幅度约为0.1-0.2个pH单位，异常的pH值会干扰细胞功能。同时某些疾病在发病前也会诱导pH出现波动，如结直肠癌，囊性纤维化等疾病均会出现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。与玻璃电极法、核磁共振法、吸收光谱法等其他检测pH值的方法相比，借助于荧光探针测量pH值，由于其对细胞没有损害性，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更显著的优势。
罗丹明及其衍生物是一类重要的荧光染料，比较典型的罗丹明有罗丹明6G，罗丹明B，四甲基罗丹明TMR，罗丹明101，罗丹明110等。与其他类别的荧光染料相比，罗丹明染料具有优异的光稳定性、良好的水溶性、光谱可调节范围宽(500-700nm)、较高的发光效率(Φ＝0.9-0.5)。在以往的研究中，基于罗丹明的荧光探针都是以五元螺酰亚胺环结构为基础进行的研究与报道，尚未有设计合成六元环的罗丹明pH荧光探针。六元环是有机化合物中最常见的结构，六元环化学键的角张力更小，分子内部的热运动减少，与五元环结构相比更稳定。同时，螺环的扩展也有可能提高指示剂的灵敏度。因此六元环结构的罗丹明荧光指示剂拥有广阔的应用前景。
发明内容
本发明的目的是提供一种可以快速响应、高灵敏度检测pH变化的荧光指示剂。
本发明采用的技术方案是：一种含脲结构的六元螺环罗丹明pH荧光指示剂，所述含脲结构的六元螺环罗丹明pH荧光指示剂为Rh-NH，具有如(Ⅰ)所示的结构通式：
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至R4构成-(CH2)3-，R2至R3构成-(CH2)3-。
含脲结构的六元螺环罗丹明pH荧光指示剂的制备方法，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，去除溶剂1,2-二氯乙烷；然后加入叠氮化钠的丙酮水溶液，室温下搅拌24小时，所得反应液用二氯甲烷萃取，取下层液，干燥，减压蒸除溶剂后，将所得反应液用干燥的乙腈溶解后移入反应釜中，加入过量氨水，100℃油浴反应10小时；所得产物用二氯甲烷萃取，取下层液，干燥后，经硅胶柱层析提纯，得目标产物Rh-NH。反应式如下：
优选地，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。
优选地，按摩尔比，罗丹明类化合物:三氯氧磷:叠氮化钠＝1:3:1.3。
上述的含脲结构的六元螺环罗丹明pH荧光指示剂在检测浮游动物消化道中pH的应用。
优选地，所述浮游动物为大型蚤。
本发明的有益效果是：本发明制备的荧光指示剂分子对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pH进行在线实时的检测，能够用于大型蚤肠道的荧光指示。
附图说明
图1是实施例1制备的Rh-NH1在不同pH下的荧光光谱图。
图2是实施例1制备的Rh-NH1在不同pH下的荧光分散点图。
图3是实施例1制备的Rh-NH1在不同pH下的吸收光谱图。
图4是实施例1制备的Rh-NH1在不同pH下的吸收分散点图。
图5是实施例1制备的Rh-NH1的可逆性测试。
图6是实施例1制备的Rh-NH1在不同pH下的照片；
其中，a是pH＝2.0的Rh-NH1乙醇水溶液与pH＝7.0的Rh-NH1乙醇水溶液在紫外光下的荧光颜色变化；b是pH＝2.0的Rh-NH1乙醇水溶液与pH＝7.0的Rh-NH1乙醇水溶液在日常光下的吸收颜色变化。
图7是实施例1制备的Rh-NH1对大型蚤的染色成像；
其中，a为黄绿光激发下的大型蚤染色成像；b为亮场下的大型蚤染色成像。
具体实施方法
实施例1 含脲结构的六元螺环罗丹明pH荧光指示剂——Rh-NH1
