CN108299402B - 一种多功能超灵敏Zn2+双光子检测荧光分子探针的制备方法及应用 - Google Patents
一种多功能超灵敏Zn2+双光子检测荧光分子探针的制备方法及应用 Download PDFInfo
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Abstract
本发明涉及一种多功能超灵敏Zn2+双光子检测荧光分子探针的制备方法及应用。首先,由2,4‑二羟基苯甲醛、戊烯二酸二乙酯和无水哌啶在乙醇中反应制得产物1,产物1与乙酸酐在无水吡啶中反应得产物2,产物2与四氧化锇、高碘酸钠在四氢呋喃反应得产物3,产物3与无水碳酸钾在甲醇中反应得产物4;最后,将产物4溶解于乙醇中,加入二肼吡啶、冰乙酸回流,重结晶等步骤,即得多功能超灵敏Zn2+双光子检测荧光分子探针。适用于生物样本中Zn2+的定性、定量分析,检测灵敏、准确、快捷;可应用于分析化学、生命有机分析化学、疾病预诊及医学临床检测等相关领域。
Description
技术领域
本发明属于分析化学领域,涉及一种多功能超灵敏Zn2+双光子检测荧光分子探针的制备方法及应用。
背景技术
锌是生物体必不可少的重要元素之一,在人体的含量仅次于铁,是第二多的过渡金属元素。锌离子广泛存在于人体的细胞内,并在细胞新陈代谢、基因表达、机体免疫和神经传输等众多生理活动中起重要作用。当前,锌离子检测方法主要以荧光分子探针检测法为主;所报导的方法虽然有各自的优点,但总的来说其检测响应倍率(10~60倍)还有待改善,定量方式以及探针结构带来的一些检测局限性。这会导致现有检测方法检测限度低、结果定量不准确、检测方式单一、不利于肉眼检测以及成像研究的局限等问题。这些问题对于细胞中痕量锌离子的检测来说是非常受限制的。
发明内容
本发明针对现有技术中存在的上述问题,提供一种多功能超灵敏锌离子双光子检测荧光分子探针的制备方法,制备得到的分子探针可应用与生物样本检测,具有超灵敏检测、响应倍率大、现象明显易观察和准确度高等优点,设备便捷操作,可实施性强,特别适合大批量样本组合筛选等大数据研究。
本发明还提供了一种上述制备的多功能超灵敏锌离子双光子检测荧光分子探针的应用。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种多功能超灵敏Zn2+双光子检测荧光分子探针的制备方法,其特征在于:首先,由2,4-二羟基苯甲醛、戊烯二酸二乙酯和无水哌啶在乙醇中反应制得产物1,产物1与乙酸酐在无水吡啶中反应得产物2,产物2与四氧化锇、高碘酸钠在四氢呋喃反应得产物3,产物3与无水碳酸钾在甲醇中反应得产物4;最后,将产物4溶解于乙醇中,加入二肼吡啶、冰乙酸回流,经重结晶,即得多功能超灵敏Zn2+双光子检测荧光分子探针。
上述制备方法的具体步骤为:
(1)将2,4-二羟基苯甲醛溶解于乙醇中,溶解浓度0.02~0.06g/mL,然后加入戊烯二酸二乙酯,溶解浓度0.067g/mL,混合均匀后滴加无水哌啶 2~5 mL,回流24h,冷却,析出黄色固体,固体用无水乙醇重结晶,得产物1;
(2)将产物1溶解于无水吡啶中,溶解浓度0.025g/mL,然后加入乙酸酐,加入浓度1mol/L,搅拌0.5h,加入40倍质量的碎冰,搅拌10min后析出灰白色固体,固体用体积比1:5的乙腈-二氯甲烷洗脱剂洗脱,旋蒸,得产物2;
