CN113004886A - 一种苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针的制备方法及其应用 - Google Patents
一种苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针的制备方法及其应用,其化学结构式如下:
Description
技术领域
本发明涉及的是化学分析检测技术领域,具体涉及一种苯并喹啉类的比率型近红外荧光分子探针的制备方法以及该荧光分子探针在检测氟离子方面的应用。
背景技术
氟离子是人体所必须的元素之一,同时也是最小的阴离子,具有强腐蚀性等独特性质。适量的氟化物可以预防龋齿和治疗骨质疏松症,因此被广泛应用在牙膏、药物甚至引用水中。然而,由于氟离子在人体内的代谢极慢,摄入过量的氟将会对人体造成永久性、不可逆的伤害,如氟中毒、尿石症,甚至死亡。美国环境保护署(EPA)设定了饮用水中氟离子的最大污染物水平(MCL)为4 mgL-1(4 ppm或211 mM)。因此,开发快速有效的氟离子检测分析方法在环境和生物系统中非常重要。
迄今为止,已有氟离子选择电极法、氟试剂比色法、离子色谱法等方法用于氟离子的分析检测,但这些检测方法存在分析时间长,操作方法复杂,误差大等问题,限制了其进一步使用。相比之下,荧光分析法由于具有高选择性、高灵敏度、方便快捷、成本低廉、可以实时原位检测等优点,受到了越来越多的关注和应用。如CN 104449677 B公开了一种基于2-( 2-羟基苯基)苯并噻唑母核的可用于氟离子检测的荧光分子探针,该探针及相应氟离子含量检测过程均不受生物体系基质及杂质的干扰,可用于各种生物体系中氟离子含量的定量测定。该探针分子在乙腈溶液中最大发射波长为370 nm。但该探针发射波长较短,处于可见光区,易引起荧光自淬灭和信噪比低,从而导致荧光检测误差大和灵敏度低等缺点。同时,该探针水溶性差、生物相容性差,这些都限制了其在生物体内的进一步应用。同时,相比于增强型荧光探针,比率型荧光探针具有双波长发射(或激发)的特征,该波长比率值的变化独立于探针浓度和光源强度,可大大降低其他检测条件的干扰。因此设计一类具有水溶性好,并可减少对生物体的损伤等优势的比率型近红外荧光探针以用于不同体系中氟离子的灵敏特异性检测有重要意义。
发明内容
对于上述情况,本发明的目的之一提供一种合成简单、反应条件温和、成本较低的荧光探针合成方法;目的之二是提供一种水溶性好、特异性强、灵敏度高、能够比率型检测氟离子的荧光分子探针;目的之三在于提供上述荧光分子探针在纯水体系和生物体内氟离子的检测应用。
本发明使用的苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针,采用10-羟基苯并[H]喹啉类衍生物和叔丁基二甲基氯硅烷在二氯甲烷中合成,叔丁基二甲基硅醚作为识别基团。
为了实现上述目的,本发明解决问题采取的技术方案为,一种苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针,具有式I结构:
式I
具体制备方法包括以下步骤:
(1)将10-羟基苯并[H]喹啉和六甲基次胺加入的圆底烧瓶中,再向反应体系中加入三氟乙酸,升温至90 oC加热回流,反应结束后,取下反应,放冷后将其倒入冰水中,用1 N的NaOH溶液调节pH至6左右,析出大量沉淀,抽滤、收集并洗涤滤饼,真空干燥后,经柱层析纯化,可得化合物1;
(2)在圆底烧瓶将化合物1加入10mL乙醇中,充分溶解后再向反应体系中加入2-甲基吡啶盐和哌啶,升温至90 oC加热回流。反应结束后放冷浓缩,柱层析纯化,可得化合物2;
(3)在氮气保护、冰浴下将化合物2溶于装有无水二氯甲烷的圆底烧瓶中,向反应体系中滴加三乙胺并缓慢滴入溶有叔丁基二甲基氯硅烷的二氯甲烷溶液,0oC下搅拌,TLC监测反应直至反应完全。分别用水、饱和NaHCO3溶液及饱和食盐水萃取,合并有机层,用无水硫酸镁干燥后浓缩,可得目标荧光分子探针。
本发明的合成如下所示:
在本说明书的实施例中更详细地说明了该探针的合成和检测方法。本发明的荧光分子探针使用方法没有特殊限制。通常可以将探针分子溶解在PBS缓冲溶液(10mM,pH 7.4)中,于室温下进行测试。由于叔丁基二甲基硅醚阻碍了探针的激发态分子内质子转移效应(excited-state intramolecular proton transfer,ESIPT),因此探针只产生烯醇式发射,在498 nm有荧光发射,当加入氟离子后,由于氟离子能选择性切割分子探针中的叔丁基二甲基硅醚,释放出羟基,探针的ESIPT作用得以恢复,从而使探针恢复酮式发射,使得体系在498 nm处荧光减弱,637 nm处荧光显著增强,呈现比率型检测。检测机理如图所示:
相对现有技术,本发明的技术方案带来的有益技术效果:
本发明所涉及的一种苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针,具有以下优势:
(1)本发明荧光探针合成简单、原料易得,产率较高,成本较低,有利于大规模推广应用;
(2)本发明荧光探针的灵敏度高、特异性强、水溶性好、光学性能稳定、细胞膜渗透性好、细胞毒性小,且具有较强的红光发射,能有效避免生物自发荧光的干扰,使得该荧光探针在环境科学,生物化学等领域具有实际的应用价值。
附图说明
【图1】本发明实施中荧光探针的荧光强度随氟离子浓度变化的发射光谱图,横坐标为波长,纵坐标为荧光强度;
【图2】本发明实施中荧光探针的荧光强度比值(I637 nm/I498 nm)和氟离子浓度的线性关系,图横坐标为氟离子的浓度,纵坐标为荧光强度比值(I637 nm/I498 nm);
【图3】本发明实施中荧光探针对氟离子的选择性图,横坐标为波长,纵坐标为荧光强度;
【图4】本发明实施中荧光探针在MCF-7细胞内荧光成像图。
具体实施方式
实施例1
化合物1的合成
将10-羟基苯并[H]喹啉(585.6 mg, 2.5mmol)和六甲基次胺 (1680 mg, 10mmol)加入25mL的圆底烧瓶中,再向反应体系中加入25mL三氟乙酸,升温至90oC加热回流,反应结束后,取下反应,放冷后将其倒入500mL冰水中,用1 N的NaOH溶液调节pH至6左右,析出大量沉淀,抽滤、收集并洗涤滤饼,真空干燥后,经柱层析纯化,得产物512.4 mg,产率 76.5 %。
化合物2的合成
在25mL的圆底烧瓶将化合物1 (446.4 mg, 2mmol)加入10mL乙醇中,充分溶解后再向反应体系中加入2-甲基吡啶盐 (702 mg, 2.75mmol)和1mmol 哌啶,升温至90oC加热回流。反应结束后放冷浓缩,柱层析纯化,得产物 450.1 mg, 产率57.2 %。1H-NMR (400 MHz,DMSO-d6, TMS): δ4.39(s,3H), 5.35 (s, 1H), 6.82 (d, 1H, J=16.9 Hz) 7.22 (d,1H, J=15.4 Hz), 7.35-7.39 (m, 2H), 7.58-7.62 (m, 2H), 7.81 (d, 1H, J=7.9 Hz),8.06 (d, 1H, J=7.9 Hz), 8.10(t,1H, J=8.0 Hz), 8.37 (d, 1H, J=7.68 Hz), 8.83(d, 1H, J=7.68 Hz), 8.93 (d, 1H, J=8.2 Hz)。
目标荧光分子探针的合成
在氮气保护、冰浴下将化合物2(440.3 mg,1mmol)溶于装有10mL无水二氯甲烷的25mL圆底烧瓶中,向反应体系中滴加三乙胺并缓慢滴入溶有1mmol叔丁基二甲基氯硅烷的二氯甲烷溶液,0oC下搅拌,TLC监测反应直至反应完全。分别用水、饱和NaHCO3溶液及饱和食盐水萃取,合并有机层,用无水硫酸镁干燥后浓缩,可得目标荧光探针339.9 mg,产率61.3 %。1H-NMR (400 MHz, DMSO-d6, TMS): δ0.32 (s, 6H), 0.98(s, 9H), 4.39(s,3H), 6.80(d, 1H, J=16.4 Hz) 7.23 (d, 1H, J=14.2 Hz), 7.29 (d, 1H, J=7.8 Hz), 7.36-7.38(m, 1H), 7.60-7.63 (m, 2H), 7.81 (d, 1H, J=7.8 Hz), 8.05 (d, 1H, J=7.8 Hz),8.12 (t,1H, J=8.2 Hz), 8.39 (d, 1H, J=7.6 Hz), 8.86-8.90 (m, 2H)。HR-MS (ESI,positive), m/z: calculated [M-I]+: 427.63279, found [M-I]+: 427.61572.
