CN101368945A - 检测细胞内汞离子的荧光探针及合成方法和用途 - Google Patents
检测细胞内汞离子的荧光探针及合成方法和用途 Download PDFInfo
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- CN101368945A CN101368945A CNA2007101203094A CN200710120309A CN101368945A CN 101368945 A CN101368945 A CN 101368945A CN A2007101203094 A CNA2007101203094 A CN A2007101203094A CN 200710120309 A CN200710120309 A CN 200710120309A CN 101368945 A CN101368945 A CN 101368945A
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- rhodamine
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Abstract
本发明涉及检测细胞内汞离子的荧光探针及其合成方法和用途。该荧光探针对汞离子具有良好的选择性,对细胞渗透性好,本身没有毒副作用,适用于细胞内汞离子检测。将3位带有羧基的1摩尔罗丹明溶于干燥有机溶剂中,搅拌下缓慢滴加1~2摩尔POCl3,加热回流,除去有机溶剂,得到的罗丹明酰氯粗品;将1摩尔2-巯基乙胺盐酸盐或者胱胺二盐酸盐溶于干燥有机溶剂中,加入4~8摩尔缚酸剂,在冰浴条件下缓慢滴加上述所得罗丹明酰氯,除去有机溶剂,得到X为O的式(I)或式(II)的目标产物;将等摩尔上述目标产物和劳森试剂溶于干燥有机溶剂中,惰性气体保护下加热回流,柱层析分离得到X为S的式(I)或式(II)的目标产物。
Description
技术领域
本发明涉及高选择性检测汞离子、尤其涉及检测细胞内汞离子的荧光探针及其合成方法和用途。
背景技术
众所周知,汞是一种危害人体健康的重金属,由于其具有持久性、易迁移性和高度的生物富集性,使其成为目前全球最引人关注的环境污染物之一。汞污染可以通过许多天然的途径和人类的社会活动而产生,由此引起许多人类的健康和环境问题。汞离子会沉积在脑、肝和其他器官中,产生慢性中毒,损害肾、脑胃和肠道,甚至引起死亡。另外,汞元素及其化合物能够通过皮肤被吸收,引起口腔炎、齿龈炎和神经紊乱等疾病。汞离子对人体含有S原子的配合基显现出了很强的亲和力,能引起蛋白质、酶和膜的巯基(-SH)块结。汞能够在海洋生物体内累积,水中的微生物可以通过食物链的循环将无机的汞离子转变为甲基汞,这种甲基汞很容易进入人体,会对人体的中枢神经系统造成极大的损伤。汞中毒会对整个社会产生极其恶劣的影响,现在汞被优先列在全球环境监控系统(GEMS)清单上,全世界都花费大量的人力、物力和财力在开发和研究新型的检测汞离子方法。
2003年2月3日,联合国环境规划署(UNEP)在内罗毕发表了“全球汞状况评估”的报告。该报告指出,自工业革命以来,汞在全球大气、水和土壤中的含量已增加了3倍左右,工业区附近汞的含量更高。因此,迫切需要发展能高度选择性检测汞离子的化学传感器。