(3)将产物2溶解于四氢呋喃中,溶解浓度0.011 g/mL,然后加入质量分数4%的四氧化锇水溶液,搅拌0.5h,然后加入高碘酸钠,加入浓度0.017g/mL,室温下搅拌5~6天,减压蒸馏除去四氢呋喃加入二氯甲烷100/mL,水洗,有机层干燥除水,得到白色固体,固体用体积比为从1:0~5的二氯甲烷-乙腈体系洗脱剂梯度洗脱,旋蒸,得产物3;
(4)将产物3溶解于甲醇中,溶解浓度0.012 g/mL,然后加入无水碳酸钾,溶解浓度0.015 g/mL,室温下搅拌0.5h,TLC分析原料消耗完毕后,加入1当量浓度的盐酸调节pH至3~4,析出黄色固体,过滤,滤饼用水洗涤,真空干燥,得产物4;
(5)将产物4溶解于乙醇中,溶解浓度0.002 g/mL,然后加入2肼吡啶,溶解浓度0.002 g/mL,混合均匀后加入2~3滴冰乙酸,回流4~5h,减压蒸馏浓缩溶剂,冷却析出深黄色固体,固体用重结晶的方法纯化后得到金黄色絮状晶体,即得多功能超灵敏Zn2+双光子检测荧光分子探针。
进一步的,所述多功能超灵敏Zn2+双光子检测荧光分子探针效果判断指标如下:
检测灵敏度:检测限8.2nmol/L;
检测响应倍率:检测时荧光增强,最高可达338.36倍;
检测速度:1秒完成检测;
颜色变化:日光灯下表现为由淡黄色变为黄绿色;紫外灯下表现由微弱荧光变为亮绿色;
光学机理指标:ICT 机理的锌离子荧光探针
本发明还提供了一种上述方法制备的多功能超灵敏Zn2+双光子检测荧光分子探针的应用,适用于生物样本中锌离子的定性、定量分析;其中生物样本包括血清、活细胞,可应用于分析化学、生命有机分析化学、疾病预诊及医学临床检测相关领域。
进一步的,所述多功能超灵敏Zn2+双光子检测荧光分子探针定量分析生物样本中锌离子时,适用于检测血清中锌离子含量;定性检测生物样本中锌离子时,适用于血清样品中锌离子的检测和活细胞内锌离子的检测。
本发明提供的荧光分子探针检测血清中锌离子含量的方法,包括以下步骤:
1)配制溶液
探针储备液:准确称取多功能超灵敏Zn2+双光子检测荧光分子探针溶于无水DMSO,配制为浓度100μM的探针储备液;
锌离子储备液:准确称取待测目标物0.0029g硫酸锌溶于10ml蒸馏水中,配制为浓度1000μM的锌离子储备液;
2)建立血清-锌离子标准品的线性方程
将步骤1)配制的锌离子储备液用蒸馏水稀释得到梯度浓度为0~40μM的锌离子标准溶液,然后分别取200 μL锌离子标准溶液与100μL步骤1)配制的探针储备液和650μL血清储备液混合后,加入50 μL 浓度为10 mM、 pH 7.42的Tris-盐酸缓冲液,充分振荡,使体系混合均匀,在25℃下放置50min,然后经荧光分光光度计检测,建立血清-锌离子浓度与荧光信号强度的线性方程;
3)荧光检测待测血清样品中锌离子的含量
将1000μL待测样品加入到石英比色皿后,在荧光检测仪内进行扫描检测,收集荧光发射位置的强度数据代入血清-锌离子浓度与荧光信号强度的线性方程,计算得待测血清样品中锌离子含量;
进一步的,本发明利用探针检测待测血清样品时,以荧光检测的方法对待测物进行多次平行检测,并用锌离子标准品溶液进行校准,得到荧光检测的最优检测范围,从而根据不同样品所含待测物的浓度范围来选择荧光检测手段进行定量。
进一步的,所述荧光检测范围为,0-40μM.
