CN107312524B - 一种用于检测碱性磷酸酶的荧光探针及其制备方法 - Google Patents
一种用于检测碱性磷酸酶的荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种用于检测碱性磷酸酶的荧光探针及其制备方法。所述探针是2‑磷酸基萘甲醛缩氨基萘，分子式为C21H14NO5P。制备方法：将邻羟基萘甲醛与三氯氧磷加入反应器中，用吡啶溶解，室温搅拌一段时间后将反应液倒入冰水中继续反应，得2‑磷酸基萘甲醛；然后将2‑磷酸基萘甲醛和1‑氨基萘在有机醇里加热回流，可得目标化合物。该探针在pH 7.0缓冲溶液中对碱性磷酸酶具有选择性荧光识别功能，发出橙色荧光，检出限为0.002U/mL。该探针化学性质稳定，合成简单，具有良好的生物相容性，能用于细胞内碱性磷酸酶的检测。
Description
技术领域
本发明涉及碱性磷酸酶的检测技术，具体为一种用于检测碱性磷酸酶的荧光探针及其制备方法。
背景技术
碱性磷酸酶(ALP)属于一种水解酶，具有促使核苷酸、蛋白质、糖类等不同底物去磷酸化功能。人血清里ALP的正常浓度在40-150mU/mL范围内。浓度出现异常时常常是一些疾病的先兆，比如肝炎、前列腺癌、骨质疏松和骨癌等。在医学诊断上通常被认为是一种重要的生物信号分子，某种程度上ALP的活性代表了细胞的活性。因此，实时监测ALP浓度对于区分正常细胞和异常细胞(如增生)具有重要意义。
已有一些文献利用荧光探针和量子点等手段来检测ALP的活性。其基本原理是利用ALP水解酶的去磷酸化活性，将对硝基苯磷酸基水解为对硝基酚，产生荧光。但这些探针的灵敏度低，合成过程繁杂，响应机理复杂。最近，具有聚集诱导发光特性(AIE)的荧光分子已发展成为一种新型的荧光材料，这类分子的荧光强度和灵敏度要远大于普通荧光分子和量子点，在发光材料和生物传感领域得到极大重视。自由AIE分子并不发光，在聚集态下会发出极强的荧光。AIE材料中，希夫碱类的AIE分子合成简单，发光范围宽广。最近有一些关于AIE分子在识别ALP方面的报道，但这些探针的发光都在绿色和蓝色荧光范围，在生物领域并不实用。
发明内容
本发明的目的在于提供一种检测碱性磷酸酶的荧光探针及其制备方法。该探针能对碱性磷酸酶选择性荧光识别，且灵敏度高，化学性质稳定，合成方法简单，具有良好的生物相容性，能用于检测细胞内碱性磷酸酶。
本发明提供的一种用于检测碱性磷酸酶的荧光探针，其特征在于，分子式为C21H14NO5P，结构式为：
本发明提供的一种用于检测碱性磷酸酶的荧光材料的制备方法，包括如下步骤：
1)按摩尔比1:1.5～2.5将邻羟基萘甲醛缓慢加入三氯氧磷的吡啶溶液中，室温搅拌4～6小时后，将反应液倒入冰水中继续搅拌12～16小时；反应结束后，减压蒸馏，洗涤，硅胶柱色谱分离，得化合物2-磷酸基萘甲醛；
2)按摩尔比为1:1将2-磷酸基萘甲醛和1-氨基萘在有机醇里加热回流至少0.5～1.0小时，冷却，过滤，洗涤，硅胶柱色谱分离，得目标化合物2-磷酸基萘甲醛缩氨基萘(PN)。
步骤1)中所述的室温搅拌时间为4小时。
步骤1)中所述的冰水浴搅拌时间为12小时。
步骤1)中所述的邻羟基萘甲醛和三氯氧磷的摩尔比为1:2。
步骤2)中所述的有机醇可以为1个碳至5个碳的有机醇，优选甲醇或乙醇。
本发明荧光探针对碱性磷酸酶的识别体系为pH 7.0的缓冲溶液(37℃)，包括磷酸缓冲溶液、HEPES缓冲溶液或Tris-HCl缓冲溶液，可在溶液和细胞内实现对碱性磷酸酶的荧光响应。
本发明荧光探针对碱性磷酸酶的检出限为0.002U/mL。
本发明合成的荧光探针PN在水溶液中并不发光，这是由于2位磷酸基的存在阻止了分子内电荷转移(ICT)效应；在ALP的作用下，磷酸基被水解为羟基，羟基ICT效应被激活，探针就会在水溶液中发生AIE聚集发光现象，发出极强橙色荧光，便于裸眼识别，从而提高对碱性磷酸酶的检测效果。
与传统生物试剂比较，该荧光探针响应时间短，灵敏度高，抗干扰能力强，化学性质稳定，能长时间保存，且具有生物相容性好、对细胞毒性小等优点。该探针可用于研究溶液中及细胞内碱性磷酸酶的含量及在细胞中的定位，对相关疾病如癌症、艾滋病等的诊断具有重要意义。
附图说明
图1荧光探针PN的合成路线。
图2荧光探针PN的电喷雾质谱图。
图3荧光探针PN对碱性磷酸酶选择性识别的荧光光谱，HEPES缓冲溶液(pH 7.0)，[PN]＝10μmol·L-1，[蛋白]＝(2.0U/mL)，λex＝480nm,t＝20min,37℃。