光化学传感器是近年来迅速发展起来的一个新的科学问题,它的出现显然和超分子科学的进展诸如分子组装、主客体化学、非共价相互作用、氢键作用、疏水作用等以及光诱导电子转移(PET)过程、分子内共扼的电荷转移化合物结构及其发光特性等研究密切相关:它的发展也和许多科学技术领域诸如生物化学、临床医学、药物化学以及环境科学中提出的大量实际问题密切相关。由于上述种种原因,有力的推动了光化学传感器的研究进展。光化学传感器按其信号检测方法的不同,主要分为荧光化学传感器(FluorescentChemosensor)和比色化学传感器(Colorimetric Chemosensor)。荧光化学传感器主要是依靠荧光信号为检测手段,通常有荧光的增强、碎灭或者发光波长的移动,而比色化学传感器主要是借助于色调的变化,通过肉眼观察就可以检测,方便实际应用。用于对外来物种进行检测的荧光化学传感器的设计和研究,是近年来受到广泛关注的一个科学问题。荧光传感器的检测过程主要是通过器件接受体(Receptor)部分对外来物种(包括阳离子,阴离子及中性分子等)的选择性接纳,然后经不同的作用机制,如光诱导电子转移(PET)或能量转移、金属—配体电荷转移(MLCT)、分子内电荷转移(ICT),再通过器件的信号报告部分给出有关器件在接纳物种过程中的信息变化。荧光化学传感器由于其有选择性好、灵敏度高、响应时间快等优点,目前在汞离子的痕量检测中受到了人们广泛的关注。对于大多数汞离子的荧光化学传感器的设计,主要包括两个部分:一个是用来输出荧光信号的发光基团,另一个是接受体,通常含有氮或硫等配位原子,可以和汞离子发生较强烈的作用,以达到接受汞离子的目的(Angew.Chem.,Int.Ed.2004,43,4300-4302.;J.Am.Chem.Soc.2003,125,3418-3419.)。
罗丹明类化合物因其较大的摩尔消光系数和高的荧光量子产率,已作为荧光标记物在生物技术方面有广泛应用,但把它作为直接用来识别特定客体的化学传感分子,还不是很多。近期报道了基于罗丹明B螺环内酰胺结构,对Cu2+的Pb2+有选择性识别的化学传感分子。这类传感分子的设计原理是,金属离子通过与受体的配位作用,诱导了螺环内酰胺开环结构的形成。根据这个过程实现对离子的识别,有这样的优点:传感分子对某种金属离子有识别作用时,不仅使吸收和荧光的强度有较大的增强,而且会使溶液出现由无色到有色的变化,这一过程可方便的实现肉眼检测。
目前用来测定汞离子的化学传感器,大多数在实际应用中都存在一些问题,比如各种竞争的金属离子对测定的干扰,主体化合物在水中的溶解性较差,以及响应时间较长等等,特别是用于生物体系的检测中,生物本体自荧光干扰严重,对pH变化比较敏感,使其受到了一定的限制。
发明内容
本发明的目的之一在于克服现有技术荧光探针的性能和结构上的不足之处,提供一种性能优良、适用于汞离子检测的以罗丹明为母体的检测细胞内汞离子的荧光探针。
本发明的目的之二是提供检测细胞内汞离子的荧光探针的合成方法。
本发明的目的之三是提供检测细胞内汞离子的荧光探针的用途。
本发明的检测细胞内汞离子的荧光探针具有如式(I)或式(II)所示的结构:
其中式(I)或式(II)中的R1、R2、R3、R4可以分别或者同时选自氢、1~5个碳原子烷基或3~8个碳原子环烷基中的一种;R5、R6、R7、R8可以分别或者同时选自氢或1~5个碳原子烷基中的一种;X为O或S。
R1和R7可以共同拥有2至5个碳原子的烷基链,其将R7的碳链连接在R1所连接的氮上;R2和R5可以共同拥有2至5个碳原子的烷基链,其将R5的碳链连接在R2所连接的氮上;R3和R8可以共同拥有2至5个碳原子的烷基链,其将R8的碳链连接在R3所连接的氮上;R4和R6可以共同拥有2至5个碳原子的烷基链,其将R6的碳链连接在R4所连接的氮上;可得到如下结构:
上述结构式中的X为O或S。
本发明的检测细胞内汞离子的荧光探针的合成方法包括以下步骤:
(1)将3位带有羧基的1摩尔罗丹明溶于干燥有机溶剂中,搅拌下缓慢滴加1~2摩尔POCl3,加热回流反应,反应完成后,冷却至室温,减压除去有机溶剂,得到的罗丹明酰氯粗品直接用作下步反应;