本发明利用探针进行血清样品中锌离子的检测时,方法为:待测血清样品与无水DMSO按体积比5:1混合后,5000 rpm离心20min,取出上清液过透析膜进行处理,然后取200μL透析后上清液依次加入100μL探针储备液和200 μL锌离子储备液,用pH 7.42的Tris-盐酸缓冲液定容至1000μL后,于25℃保存50min,根据颜色判断血清样品中是否含有锌离子,判断标准为:紫外灯下为黄绿色则血清样品中含有锌离子。
本发明利用探针进行活细胞内锌离子的检测时,方法为:将待测活细胞样品在培养基中培育18~26h,待测活细胞接种量为2×107~9×107个/mL,然后加入多功能超灵敏Zn2+双光子检测荧光分子探针,探针浓度为1 μM,于25 ℃培育10~12h,以pH 7.42的Tris-盐酸缓冲液洗多次,然后于荧光显微镜下观察细胞成像,根据发光情况判断待测活细胞中是否含有锌离子,判断标准为:紫外灯下为绿色则活细胞样品中含有锌离子;所述的活细胞优选为肝癌细胞,培养基优选为DMEM培养基。
本发明成功合成一个新型高准确度、超灵敏多功能荧光探针,并用于检测锌离子,并且系统地在试管中和细胞体内进行研究。探针设计思路为合成7-羟基香豆素(7-hydroxycoumarin)作为基础母体环,先后引入醛基并接二肼基吡啶(2-hydrazinylpyridine),构成多功能超灵敏双光子检测荧光探针分子HMCN((E)-7-hydroxy-3-((2-(pyridin-2-yl)hydrazono)methyl)-2H-chromen-2-one)。实施检测时,加入待测物锌离子后,探针分子HMCN的氮迅速与锌配位,释放超强荧光信号。该探针呈现出优秀的特异性、灵敏度、准确性,同时该探针具有双光子性质,可以进行高分辨荧光成像和双光子成像。此外,该探针以应用于检测锌离子在细胞环境中动态检测。这些特征都使所得分子探针成为探索生命体系锌离子代谢过程的重要工具。
本发明制备的多功能超灵敏Zn2+双光子检测荧光分子探针检测机理,如图1所示。
本发明技术方案有益效果为:1)灵敏度高检测速度快:探针对待测物响应的超灵敏,1秒左右响应倍率可达300倍;2)在不加任何其他附加材料的条件下,提高了检测灵敏性,并且避免了填加附加材料,减少附加材料的消耗以及减少了在检测中的误差来源;3)生物样品成像多种多样化:本发明在检测中成功检测了待测物在血清、活细胞中双光子成像,这种方法是在之前的方法中没有能够做到的。双光子成像的实现对于锌离子这一生物标志物的深入研究起到很大的推动作用。
附图说明
图 2 为本发明实施例1制备的多功能超灵敏Zn2+双光子检测荧光分子探针的H谱;
图 3为本发明实施例1制备的多功能超灵敏Zn2+双光子检测荧光分子探针的C谱;
图 4为荧光定量的线性方程;
图 5为细胞成像实验
图6为多功能超灵敏Zn2+双光子检测荧光分子探针(1μM)与锌离子(5μM)在pH 范围为2.47~12.1内的荧光响应;
图7为温度对多功能超灵敏Zn2+双光子检测荧光分子探针(1μM)与锌离子(5μM)反应的荧光信号影响;
图8为多功能超灵敏Zn2+双光子检测荧光分子探针(1μM)对待测目标物锌离子梯度浓度的荧光响应图(锌离子浓度:0~40μM)
图9为多功能超灵敏Zn2+双光子检测荧光分子探针对待测物锌离子及其他物质选择性对照实验(其他物质)。
具体实施方式