实施例2
荧光探针的荧光强度与氟离子的浓度关系实验
准确称量554.5 mg实施例1制备的纯度为99 %以上的目标荧光分子探针溶于乙腈溶液中,制备成1mM的探针母液。取荧光探针母液,分8组,每组10mL,分别加入不同浓度的氟离子溶液,调节到溶液中含有探针分子的浓度为10μM,氟离子的浓度则分别为1μM、2μM、5μM、10μM、20μM、30μM、40μM、50μM。室温孵育20 min后,分别在10 mm的比色皿中测试不同体系的荧光光谱。结果如附图1显示:当加入氟离子后,检测体系在498 nm处的荧光强度逐渐减弱,637 nm处的荧光强度逐渐增强,且其荧光强度与浓度成线性关系。线性拟合图(附图2)表明当氟离子的浓度处于0-20μM的范围区间时,I637 nm/I498 nm与过氧化氢的浓度有着良好的线性关系,回归线性方程为y=0.1166x+0.2655,线性相关系数为:0.9953,并计算出检测限(LOD)为0 .42μM (S/N=3),表明该荧光探针具有良好的灵敏度。
实施例3
荧光探针对氟离子的选择性实验
取荧光探针溶液,分10组,每组10mL,其中1组不加待测物,9组分别加入含有CO3 2-、SO4 2-、NO3-、Cl-、I-、Br-、CH3COO-、PO4 3-、F-溶液,使得每组溶液中含有探针分子的浓度为10μΜ,F-浓度为50μM,其他阴离子浓度为100μM。室温孵育20 min后,分别在10 mm的比色皿中测试不同体系的荧光光谱,记录637 nm和498 nm出的荧光强度并计算I637 nm/I498 nm的值。结果如附图2显示:发现只有加入氟离子时,I637 nm/I498 nm的值出现明显变化,而加入其它测试物时,只有微弱的荧光变化或者没有变化。表明该荧光探针具有很高的选择性,能够有效地避免其它活性分子的干扰。
实施例4 细胞成像实验
将MCF-7细胞在含有10 %牛胎儿血清的1mL细胞培养基培养12小时,然后用100μM的氟离子处理10分钟,再用10μM的本发明的荧光探针处理30分钟。置于荧光显微镜下观察其荧光变化,结果如附图4所示。实验表明进入细胞体内的探针分子和氟离子发生了反应,因此该荧光探针对细胞中的氟离子有良好的成像作用,可用于检测生物体内的氟离子。
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对发明范围的限制,本领域的相关技术人员能从发明公开的内容不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围之内。
Claims (4)
1.一种苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针,其特征是具有式I中的化学结构式。
2.根据权利要求1所述的苯并喹啉类可用于氟离子检测的比率型近红外荧光分子探针,其特征在于合成方法按如下步骤进行;
将10-羟基苯并[H]喹啉和六甲基次胺加入的圆底烧瓶中,再向反应体系中加入三氟乙酸,升温至90 oC加热回流,反应结束后,取下反应,放冷后将其倒入冰水中,用1 N的NaOH溶液调节pH至6左右,析出大量沉淀,抽滤、收集并洗涤滤饼,真空干燥后,经柱层析纯化,可得化合物1,所述化合物1的结构式如下所示:
在圆底烧瓶将化合物1加入乙醇中,充分溶解后再向反应体系中加入2-甲基吡啶盐和哌啶,升温至90 oC加热回流,反应结束后放冷浓缩,柱层析纯化,可得化合物2,所述化合物2的结构式如下所示:
在氮气保护、冰浴下将化合物2溶于装有无水二氯甲烷的圆底烧瓶中,向反应体系中滴加三乙胺并缓慢滴入溶有叔丁基二甲基氯硅烷的二氯甲烷溶液,0 oC下搅拌,TLC监测反应直至反应完全;分别用水、饱和NaHCO3溶液及饱和食盐水萃取,合并有机层,用无水硫酸镁干燥后浓缩,可得目标荧光分子探针。
3.根据权利要求1和2所述的氟离子荧光分子探针,其特征在于,其能不受CO3 2-、SO4 2-、NO3-、Cl-、I-、Br-、CH3COO-、PO4 3-等干扰。
4.权利要求1-3所述荧光探针的用途,其特征在于,用于环境体系和生物样品中氟离子的检测。
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