(2)将1摩尔2-巯基乙胺盐酸盐或者胱胺二盐酸盐溶于干燥有机溶剂中,加入4~8摩尔缚酸剂,在冰浴条件下,缓慢滴加步骤(1)所得罗丹明酰氯,室温反应,减压除去有机溶剂,加水析出沉淀,过滤后真空干燥得到X为O的式(I)或式(II)的目标产物;
(3)将等摩尔步骤(2)所得产物和劳森(Lawesson’s)试剂溶于干燥有机溶剂中,惰性气体(如氮气、氩气等)保护下加热回流,减压除去有机溶剂,柱层析分离得到X为S的式(I)或式(II)的目标产物。
所述的步骤(1)的加热回流反应时间是2~8小时。
所述的步骤(2)的室温是0℃~30℃反应,所述的反应时间是8~12小时。
所述的步骤(3)加热回流时间是4~8小时。
所述的干燥有机溶剂是1,2-二氯乙烷、乙腈、丙腈、苯或甲苯。
所述的缚酸剂是吡啶或三乙胺。
本发明的检测细胞内汞离子的荧光探针(近红外荧光探针)可用于化学模拟生物体系中汞离子的检测,生物活细胞和活组织内的汞离子的分析检测和荧光成像检测,以及临床医学上病变组织中汞离子的检测。本发明的荧光探针在使用时没有特殊的限制,可以将探针分子溶解在生理盐水、缓冲液或乙醇、乙腈或二甲亚砜等水溶性有机溶剂中,然后加入含有细胞组织的适当缓冲液中进行测试。
细胞内汞离子检测的一般方法是将培养好的细胞放于含有探针分子的缓冲溶液中孵化,将一部分细胞用激光共聚焦显微镜成像得到空白图像,然后将剩下的一部分细胞浸入含有汞离子的载体中孵育,分别用培养液冲洗后,进行激光共聚焦显微成像。
本发明提供了一种以罗丹明为母体、用于检测细胞内汞离子的荧光探针。该荧光探针对汞离子有很好的选择性,锂、钠、钾、钙、镁、铁、铜、铅、锌、镉等金属离子对检测没有影响,溶液的荧光强度与Hg2+的浓度在1×10-7M到1×10-5M的范围内有一定的线性关系,表现出了它良好的实际应用性;该荧光探针分子细胞渗透性好,响应时间极短,测定灵敏度极其高,对细胞本身没有毒副作用,适于细胞内汞离子浓度变化的检测;另外该系列探针分子结构简单,合成方法简单且效率高,使得极易推广实际应用。
附图说明
图1.本发明实施例3中荧光探针(II)—2对汞离子的选择性;横坐标为波长(nm),纵坐标为荧光强度。
图2.本发明实施例3中荧光探针(II)—2在其他离子共存时的荧光强度;横坐标为共存离子,纵坐标为在其他离子存在下加入汞离子后的荧光强度。
图3.本发明实施例3中荧光探针(II)—2的荧光强度和汞离子浓度的线性关系;横坐标为汞离子浓度,纵坐标为荧光强度。
图4.本发明实施例3中荧光探针(II)—2在人肾近曲小管上皮细胞(HK-2细胞)中对汞离子的共聚焦显微镜荧光成像。
具体实施方式
实施例1
(1)罗丹明B酰氯的合成
将1g罗丹明B溶于15mL干燥的1,2-二氯乙烷中,搅拌下缓慢滴加0.5gPOCl3,加热回流反应4小时,冷却至室温,减压除去溶剂,得到的罗丹明B酰氯粗品直接用作下步反应;
(2)化合物(I)—1的合成
将0.5g2-巯基乙胺盐酸盐溶于15mL干燥的乙腈中,加入1.2g三乙胺,在冰浴条件下,缓慢滴加30mL罗丹明B酰氯的乙腈溶液,缓慢升至室温反应12小时,减压除去溶剂,加水析出沉淀,过滤后真空干燥得产品0.58g,产率50.4%。
1HNMR(CDCl3,400MHz)δ(ppm):1.12(12H,t,J=7.0Hz),1.59(1H,s),2.25(2H,m),3.21(2H,m),3.28(8H,q,J=7.0),6.17(2H,m),6.32(2H,s),6.35(2H,m),7.09(1H,m),7.43(2H,m),7.88(1H,m)。
13CNMR(CDCl3,400MHz)δ(ppm):12.7,22.9,44.4,64.8,97.8,105.5,108.2,122.9,123.8,128.1,128.9,131.0,132.6,148.9,153.3,153.6,168.2。