下面结合具体实施例进一步说明本发明技术方案,在围绕本发明所描述技术思想情况下,根据一般的技术知识和通常用的技术手段研究的多种方式替换或变更,都属于本发明的范围内。
本发明下述实施例中:
荧光检测是利用日立Hitachi F-7000荧光光谱仪来进行,激发波长为410nm,发射波长为510nm,激发和发射狭缝宽度均为 10.0 nm,电压400V,扫描速度2400纳米/分;
荧光成像观测是通过Olympus, FV1100(Japan) 荧光共聚焦显微镜来进行;
化合物的分离提纯是采用薄层色谱硅胶柱来实现。
实施例1:制备多功能超灵敏Zn2+双光子检测探针
(1)将2,4-二羟基苯甲醛溶解于乙醇中,溶解浓度0.02~0.06g/mL,然后加入戊烯二酸二乙酯,溶解浓度0.067g/mL,混合均匀后滴加无水哌啶 2~5 mL,回流24h,冷却,析出黄色固体,固体用无水乙醇重结晶,得产物1;
(2)将产物1溶解于无水吡啶中,溶解浓度0.025g/mL,然后加入乙酸酐,加入浓度1mol/L,搅拌0.5h,加入40倍质量的碎冰,搅拌10min后析出灰白色固体,固体用体积比1:5的乙腈-二氯甲烷洗脱剂洗脱,旋蒸,得产物2;
(3)将产物2溶解于四氢呋喃中,溶解浓度0.011 g/mL,然后加入质量分数4%的四氧化锇水溶液,搅拌0.5h,然后加入高碘酸钠,加入浓度0.017g/mL,室温下搅拌5~6天,减压蒸馏除去四氢呋喃加入二氯甲烷100/mL,水洗,有机层干燥除水,得到白色固体,固体用体积比为从1:0~5的二氯甲烷-乙腈体系洗脱剂梯度洗脱,旋蒸,得产物3;
(4)将产物3溶解于甲醇中,溶解浓度0.012 g/mL,然后加入无水碳酸钾,溶解浓度0.015 g/mL,室温下搅拌0.5h,TLC分析原料消耗完毕后,加入1当量浓度的盐酸调节pH至3~4,析出黄色固体,过滤,滤饼用水洗涤,真空干燥,得产物4;
(5)将产物4溶解于乙醇中,溶解浓度0.002 g/mL,然后加入2肼吡啶,溶解浓度0.002 g/mL,混合均匀后加入2~3滴冰乙酸,回流4~5h,减压蒸馏浓缩溶剂,冷却析出深黄色固体,固体用重结晶的方法纯化后得到金黄色絮状晶体,即得多功能超灵敏Zn2+双光子检测荧光分子探针。
所述的多功能超灵敏Zn2+双光子检测荧光分子探针,反应的中间产物以及目标产物合成方法的改进,有利于提高合成过程中间体及目标产物生成产率。
制备得到的多功能超灵敏Zn2+双光子检测荧光分子探针的C谱及H谱图见图2和图3,效果判断指标如下:
检测灵敏度:检测限8.2nmol/L;
检测响应倍率:检测时荧光增强,最高可达338.36倍;
检测速度:1秒完成检测;
颜色变化:日光灯下表现为由淡黄色变为黄绿色;紫外灯下表现由微弱荧光变为亮绿色;
光学机理指标:ICT 机理的锌离子荧光探针
实施例1制备的探针与锌离子反应可行性验证:取0.1克多功能超灵敏Zn2+双光子检测荧光分子探针溶于80mLDMSO中,向其中加入2倍当量锌离子并在室温下搅拌10min,得产物。
对比例1
步骤(1)-(4)同实施例1;
步骤(5):将产物4溶解于乙醇中,溶解浓度0.002 g/mL,然后加入2,4-二硝基苯肼,溶解浓度0.002 g/mL,混合均匀后加入2~3滴冰乙酸,回流4~5h,减压蒸馏浓缩溶剂,冷却析出深黄色固体,固体用重结晶的方法纯化后得到产物。
该产物仅能检测次氯酸根,并不能与锌离子作用。
效果实施例(一)
