CN104710380B - 用于检测铅离子的囊泡探针、制备方法及应用 - Google Patents
用于检测铅离子的囊泡探针、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种用于检测铅离子的囊泡探针、制备方法及应用,本发明的囊泡探针在4‑羟乙基哌嗪乙磺酸缓冲液中呈蓝色且无荧光,与铅离子发生反应后变为红色并发出荧光(荧光发射波长约550nm),灵敏度高,易于操作,采用该探针的荧光检测方法在生物医药领域有极大的应用前景。该探针由重量比为7:3的10,12‑二炔二十五酸和式Ⅰ探针前体聚合而成,
Description
技术领域
本发明涉及一种用于检测铅离子的囊泡探针、制备方法及应用。
背景技术
铅离子快速检测技术的发展,对于促进人体健康以及环境保护具有重大意义。随着工业和其他行业的迅速发展,含铅物质作为电池,汽油,颜料和一系列工业以前的和现在的重要原料之一,铅离子的污染向水,大气,食品,医药,以及我们的日常生活环境迅猛扩散。
铅离子,最有毒的重金属阳离子之一,对人的身心健康有着极大的危害,它可经消化道,呼吸道进入人体内,对人体的多个器官和生理系统产生极大的危害。它对于人的神经系统危害尤其严重。铅离子随着血液流入脑组织,损伤小脑和大脑皮质细胞,干扰神经细胞的代谢活动,造成记忆丧失,贫血,以及脑水肿。同时也会造成肝脏以及肾脏系统的病变及萎缩,危及人体健康及生命。同时它作为有毒重金属离子对儿童影响尤其严重。儿童血铅过高还和小儿多动症、注意力不集中、学习困难、攻击性行为、以及成年后的犯罪行为有密切关系。儿童的体格生长也受到铅的影响。据国内外的研究报道,高血铅的儿童,其身材矮小的可能性增大。高血铅儿童的身材往往低于正常的儿童。此外,严重的儿童铅中毒还导致贫血等。世界卫生组织对饮用水中的铅离子的规定最大为10mg·L-1。
传统的检测铅的方法有原子吸收光谱法和电感耦合等离子体质谱法。但由于昂贵检测花费,苛刻的检测要求,以及漫长的检测耗时都限制了对铅离子的快速检测。因此,简单和快速的方法的铅离子检测方法是目前迫切需要的。同时应用荧光探针的检测方法具有良好的灵敏性,应当由此角度入手,研发更好的检测方法。
发明内容
本发明所要解决的技术问题是:克服现有技术存在的问题,提供用于检测铅离子的囊泡探针,同时提供其制备方法及应用。
本发明技术构思如下:1-氮杂-18-冠-6-醚与铅离子具有较强的配位作用,只要制备出含有1-氮杂-18-冠-6-醚头基的聚丁二炔囊泡与铅离子配位反应产生荧光,即可达到对铅离子的检测。
本发明解决其技术问题的技术方案如下:
本发明的用于检测铅离子探针前体,其具有如式Ⅰ所示的结构:
本发明上述探针前体的制备方法,其包括以下步骤:
第一步、将10,12-二炔二十五酸与氯化亚砜反应得到式Ⅱ化合物;
第二步、将式Ⅱ化合物与三乙二醇反应得到式Ⅰ所示探针前体。
本发明上述探针前体的制备方法,其进一步的技术方案是第一步的具体过程为:先将1份重量份的10,12-二炔二十五酸溶解于二氯甲烷、再加入1-10份的氯化亚砜得到反应液,在氮气保护下持续搅拌该反应液直至反应结束;然后,减压蒸馏去除溶剂,即得式Ⅱ化合物。
本发明上述探针前体的制备方法,其进一步的技术方案还可以是第二步的具体过程为:先将式Ⅱ化合物溶解于乙腈中得到式Ⅱ化合物溶液,再将式Ⅱ化合物溶液加到含有1-氮杂-18-冠-6-醚的乙腈溶液中,得到反应液,在氮气保护下持续搅拌该反应液直至反应结束;然后,减压蒸馏去除溶剂获得粗产物,并将粗产物以层析柱纯化,即得式Ⅰ所示探针前体。其中式Ⅱ化合物与1-氮杂-18-冠-6-醚的摩尔比优选为1:1。更进一步的技术方案是第二步中在层析柱纯化过程中采用体积比100:2的二氯甲烷:甲醇为洗脱液。
本发明的用于检测铅离子的囊泡探针,其由重量比为10:1-1:10的10,12-二炔二十五酸和式Ⅰ探针前体聚合而成。
本发明上述囊泡探针的制备方法,其包括以下步骤:
将10,12-二炔二十五酸和式Ⅰ探针前体溶解于二甲基亚砜得到DMSO溶液,将DMSO溶液加至温度为至少80℃的4-羟乙基哌嗪乙磺酸缓冲液中得到反应液,冷却反应液,以紫外光照射反应液进行聚合反应,即得囊泡探针。
本发明上述囊泡探针的制备方法,其进一步的技术方案是冷却反应液前将反应液超声、并用滤器过滤;所述的10,12-二炔二十五酸和式Ⅰ探针前体重量比为7:3。