TOF-HRMS found[M+H]+:m/z,501.2445,Calcd.501.2450。
实施例2
(1)罗丹明B酰氯的合成
将1g罗丹明B溶于15mL干燥的1,2-二氯乙烷中,搅拌下缓慢滴加0.5mLPOCl3,加热回流反应4小时,冷却至室温,减压除去溶剂,得到的罗丹明B酰氯粗品直接用作下步反应;
(2)化合物(II)—1的合成
将0.5g胱胺二盐酸盐溶于15mL干燥的乙腈中,加入1.2mL三乙胺,在冰浴条件下,缓慢滴加30mL罗丹明B酰氯的乙腈溶液,缓慢升至室温反应12小时,减压除去溶剂,加水析出沉淀,过滤后真空干燥得产品0.64g,产率55.6%。
1HNMR(CDCl3,400Hz)δ(ppm):1.17(24H,t,J=7.0Hz),2.27(4H,q),3.29(4H,m),3.35(16H,q,J=7.0),6.28(4H,d),6.38(4H,s),6.45(4H,d),7.07(2H,m),7.43(4H,m),7.90(2H,m)。
13CNMR(CDCl3,400MHz)δ(ppm):12.7,35.9,40.0,44.4,65.0,97.9,105.5,108.0,122.7,124.0,128.1,128.8,131.8,132.4,149.0,153.7,167.5。
TOF-HRMS found[M+H]+:m/z,1000.4746,Calcd.1000.4743。
实施例3
(1)罗丹明B酰氯的合成
将1g罗丹明B溶于15mL干燥的1,2-二氯乙烷中,搅拌下缓慢滴加0.5mLPOCl3,加热回流反应4小时,冷却至室温,减压除去溶剂,得到的罗丹明B酰氯粗品直接用作下步反应;
(2)化合物(II)—1的合成
将0.5g胱胺二盐酸盐溶于15mL干燥的乙腈中,加入1.2mL三乙胺,在冰浴条件下,缓慢滴加30mL罗丹明B酰氯的乙腈溶液,缓慢升至室温反应12小时,减压除去溶剂,加水析出沉淀,过滤后真空干燥得产品0.64g,产率55.6%;
(3)化合物(II)—2的合成
将1.3g化合物(II)—1和0.52g劳森(Lawesson’s)试剂溶于15mL干燥的甲苯中,氮气气体保护下加热回流4小时,减压除去有机溶剂,柱层析分离得到产品0.28g,产率20.8%。
1HNMR(CDCl3,400Hz)δ(ppm):1.12(24H,t,J=6.5),2.39(4H,m),3.28(16H,m),3.54(4H,m),6.26(12H,m),7.09(2H,m),7.49(4H,m),8.12(2H,m)。
13CNMR(CDCl3,400MHz)δ(ppm):12.7,29.8,35.0,44.5,73.6,98.1,103.4,108.2,123.5,124.9,128.7,132.4,138.6,149.3,150.1,153.5,190.0。
TOF-HRMS found[M+H]+:m/z,1033.462,Calcd.1033.44。
请参照附图1~4。探针对汞离子选择性
使用实施例3中的化合物(II)—2评价对汞离子的选择性。图1是在化合物(II)—2(5×10-6M)的乙醇/水(体积比为80/20)溶液中(pH=7),加入相当于10倍浓度的各种金属离子时溶液荧光强度的变化。探针激发波长为510nm,发射波长为580nm。当10倍浓度的Hg2+加入到5μM的化合物(II)—2的溶液中时,溶液的荧光增强近十倍。10倍浓度的其他金属离子的加入,如锂、钠、钾、钙、镁、铁、铜、铅、锌、镉等,对溶液荧光强度的影响很小。图2是当只有Hg2+存在和Hg2+与相当于其10倍浓度的其他金属离子共存时,化合物在λmax=580nm处的荧光强度变化情况。结果表明,其他常见金属离子的存在对Hg2+的响应没有影响,该探针对Hg2+有良好的选择性识别和实际应用性。