实施例1制备的多功能超灵敏Zn2+双光子检测荧光分子探针定量分析生物样本中锌离子:检测血清中锌离子含量
1)配制溶液
探针储备液:准确称取多功能超灵敏Zn2+双光子检测荧光分子探针溶于无水DMSO,配制为浓度100μM的探针储备液;
锌离子储备液:准确称取待测目标物0.0029g硫酸锌溶于10ml蒸馏水中,配制为浓度1000μM的锌离子储备液;
2)建立血清-锌离子标准品的线性方程
将步骤1)配制的锌离子储备液用蒸馏水稀释得到梯度浓度为0~40μM的锌离子标准溶液,然后分别取200 μL锌离子标准溶液与100μL步骤1)配制的探针储备液和650μL血清储备液混合后,加入50 μL 浓度为10 mM、 pH 7.42的Tris-盐酸缓冲液,充分振荡,使体系混合均匀,在25℃下放置50min,然后经荧光分光光度计检测,建立血清-锌离子浓度与荧光信号强度的线性方程,线性方程分为检测锌离子浓度范围0~5μM (图4-A)、5~40μM (图4-B)以及0~40μM的叠加(图4-C)。
3)荧光检测待测血清样品中锌离子的含量
将1000μL待测样品加入到石英比色皿后,在荧光检测仪内进行扫描检测,收集荧光发射位置的强度数据代入血清-锌离子浓度与荧光信号强度的线性方程,计算得待测血清样品中锌离子含量;
将检测待测血清样品,以荧光检测的方法对待测物进行10次平行检测,并用锌离子标准品溶液进行校准,得到荧光检测的最优检测范围,从而根据不同样品所含待测物的浓度范围来选择荧光检测手段进行定量。
效果实施例(二)
定性检测生物样本中锌离子
活细胞内锌离子的检测,方法为:将肝癌细胞置入DMEM培养基中培育24h,等到肝癌细胞在培养基中接种量为2×107~9×107个/mL,然后加入1µM的多功能超灵敏Zn2+双光子检测荧光分子探针于25℃共培育12h,用缓冲液(Tris-盐酸)pH:7.42洗涤三次,置入荧光共聚焦显微镜下观测成像,探针的荧光颜色为:绿色,可由此判断血清样品中含有锌离子。
图5a-图5e分别是增加锌离子的浓度,该图中的A、B分别是500nm-560nm收集光区图和明场图。
对比试验分析:在410nm激发下,肝癌细胞分别与空白、探针 (1μM)和锌离子(5μM)共培育后,进行检测,并未检测出荧光发射, 这表明探针对于细胞中的各种物质对荧光没有造成干扰。而后,加入10倍量的待测物锌离子,在紫外灯下检测,检测出明显的发射效果,绿色荧光的成像得以顺利观察到。这说明探针可以应用于细胞成像方面。
本发明多功能超灵敏Zn2+双光子检测荧光分子探针各项技术指标的实验验证,具体如下:
本发明技术方案实验条件优化试验
1、反应体系pH值的优化
一般而言,pH 值影响有机分子探针的荧光性质,因而在反应中一般利用缓冲溶液加以调节pH从而适应实验的要求。针对本发明要探测的锌离子本身特点,研究了在生理环境下可能达到的pH值(2.47~12.10)。从图6中可见,由于锌离子与酸碱的作用,在生理环境范围内pH值的波动对于目标锌离子与探针的混合溶液所表现的荧光强度响应产生了一定的影响。因此,在一般的生物环境体系中,当 pH值为7.42时,探针与锌离子的反应达到最优化。
2、反应温度的优化
在化学反应中,温度的影响是非常重要,对于本发明所研究的生物样本如活细胞、组织体系更是如此。不同温度下探针对待测目标物有较好的反应是整个实验成败的关键。如图7所示,对温度在20~45 ℃范围的反应具有的荧光响应进行了研究。由实验很容易发现,本发明所研究的探针在所取的温度范围内与待测物反应都具有比较好的荧光反应,并进一步证实该体系能够更好地应用于生物样本的检测。