反应式如下：
将1摩尔量的罗丹明B与3摩尔量的POCl3加入到干燥的1,2-二氯乙烷中，90℃加热回流反应3-5小时，冷却至室温后，去除溶剂1,2-二氯乙烷，然后加入叠氮化钠的丙酮水溶液(将含有1.3摩尔量的叠氮化钠的水溶液0.5mL溶解于30mL丙酮中)，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥，减压蒸去溶剂后将反应物用干燥的乙腈溶解后移入反应釜中，加入过量氨水，100℃油浴反应10小时，所得反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥，经硅胶柱层析提纯，得目标产物Rh-NH1。HRMS：456.2525。
制备的荧光指示剂Rh-NH1的pKa分别为4.75(±0.09)(Abs)和5.27(±0.04)(Fl)，当pH大于6.5时Rh-NH1溶液无颜色和荧光；随着pH的降低，Rh-NH1溶液呈现粉红色，溶液荧光也由无色变为红色，并不断增强，最大吸收峰和最大荧光峰分别在566nm和586nm。另外，该指示剂还具有较好的pH可逆性。使用该试剂对大型蚤进行染色后，发现指示剂Rh-NH1能够定位于大型蚤的肠道内，并指示其显酸性。
实施例2 含脲结构的六元螺环罗丹明pH荧光指示剂——Rh-NH2
将1摩尔量罗丹明6G与3摩尔量的POCl3加入到干燥的1,2-二氯乙烷中，90℃加热回流反应3-5小时，冷却至室温后，去除溶剂1,2-二氯乙烷，然后加入叠氮化钠的丙酮水溶液(将含有1.3摩尔量的叠氮化钠的水溶液0.5mL溶解于30mL丙酮中)，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥，减压蒸去溶剂后将反应物用干燥的乙腈溶解后移入反应釜中，加入过量氨水，100℃油浴反应10小时，所得反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥。经硅胶柱层析提纯，得到目标产物Rh-NH2。HRMS：428.2212。
实施例3 含脲结构的六元螺环罗丹明pH荧光指示剂——Rh-NH3
将1摩尔量四甲基罗丹明TMR与3摩尔量的POCl3加入到干燥的1,2-二氯乙烷中，90℃加热回流反应3-5小时，冷却至室温后，去除溶剂1,2-二氯乙烷，然后加入叠氮化钠的丙酮水溶液(将含有1.3摩尔量的叠氮化钠的水溶液0.5mL溶解于30mL丙酮中)，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥，减压蒸去溶剂后将反应物用干燥的乙腈溶解后移入反应釜中，加入过量氨水，100℃油浴反应10小时。反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥。经硅胶柱层析提纯，得到目标产物Rh-NH3。HRMS：400.1899。
实施例4 含脲结构的六元螺环罗丹明pH荧光指示剂——Rh-NH4
将1摩尔量罗丹明110与3摩尔量的POCl3加入到干燥的1,2-二氯乙烷中，90℃加热回流反应3-5小时，冷却至室温后，去除溶剂1,2-二氯乙烷，然后加入叠氮化钠的丙酮水溶液(将含有1.3摩尔量的叠氮化钠的水溶液0.5mL溶解于30mL丙酮中)，室温下搅拌24小时，反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥，减压蒸去溶剂后将反应物用干燥的乙腈溶解后移入反应釜中，加入过量氨水，100℃油浴反应10小时。所得反应液用二氯甲烷萃取，取下层液，用无水硫酸镁干燥。经硅胶柱层析提纯，得到目标产物Rh-NH4。HRMS：344.1273。
实施例5 含脲结构的六元螺环罗丹明pH荧光指示剂——Rh-NH5