探针的荧光强度和汞离子浓度的线性关系
使用实施例3中的化合物(II)—2评价探针的荧光强度和汞离子浓度的线性关系。图3是在λmax=580nm处的荧光强度随Hg2+浓度的变化曲线,结果表明溶液的荧光强度与Hg2+的浓度在1×10-7M到1×10-5M的范围内有一定的线性关系(R2=0.99873)。Hg2+的10-7M的数量级浓度是对人体安全的浓度限值,并且在包含这个数值的两个数量级范围内探针分子可与Hg2+保持线性相关性,表现出了它良好的实际应用性。
细胞培养
人肾近曲小管上皮细胞(HK-2细胞)细胞以DMEM/F12为培养基,在25cm2的细胞瓶中培养,置于37℃湿度可控的孵育箱中,每两天换一次新鲜的培养基;将细胞培养在六孔板中,控制每孔中的细胞密度在104;孵育24小时后细胞绝大多数仍呈贴壁状态,实验时更换一次新鲜培养基。加入20μM探针的缓冲液中,37℃中孵育30分钟,吸出培养液并用PBS缓冲溶液(NaH2PO4-Na2HPO4)冲洗三次,然后将一部分细胞用激光共聚焦显微镜成像得到空白图像,然后将剩下的一部分细胞20μM的Hg2+,孵育30分钟,吸出培养液并用PBS缓冲溶液冲洗三次,进行激光共聚焦显微成像。
激光共聚焦显微成像
利用激光共聚焦显微镜成像技术,研究人肾近曲小管上皮细胞(HK-2细胞)内探针对汞离子的识别作用。图4(a)是探针(II)—2孵育过的HK-2细胞,几乎看不到荧光;图4(b)是加入汞离子后可以观察到细胞质内有很强的荧光;图4(c)为亮场图像,表明细胞在整个实验过程中仍保持良好的细胞活力。
DMEM/F12细胞培养基是直接购买的生物试剂。
DMEM/F12细胞培养基
成分(mg/L) | MD206 | MD207 | 成分(mg/L) | MD206 | MD207 |
无水氯化钙 | 116.6 | 116.6 | L-色氨酸 | 9.02 | 9.02 |
五水硫酸铜 | 0.0013 | 0.0013 | L-酪氨酸 | 38.4 | 38.4 |
九水硝酸铁 | 0.05 | 0.05 | L-缬氨酸 | 52.85 | 52.85 |
七水硫酸亚铁 | 0.417 | 0.417 | D-葡萄糖 | 3151 | 3151 |
氯化钾 | 311.8 | 311.8 | HEPES | 3574.5 | — |
氯化镁 | 28.64 | 28.64 | 次黄嘌呤 | 2 | 2 |
无水硫酸镁 | 48.84 | 48.84 | 亚油酸 | 0.042 | 0.042 |
氯化钠 | 6999.5 | 6999.5 | 硫辛酸 | 0.105 | 0.105 |
无水磷酸二氢钠 | 54.35 | 54.35 | 酚红 | 8.1 | 8.1 |
磷酸氢二钠 | 71.02 | 71.02 | 1,4-丁二胺二盐酸盐 | 0.081 | 0.081 |
七水硫酸锌 | 0.432 | 0.432 | 丙酮酸钠 | 55 | 55 |
L-精氨酸盐酸盐 | 147.5 | 147.5 | 维生素H | 0.0035 | 0.0035 |
L-胱氨酸盐酸盐 | 31.29 | 31.29 | D-泛酸钙 | 2.24 | 2.24 |
L-谷氨酰胺 | 365 | 365 | 氯化胆碱 | 8.98 | 8.98 |
甘氨酸 | 18.75 | 18.75 | 叶酸 | 2.65 | 2.65 |
L-组氨酸盐酸盐 | 31.48 | 31.48 | i-肌醇 | 12.6 | 12.6 |