图4荧光探针PN与碱性磷酸酶作用的荧光光谱，[PN]＝10μmol·L-1，[ALP]＝(0,0.01,0.25,0.5,0.75,1.0,1.25,1.5,1.75,2.0U/mL)，HEPES缓冲溶液(pH 7.0)，λex＝480nm，t＝20min,37℃。
图5细胞SW480荧光成像：a为细胞空白实验，不加探针；b细胞加探针；c是a的明场,d是b的明场。
具体实施方式
实施例1
荧光探针PN的合成(合成路线见图1)：
将邻羟基萘甲醛(0.17g，1.0mmol)缓慢加入溶解有三氯氧磷(0.2mL,2.0mmol)的20mL吡啶溶液中，在圆底烧瓶中室温搅拌反应4小时；再将反应液倒入含有50mL冰水的圆底烧瓶中，继续搅拌12小时，过滤，硅胶柱色谱分离，得化合物2-磷酸基萘甲醛(0.25g)。
将1-氨基萘(0.14g，1.0mmol)和2-磷酸基萘甲醛(0.25g，1.0mmol)在20mL甲醇里加热回流0.5小时，冷却，过滤，洗涤，硅胶柱色谱分离，得目标化合物PN(0.37g)。
由电喷雾质谱表征可知化合物PN：实验值m/z＝375.3[M+H]+，理论值m/z＝375.3[M+H]+(附图2)。核磁1H-NMR(DMSO)，δ(ppm,300MHz，TMS)：δ7.86(s，1H,Phen-H),7.93(s，1H，Phen-H)，7.63(s，1H，Phen-H)，7.21(s，1H,Phen-H)，7.30(m，5H，Phen-H)，7.53(s，1H)，7.7(t，3H，Phen-H)，8.39(1H，-HC＝N)。元素分析(％C)：理论值67.20，H：3.76，N：3.73，实验值C：67.25，H：3.66，N：3.70。
实施例2
荧光探针PN在HEPES(pH 7.0)缓冲溶液中首先可以实现对溶液中碱性磷酸酶(ALP)的荧光响应，如附图3。将2.0U/mL的蛋白ALP,GDP，ACP，PDE，GAL，AChE，GoX和Trysin分别加入10μmol·L-1PN的HEPES(pH 7.0)缓冲溶液中。由附图3可知，ALP的加入使得溶液在523，550和600nm处的荧光大幅度增强(λex＝480nm)，溶液的颜色逐渐变为橙色。其他常见蛋白分子GDP，ACP，PDE，GAL，AChE，GoX和Trysin的加入基本不影响到荧光探针PN的荧光强度，表明探针PN在HEPES(pH 7.0)缓冲溶液中对ALP具有选择性识别功能，其他常见蛋白基本不会对ALP的识别产生干扰作用。附图4为不同浓度的ALP对探针PN的荧光滴定图。由图可见，随着ALP的加入，探针PN溶液的荧光逐渐增强。经DL＝3Sb/S公式计算，探针PN对ALP蛋白的检出限为0.002U/mL。
实施例3
探针PN可以实现对细胞内源ALP的传感。图5中a为空白实验，不加探针。图5中b为将10μmol·L-1探针PN与SW480cells在细胞培养液中37℃孵育0.5小时。在荧光显微镜下可以清楚地看到，细胞在加入探针PN后，逐渐发出橙色的荧光。图5中c和d分别为图5中a和b相应的明场图片。该实验表明探针PN对细胞内的内源ALP具有良好的荧光识别功能。
Claims (9)
1.一种用于检测碱性磷酸酶的荧光探针，其特征在于，分子式为C21H16NO4P，结构式为：
2.根据权利要求1所述的荧光探针的制备方法，其特征在于，包括如下步骤：
1)按摩尔比1:1.5～2.5将邻羟基萘甲醛缓慢加入三氯氧磷的吡啶溶液中，室温搅拌4～6小时后，将反应液倒入冰水中继续搅拌12～16小时；反应结束后，减压蒸馏，洗涤，硅胶柱色谱分离，得化合物2-磷酸基萘甲醛；
2)按摩尔比为1:1将2-磷酸基萘甲醛和1-氨基萘在有机醇里加热回流至少0.5小时，冷却，过滤，洗涤，硅胶柱色谱分离，得目标化合物2-磷酸基萘甲醛缩氨基萘。
3.根据权利要求2所述的荧光探针的制备方法，其特征在于，步骤2)中所述的加热回流时间至少1.0小时。
4.根据权利要求2所述的荧光探针的制备方法，其特征在于，步骤1)中所述的邻羟基萘甲醛与三氯氧磷的摩尔比为1:2。
5.根据权利要求2所述的荧光探针的制备方法，其特征在于，步骤1)中所述的室温搅拌时间为4小时。
6.根据权利要求2所述的荧光探针的制备方法，其特征在于，步骤1)中所述的冰水浴搅拌时间为12小时。
7.根据权利要求2所述的荧光探针的制备方法，其特征在于，步骤2)中所述的有机醇为甲醇或乙醇。
8.根据权利要求1所述的荧光探针在制备检测碱性磷酸酶试剂中的应用。
9.根据权利要求1所述的荧光探针在制备检测细胞内碱性磷酸酶试剂中的应用。
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