将1摩尔量的罗丹明101与3摩尔量的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，去除溶剂1,2-二氯乙烷，然后加入叠氮化钠的丙酮水溶液(将含有1.3摩尔量的叠氮化钠的水溶液0.5mL溶解于30mL丙酮中)，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，减压蒸去溶剂后将反应物用干燥的乙腈溶解后移入反应釜中，加入过量氨水，100℃油浴反应10小时。反应液用二氯甲烷萃取，取下层液，无水硫酸镁干燥。经硅胶柱层析提纯，得到目标产物Rh-NH5。HRMS：504.2525。
实施例6 应用试验
本应用试验采用实施例1制备的Rh-NH1荧光指示剂进行。
[1]荧光光谱的测定
取实施例1制备的含脲结构的六元螺环罗丹明pH荧光指示剂(Rh-NH1)溶于乙醇水溶液中(按体积比，乙醇:水＝5:5)，配制浓度为2×10-5mol/L的Rh-NH1指示剂水溶液。
取Rh-NH1指示剂水溶液分别用氢氧化钠和盐酸调节pH，分别调至pH范围在3.5-8.0的不同pH值的溶液，分别测试吸收光谱和荧光光谱。图1和图3是Rh-NH1在不同pH下的荧光光谱图和吸收光谱图。选取最大荧光和最大吸收强度对pH作散点图(图2、图4)，图2和图4中每个点分别代表不同pH值的最大荧光强度和最大吸收强度。结果表明，在pH 6.5以上时Rh-NH1没有颜色和荧光，随着pH值的降低，吸收和荧光强度不断增强，到pH＝3.5时分别在566nm和586nm处达到最大值。将散点图分别做拟合处理，得到Rh-NH1的pKa分别为4.75(±0.09)(Abs)和5.27(±0.04)(F1)。Rh-NH1荧光指示剂在pH处于2.0～6.5范围内较为敏感，其荧光强度增强约80倍。
[2]可逆性测试
Rh-NH1在pH在2.0和7.0荧光变化的可逆性。向pH＝7.0的2×10-5mol/L的Rh-NH1指示剂乙醇水溶液中加入HCl溶液将pH调到2.0，如图5所示，随着HCl的加入，溶液颜色迅速发生变化，变为明亮的粉色(0→1过程)，进行荧光光谱测定。再向溶液中加入NaOH溶液将溶液pH调到7.0，荧光强度恢复到原来的位置(1→2过程)，如此重复多次(2→3,3→4,4→5，5→6，6→7，7→8，8→9，9→10，10→11)，这种变化仍然存在。
图6中a为紫外光下的荧光颜色变化图，图6中b为可见光下的吸收颜色变化图。其中，pH＝2.0的Rh-NH1乙醇水溶液与pH＝7.0的Rh-NH1乙醇水溶液相比，无论是荧光颜色还是吸收颜色均有明显的增强。
上述现象表明，本发明的荧光指示剂pH响应迅速，荧光强度变化明显、稳定、可逆。
[3]大型蚤染色实验
取一定量的Rh-NH1溶于DMSO中，再加入到装有大型蚤的3mL水中，3分钟后，抓取大型蚤于载玻片。在显微镜下观察，用黄绿光激发，观察Rh-NH1在大型蚤体内的荧光染色情况，并与亮场下的对比。如图7所示，在对大型蚤进行染色后，在的亮场下可以看到大型蚤的基本结构成像(图7中b)，而在黄绿光激发下，在大型蚤的肠道内发出了明亮的荧光(图7中a)。这表明Rh-NH1能够定位于大型蚤的肠道内，并指示大型蚤的肠道为酸性。
Claims (2)
1.含脲结构的六元螺环罗丹明pH荧光指示剂在实时检测pH和在检测浮游动物消化道中pH的应用，其特征在于，所述含脲结构的六元螺环罗丹明pH荧光指示剂为Rh-NH，具有如（Ⅰ）所示的结构通式，
（I）
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至R4构成-(CH2)3-，R2至R3构成-(CH2)3-。
2.如权利要求1所述的应用，其特征在于，所述浮游动物为大型蚤。
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