L-异亮氨酸 | 54.47 | 54.47 | 烟酰胺 | 2.02 | 2.02 |
L-亮氨酸 | 59.05 | 59.05 | 盐酸吡哆醛 | 2 | 2 |
L-赖氨酸盐酸盐 | 91.25 | 91.25 | 盐酸吡哆醇 | 0.031 | 0.031 |
L-蛋氨酸 | 17.24 | 17.24 | 核黄素 | 0.219 | 0.219 |
L-苯丙氨酸 | 35.48 | 35.48 | 盐酸硫胺 | 2.17 | 2.17 |
L-丝氨酸 | 26.25 | 26.25 | 胸苷 | 0.365 | 0.365 |
L-苏氨酸 | 53.45 | 53.45 | 维生素B12 | 0.68 | 0.68 |
L-丙氨酸 | 4.45 | 4.45 | |||
L-天门冬酰胺 | 7.5 | 7.5 | pH(无碳酸氢钠) | 5.8±0.3 | 5.8±0.3 |
L-天门冬氨酸 | 6.65 | 6.65 | pH(加碳酸氢钠) | 6.8±0.3 | 6.9±0.3 |
L-半胱氨酸盐酸盐 | 17.56 | 17.56 | 渗透压(无碳酸氢钠) | 279±5% | 277±5% |
L-谷氨酸 | 7.35 | 7.35 | 渗透压(加碳酸氢钠) | 299±5% | 300±5% |
L-脯氨酸 | 17.25 | 17.25 |
Claims (10)
2.根据权利要求1所述的检测细胞内汞离子的荧光探针,其特征是:R1和R7可以共同拥有2至5个碳原子的烷基链,其将R7的碳链连接在R1所连接的氮上;R2和R5可以共同拥有2至5个碳原子的烷基链,其将R5的碳链连接在R2所连接的氮上;R3和R8可以共同拥有2至5个碳原子的烷基链,其将R8的碳链连接在R3所连接的氮上;R4和R6可以共同拥有2至5个碳原子的烷基链,其将R6的碳链连接在R4所连接的氮上。
3.一种根据权利要求1~2任一项所述的检测细胞内汞离子的荧光探针的合成方法,其特征是,所述的方法包括以下步骤:
(1)将3位带有羧基的1摩尔罗丹明溶于干燥有机溶剂中,搅拌下缓慢滴加1~2摩尔POCl3,加热回流反应,反应完成后,冷却至室温,减压除去有机溶剂,得到的罗丹明酰氯粗品直接用作下步反应;
(2)将1摩尔2-巯基乙胺盐酸盐或者胱胺二盐酸盐溶于干燥有机溶剂中,加入4~8摩尔缚酸剂,在冰浴条件下,缓慢滴加步骤(1)所得罗丹明酰氯,室温反应,减压除去有机溶剂,加水析出沉淀,过滤后真空干燥得到X为O的式(I)或式(II)的目标产物;
(3)将等摩尔步骤(2)所得产物和劳森试剂溶于干燥有机溶剂中,惰性气体保护下加热回流,减压除去有机溶剂,柱层析分离得到X为S的式(I)或式(II)的目标产物。
4.根据权利要求3所述的方法,其特征是:所述的步骤(1)的加热回流反应时间是2~8小时。
5.根据权利要求3所述的方法,其特征是:所述的步骤(2)的室温是0℃~30℃。
6.根据权利要求3或5所述的方法,其特征是:所述的步骤(2)的反应时间是8~12小时。
7.根据权利要求3所述的方法,其特征是:所述的步骤(3)加热回流时间是4~8小时。
8.根据权利要求3所述的方法,其特征是:所述的干燥有机溶剂是1,2-二氯乙烷、乙腈、丙腈、苯或甲苯。
9.根据权利要求3所述的方法,其特征是:所述的缚酸剂是吡啶或三乙胺。
10.一种根据权利要求1~2任一项所述的检测细胞内汞离子的荧光探针的用途,其特征是:所述的检测细胞内汞离子的荧光探针用于化学模拟生物体系中汞离子的检测,生物活细胞和活组织内的汞离子的分析检测和荧光成像检测,以及临床医学上病变组织中汞离子的检测。
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