CN110698409B - 一种特异性识别汞离子的反应型苯并咪唑类荧光探针及制备方法和应用 - Google Patents
一种特异性识别汞离子的反应型苯并咪唑类荧光探针及制备方法和应用 Download PDFInfo
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Abstract
Description
【技术领域】
本发明属于有机小分子荧光探针技术领域,具体涉及一种特异性识别汞离子的反应型苯并咪唑类荧光探针及制备方法和应用。
【背景技术】
汞(Hg)在化学元素周期表中位于第六周期、第IIB族,是一种易挥发的液态金属,它广泛应用于电子、化工和制药等行业。汞离子(Hg2+)作为一种毒害性强的重金属离子,可通过细菌代谢转化为毒性更强的甲基汞,然后经过食物链富集于有机体中。汞的积累会使细胞功能失常,导致认知、行为紊乱和神经损伤,甚至低浓度的Hg2+也会诱导诸如水俣病、肾功能衰竭等疾病。美国环境保护署和世界健康组织规定饮用水和食物中能检测到的Hg2+浓度必须小于2ppb(10nM)。由于化石燃料的使用、固体垃圾的焚烧、工业三废的不合理排放,以及火山爆发等等,越来越严重的汞污染对人类健康和环境造成了威胁。因此,研发有效、可靠的方法来检测环境及生物体内的Hg2+具有非常重要的意义。
传统的Hg2+检测方法有原子吸收光谱法、毛细管电泳法、比色法、电化学方法等,但这些方法需要昂贵的仪器设备,且存在耗时长、样品前处理复杂等弊端。相比之下,荧光分析法由于在检测Hg2+方面具有操作简单、响应快速、灵敏度和选择性高、可用于生物成像等优点,受到了广大研究者们越来越多的重视。
检测Hg2+的荧光探针主要有络合型和反应型两类。其中,反应型的Hg2+荧光探针主要是基于Hg2+较强的嗜硫性而设计的,Hg2+可与探针结构中硫原子发生不可逆的脱硫反应,具有响应速度快、专一性高等特点,在复杂体系中对Hg2+的检测有较好的应用价值。目前,虽然报道了不少Hg2+荧光探针,但很多存在选择性差、抗干扰能力弱等缺陷,真正实用的Hg2+荧光探针很少,开发高性能的Hg2+荧光探针是本领域技术人员亟待解决的问题。
【发明内容】
本发明的目的是克服现有技术的上述缺陷,提供一种合成简易、选择性好、灵敏度高、使用方便、抗干扰能力强、响应快的特异性识别汞离子的反应型苯并咪唑类荧光探针及其制备方法和应用。
为实现上述目的,本发明提供了一种特异性识别汞离子的反应型苯并咪唑类荧光探针,命名为探针ZY13,其特征在于其分子式为C18H20N2S2,结构式如下:
本发明还提供了所述特异性识别汞离子的反应型苯并咪唑类荧光探针的制备方法,其特征在于包括如下步骤:
(A)以醋酸为溶剂,将邻苯二胺和对苯二甲醛混合,回流,反应完毕后,向所得反应液加入70%~90%醋酸物质的量的NaHCO3粉末,调节溶液的pH,然后过滤,将固体成分用水清洗1遍,再过滤,所得固体通过柱层析法提纯,得到中间体,即2-(4-醛基苯基)苯并咪唑;
(B)以无水四氢呋喃为溶剂,将中间体、乙硫醇和BF3·Et2O溶液混合,在低温条件下密封反应,在搅拌下将反应液倒入水中,随后用0.5mol/L NaHCO3水溶液中和至pH为7,过滤,将所得固体通过柱层析法纯化,即得目标探针ZY13。
优选地,步骤(A)中所述醋酸的质量为邻苯二胺质量的100~150倍,邻苯二胺和对苯二甲醛的摩尔比为1:1~1:3,所述回流反应温度为100~120℃,反应时间为20~60分钟,所述调节溶液的pH为调节至pH 3~6,所述柱层析法提纯所用洗脱剂乙酸乙酯和二氯甲烷的体积比为5:1~3:1。
优选地,步骤(B)中所述四氢呋喃的质量为乙硫醇质量的40~60倍,中间体、乙硫醇和BF3·Et2O溶液的摩尔比为1:2:3~1:4:5,所述低温为-10~10℃,所述密封反应时间为12~24小时,所述柱层析法纯化所用洗脱剂四氢呋喃和石油醚的体积比为1:2~1:6。
本发明又提供了所述探针在Hg2+检测中的用途,其特征在于:所述用途均为通过检测所述探针的荧光强度而实现。
优选地,所述用途为用所述探针制成试纸,并可视化检测水溶液中的Hg2+,实现可视化检测水溶液中浓度低于毫摩尔每升级别的Hg2+。
优选地,所述用途为用所述探针实现活细胞中Hg2+的荧光成像。
本发明的有益效果是:
1.本发明提供的探针ZY13与Hg2+反应后,诱导探针的硫缩醛脱保护,使得反应体系的荧光强度明显增强,而且其荧光颜色由无色变为蓝色,它可实现Hg2+的特异性识别,具有较高的选择性和灵敏度,抗干扰能力强,响应快,对Hg2+的最低检测限为9.2nmol/L,可满足美国环境保护署等相关国家标准对Hg2+的检测限量要求;
2.探针ZY13对Hg2+的检测过程方便快捷,无需大型检测仪器,能实现Hg2+的可视化检测,不仅可检测水环境中一定浓度的Hg2+,还可应用于细胞中Hg2+的荧光成像,在生物和水环境监测领域将具有巨大的应用价值;
3.探针ZY13的稳定性好,可长期保存使用。
【附图说明】
图1为实施例4中探针ZY13对各种金属离子的荧光响应图;
图2为实施例5中不同金属离子对探针ZY13识别金属离子的干扰的荧光数据图;
图3为实施例6中探针ZY13与Hg2+的荧光滴定光谱图;
图4为实施例6中探针ZY13荧光强度对Hg2+浓度的线性关系图;
图5为实施例7中探针ZY13在加入Hg2+后荧光光谱随时间的变化图;
图6为实施例7中探针ZY13在加入Hg2+4分钟前(左)后(右)的荧光颜色变化图(365nm紫外灯照射);
图7为实施例8中浸泡过探针ZY13的试纸在紫外灯(365nm)下接触不同浓度Hg2+后的荧光颜色变化图(从左至右Hg2+浓度依次为0mmol/L,0.1mmol/L,1mmol/L);
图8为实施例9中探针ZY13对HeLa活细胞中Hg2+的荧光成像。
【具体实施方式】
现结合附图和实施例详细说明本发明的技术方案。应理解,以下实施例仅用于说明本发明而不用于限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1探针ZY13的合成
探针ZY13的合成路线如下:
具体步骤如下:
(1)向圆底烧瓶中加入邻苯二胺(1.08g,10mmol)和对苯二甲醛(1.34g,10mmol),再加入冰醋酸100mL,在100℃的条件下搅拌并回流20min。反应结束后,将反应液冷却至室温,加入NaHCO3粉末103g,调节溶液的pH至3左右。抽滤,用去离子水10mL洗涤滤饼,干燥。以乙酸乙酯和二氯甲烷为洗脱剂(体积比为5:1),用硅胶柱层析纯化可得中间体。产率30%;ESI-MS:223.13[M+H]+;1HNMR(DMSO-d6,400MHz,ppm):δ13.06(s,1H),10.08(s,1H),8.36(d,2H,J=4.8Hz),8.04(d,2H,J=7.6Hz),7.72(d,1H,J=7.8Hz),7.38(d,1H,J=7.6Hz),7.26(t,2H,J=6.8Hz);13CNMR(DMSO-d6,100MHz,ppm):δ192.22,151.32,142.44,135.84,128.88,127.11,122.49,118.97,111.68。
(2)将0.22g(1.0mmol)中间体溶于8mL无水四氢呋喃,再向其中加入0.12g(2.0mmol)乙硫醇和0.4mL BF3·Et2O溶液,反应体系在-10℃下密封搅拌12h。反应完毕,在搅拌条件下将反应液倒入40mL水中,然后用0.5mol/L NaHCO3水溶液中和至pH为7。过滤,粗产品以四氢呋喃和石油醚为洗脱剂(体积比为1:2),用硅胶柱层析纯化可得探针ZY13。产率37%;ESI-MS:m/z 328.44(M+);1H NMR(400MHz,CDCl3,ppm):δ1.38(t,J=7.2Hz,6H),1.38(q,J=6.8Hz,4H),4.87(s,1H),7.32–7.44(m,4H),7.86(d,J=7.6Hz,2H),8.82(d,J=8.4Hz,2H),12.32(s,1H);13C NMR(100MHz,CDCl3):δ156.2,142.5,139.4,134.1,130.3,126.8,124.6,116.8,53.4,28.1,16.6。
实施例2探针ZY13的合成
(1)向圆底烧瓶中加入邻苯二胺(1.08g,10mmol)和对苯二甲醛(2.68g,20mmol),再加入冰醋酸130mL,搅拌并回流40min。反应结束后,将反应液冷却至室温,加入NaHCO3粉末153g,调节溶液的pH至5左右。抽滤,用去离子水10mL洗涤滤饼,干燥。以乙酸乙酯和二氯甲烷为洗脱剂(体积比为4:1),用硅胶柱层析纯化可得中间体。产率38%。