3、光学性质与机理验证
本发明旨在制备一种具备新颖发光机理的多功能分子探针。探针对待测目标物锌离子梯度浓度0~40μM的荧光响应如图8所示。
4、探针分子检测锌离子的选择性分析
下述物质储备液配制方法:分别用蒸馏水溶解Br-、Cl-、F-、SO4 2-、SO3 2-、HCO3 -、NO2 -、NO3 -、SCN-、ClO3 -、CO3 2-、Zn2+、Na+、Cu2+、PO4 3-、H2PO4 -、HPO4 2-、Ca2+、Mg2+、Ag+、Cd2+、Zn+、Al3+、Fe2 +、Fe3+,得到上述各种离子的储备液(上述各物质溶液依次对应图9中标号1-25),结果如图9所示。
首先,相比较待测物锌离子而言,探针对其他各种离子未表现出响应,这是由于锌离子具有不同于其他离子的结构所导致的。其次,通过pH滴定实验发现,在pH值为2.47~12.10的区间范围,荧光的强度在6.41与7.42时达到最强,这表明该探针在生物环境即pH为7.42时是完全适用的。同时,温度实验证实了本发明的探针非常适用于生物样品。
Claims (3)
2.根据权利要求1所述的制备方法,其特征在于,具体步骤为:
(1)将2,4-二羟基苯甲醛溶解于乙醇中,溶解浓度0.02~0.06g/mL,然后加入戊烯二酸二乙酯,溶解浓度0.067g/mL,混合均匀后滴加无水哌啶 2~5 mL,回流24h,冷却,析出黄色固体,固体用无水乙醇重结晶,得产物1;
(2)将产物1溶解于无水吡啶中,溶解浓度0.025g/mL,然后加入乙酸酐,加入浓度1mol/L,搅拌0.5h,加入40倍质量的碎冰,搅拌10min后析出灰白色固体,固体用体积比1:5的乙腈-二氯甲烷洗脱剂洗脱,旋蒸,得产物2;
(3)将产物2溶解于四氢呋喃中,溶解浓度0.011 g/mL,然后加入质量分数4%的四氧化锇水溶液,搅拌0.5h,然后加入高碘酸钠,加入浓度0.017g/mL,室温下搅拌5~6天,减压蒸馏除去四氢呋喃加入二氯甲烷100mL,水洗,有机层干燥除水,得到白色固体,固体用体积比为1:0~5的二氯甲烷-乙腈体系洗脱剂梯度洗脱,旋蒸,得产物3;
(4)将产物3溶解于甲醇中,溶解浓度0.012 g/mL,然后加入无水碳酸钾,溶解浓度0.015 g/mL,室温下搅拌0.5h,TLC分析原料消耗完毕后,加入1当量浓度的盐酸调节pH至3~4,析出黄色固体,过滤,滤饼用水洗涤,真空干燥,得产物4;
(5)将产物4溶解于乙醇中,溶解浓度0.002 g/mL,然后加入2-肼吡啶,溶解浓度0.002g/mL,混合均匀后加入2~3滴冰乙酸,回流4~5h,减压蒸馏浓缩溶剂,冷却析出深黄色固体,固体用重结晶的方法纯化后得到金黄色絮状晶体,即得多功能超灵敏Zn2+双光子检测荧光分子探针。
3.根据权利要求1或2所述的制备方法,其特征在于,所述多功能超灵敏Zn2+双光子检测荧光分子探针效果判断指标如下:
检测灵敏度:检测限8.2nmol/L;
检测响应倍率:检测时荧光增强,最高可达338.36倍;
检测速度:1秒完成检测;
颜色变化:日光灯下表现为由淡黄色变为黄绿色;紫外灯下表现由微弱荧光变为亮绿色;
光学机理指标:ICT 机理的锌离子荧光探针。
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