本发明上述囊泡探针的制备方法,其进一步的技术方案还可以是以紫外光照射反应液进行聚合反应采用254nm紫外灯作为紫外光源。
本发明上述的囊泡探针在检测铅离子中的应用。
本发明具有以下有益效果:
本发明的囊泡探针在4-羟乙基哌嗪乙磺酸缓冲液中呈蓝色且无荧光,遇铅离子发生反应后变为红色并发出荧光(荧光发射波长550nm左右)。与现有检测方法相比,采用本发明囊泡探针的荧光检测方法反应速度快,灵敏度高,具有适宜的荧光发射波长(550nm左右),在生物医药领域有极大的应用前景。
附图说明
图1为本发明实施例1的探针前体1H NMR图谱
图2为本发明实施例1的探针前体13C NMR图谱
图3为本发明实施例1的探针前体HRMS(EI)图谱
图4为本发明实施例2的荧光发射光谱图。
图5为本发明实施例2的紫外可见光吸收光谱图。
图6为本发明实施例2的颜色变化图。
图7为本发明实施例2的荧光图。
图7中a、b、c、d、e、f、g、h、i、j、k、l为实例2探针分别在铅离子浓度为0μM,2μM,4μM,8μM,16μM,20μM,25μM,50μM,100μM,200μM,500μM,1000μM下的荧光图。
图8为本发明实施例2的荧光强度量化图。
具体实施方式
下面结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
以下内容中涉及的实验材料和试剂,如未特别说明则为市售品。
实施例1式Ⅰ探针前体、囊泡探针的制备
一、制备式Ⅰ探针前体
第一步、将0.375g(1mmol)10,12-二炔二十五酸溶解于20ml二氯甲烷中,然后向其中滴加2ml氯化亚砜得到反应液,在氮气保护下持续搅拌该反应液直至反应结束(通常为搅拌过夜);然后,减压蒸馏去除溶剂,即得呈无色油状物的式Ⅱ化合物。
第二步、先将式Ⅱ化合物溶解于乙腈中得到式Ⅱ化合物溶液,再将式Ⅱ化合物溶液加至含有0.264g1-氮杂-18-冠-6-醚的40ml乙腈溶液中、得到反应液,在氮气保护下持续搅拌该回流反应液直至反应结束;然后,减压蒸馏去除溶剂获得粗产物,并将粗产物以层析柱纯化(采用体积比100:2的二氯甲烷:甲醇为洗脱液),即得式Ⅰ所示探针前体。重量90.5mg,分子量为619。
式Ⅰ探针前体的1H NMR(CDC13,500MHz)δ(ppm):δ0.86-0.89(t,3H,J=13.6Hz),1.25(s,26H),1.47-1.52(t,4H,J=20.55Hz),1.60-1.63(t,2H,J=14.1Hz),2.22-2.24(t,2H,J=13.9Hz),2.31-2.34(t,4H,J=15.1),3.60-3.62(t,2H,J=9.05),3.66-3.67(t,6H,J=1.55Hz),3.69-3.73(t,2H,J=22.4),4.22-4.24(t,2H,J=9.55)。
式Ⅰ探针前体的13C NMR(CDC13,500MHz)δ(ppm):14.03,19.12,19.14,22.62,24.79,28.27,28.31,28.70,28.80,28.84,28.99,29.02,29.28,29.42,29.54,29.56,29.58,29.63,31.86,34.09,61.71,65.24,65.32,66.83,69.13,70.31,70.36,70.48,76.75,77.00,173.7。
式Ⅰ探针前体的HRMS(EI)m/z=642.4704[M+Na]+。
工艺路线如下:
二、制备囊泡探针
将10,12-二炔二十五酸和式Ⅰ化合物按重量比7:3溶解于1ml二甲基亚砜得到DMSO溶液,将DMSO溶液用注射器在5分钟内滴加至温度为80℃的4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(10mM pH=7.4)中得到反应液,此时10,12-二炔二十五酸的最终浓度为1mM;将反应液在80℃下超声25分钟,并用0.8μm滤器过滤,除去不溶性颗粒;将反应液置4℃下冷却12个小时;采用254nm紫外灯(1mW/cm2)照射反应液10分钟进行聚合反应,即得蓝色囊泡探针。如无特别说明,以下的实验均采用此囊泡探针。
实施例2囊泡探针与铅离子反应的光谱性质和颜色变化
一、囊泡探针与铅离子反应的光谱性质
分别取九份60μl 1mM的囊泡探针加至各石英皿中,然后分别加入4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(10mM pH=7.4)稀释并定容至3ml;然后,分别加入0μL,0.15μL,0.3μL,0.45μL,0.6μL,0.75μL,0.99μL,1.05μL,1.2μL,1.35μL,1.5μL,1.65μL,1.8μL,(10mM的铅离子)不同体积的铅离子(0μM,1μM,1.5μM,2μM,2.5μM,3μM,3.5μM,4μM,4.5μM,5μM,5.5μM,6μM),反应5分钟后分别测量荧光发射光谱和紫外可见光吸收光谱。荧光发射光谱测定时以492nm激发,激发与发射的狭缝宽度为5nm。
荧光发射光谱如图4所示,铅离子浓度在0-6μM范围内时,约550nm处的荧光强度随铅离子浓度的增加而增强。
紫外可见光吸收光谱如图5所示,铅离子浓度在0-6μM范围内时,随铅离子浓度的增加,640nm处的吸光度逐渐降低,545nm处的吸光度逐渐增加。
二、囊泡探针与铅离子反应的颜色变化
分别取十份100μl 1mM的囊泡探针放入96孔板中,加入4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(10mM pH=7.4)使各囊泡探针最终浓度为250μM;向各孔中分别加入0μl,0.4μl,0.8μl,1.2μl,1.6μl,2.0μl,2.4μl,2.8μl,3.2μl,3.6μl,的1mM铅离子,使其最终浓度为0μM,1μM,2μM,3μM,4μM,5μM,6μM,7μM,8μM,9μM;反应5分钟后观察颜色变化。
结果如图6所示,随铅离子浓度增加,各孔内液体逐渐由蓝色变为红色,其中,9μM的铅离子颜色变化最为明显,完全变为红色,肉眼可直接观察到。
三、囊泡探针与铅离子反应的荧光变化
分别取十二份0.2μl 1mM的囊泡探针滴入含有微孔的玻璃片上孔,加入0.2μl用4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(10mM pH=7.4)配置的不同浓度(0,4,8,16,32,40,50,100,200,400,1000,2000mM)的铅离子溶液。使各囊泡探针最终浓度为500μM,铅离子最终浓度分别为0μM,2μM,4μM,8μM,16μM,20μM,25μM,50μM,100μM,200μM,500μM,1000μM;待微量溶剂自然风干后在荧光显微镜下观察荧光变化。
结果如图7所示,随铅离子浓度增加,各孔内荧光强度逐渐增加,其中,1000μM的铅离子荧光变化最为明显,几乎囊泡完全发出红色荧光。
Claims (2)
1.一种用于检测铅离子的囊泡探针的制备方法,其特征在于包括以下步骤:
第一步、将10,12-二炔二十五酸与氯化亚砜反应得到式Ⅱ化合物;
第二步、将式Ⅱ化合物与1-氮杂-18-冠-6-醚反应得到式Ⅰ所示探针前体;
第三步:将重量比为10:1-1:10的10,12-二炔二十五酸和式Ⅰ探针前体聚合得到用于检测铅离子的囊泡探针;
其中:式Ⅰ所示的结构如下:
式Ⅱ化合物结构如下:
第一步的具体过程为:先将1份重量份的10,12-二炔二十五酸溶解于二氯甲烷、再加入1-10份的氯化亚砜得到反应液,在氮气保护下持续搅拌该反应液直至反应结束;然后,减压蒸馏去除溶剂,即得式Ⅱ化合物;
第二步的具体过程为:先将式Ⅱ化合物溶解于乙腈中得到式Ⅱ化合物溶液,再将式Ⅱ化合物溶液加到含有1-氮杂-18-冠-6-醚的乙腈溶液中,得到反应液,在氮气保护下持续搅拌该反应液直至反应结束;然后,减压蒸馏去除溶剂获得粗产物,并将粗产物以层析柱纯化,即得式Ⅰ所示探针前体;其中在层析柱纯化过程中采用体积比100:2的二氯甲烷:甲醇为洗脱液;所述的式Ⅱ化合物与1-氮杂-18-冠-6-醚的摩尔比为1:1;
第三步的具体过程为:将10,12-二炔二十五酸和式Ⅰ探针前体按重量比为7:3溶解于二甲基亚砜得到DMSO溶液,将DMSO溶液加至温度为至少80℃的4-羟乙基哌嗪乙磺酸缓冲液中得到反应液,冷却反应液,以紫外光照射反应液进行聚合反应,即得囊泡探针;其中所述的冷却反应液前将反应液超声、并用滤器过滤。
2.根据权利要求1所述的制备方法,其特征在于第三步中所述的以紫外光照射反应液进行聚合反应采用254nm紫外灯作为紫外光源。
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