(2)将0.22g(1.0mmol)中间体溶于10mL无水四氢呋喃,再向其中加入0.19g(3.0mmol)乙硫醇和0.5mL BF3·Et2O溶液,反应体系在0℃下密封搅拌18h。反应完毕,在搅拌条件下将反应液倒入40mL水中,然后用0.5mol/L NaHCO3水溶液中和至pH为7。过滤,粗产品以四氢呋喃和石油醚为洗脱剂(体积比为1:4),用硅胶柱层析纯化可得探针ZY13。产率56%。
实施例3探针ZY13的合成
(1)向圆底烧瓶中加入邻苯二胺(1.08g,10mmol)和对苯二甲醛(4.02g,30mmol),再加入冰醋酸150mL,搅拌并回流60min。反应结束后,将反应液冷却至室温,加入NaHCO3粉末198g,调节溶液的pH至6左右。抽滤,用去离子水10mL洗涤滤饼,干燥。以乙酸乙酯和二氯甲烷为洗脱剂(体积比为3:1),用硅胶柱层析纯化可得中间体。产率35%。
(2)将0.22g(1.0mmol)中间体1溶于10mL无水四氢呋喃,再向其中加入0.25g(4.0mmol)乙硫醇和0.6mL BF3·Et2O溶液,反应体系在10℃下密封搅拌24h。反应完毕,在搅拌条件下将反应液倒入40mL水中,然后用0.5mol/L NaHCO3水溶液中和至pH为7。过滤,粗产品以四氢呋喃和石油醚为洗脱剂(体积比为1:6),用硅胶柱层析纯化可得探针ZY13。产率32%。
实施例4探针ZY13对不同金属离子的选择性研究
取实施例1制备的探针ZY13溶于乙醇中,配制成浓度为100μmol/L的探针母液;用蒸馏水作溶剂,配制各种浓度均为1mmol/L的金属离子(K+,Ca2+,Na+,Mg2+,Cr3+,Mn2+,Fe3+,Fe2+,Co2+,Ni2+,Cu2+,Zn2+,Pb2+,Ag+,Cd2+,Hg2+)储备液。用移液枪分别加入物质的量比为1:4比例的探针和金属离子,然后用乙醇和蒸馏水稀释(VEtOH:VH2O=1:4),使得探针和金属离子在荧光比色皿中的最终浓度分别为10μmol/L和40μmol/L。将配制好的溶液振荡5min,然后测试它们的荧光光谱。由图1可看出,探针ZY13自身的荧光较弱,Hg2+的加入使得探针ZY13的荧光强度显著增强,其它的金属离子的加入对探针荧光几乎没有影响。因此,探针ZY13能特异性识别Hg2+,具有良好的选择性。
实施例5不同金属离子对探针ZY13识别Hg2+的干扰研究
向实施例1制备的探针ZY13溶液(1mmol/L)中加入加入10倍量1mmol/L的各种竞争金属离子(K+,Ca2+,Na+,Mg2+,Cr3+,Mn2+,Fe3+,Fe2+,Co2+,Ni2+,Cu2+,Zn2+,Pb2+,Ag+,Cd2+),测试其荧光光谱;然后再加入与竞争金属离子相同浓度的Hg2+,检测竞争反应后的荧光强度。如图2所示,加入Hg2+后溶液的荧光强度基本相同,各种竞争金属离子不会明显影响探针对Hg2 +的检测,这表明探针ZY13对Hg2+的识别具有较强的抗干扰能力。
实施例6探针ZY13与不同当量的Hg2+反应的荧光光谱变化
向实施例1制备的探针ZY13溶液(10μmol/L)中加入不同当量(0~4equiv)的Hg2+溶液,测定反应体系的荧光光谱。由图3可见,随着Hg2+浓度的增大,探针ZY13在380nm处的荧光强度逐渐增强。当荧光强度达到最大值时,比探针自身的荧光强度增强约9倍。研究发现:当Hg2+的浓度在0.15~0.78μmol/L的范围内,它与探针的荧光强度具有较好的线性关系,相对应的线性方程为:y=332.4391x+28.1752,校正相关系数为R2=0.9835,这表明探针ZY13可以定量检测Hg2+,并有较好的灵敏度(图4)。最低检测限按3σ/k计算为9.2nmol/L,该检测限可满足美国环境保护署等相关国家标准对Hg2+的检测限量要求,表明该探针分子在环境领域可能具有较大的应用价值。
实施例7探针ZY13对Hg2+的响应时间测试
向实施例1制备的探针ZY13溶液(1mmol/L)中加入4倍量的Hg2+溶液(1mmol/L),每1min测一次溶液的荧光强度,一直测到第6min,荧光谱图见图5。实验结果表明,探针ZY13与Hg2+反应4分钟后其荧光强度就已经趋于稳定。将此时的反应液放在365nm紫外灯下照射,溶液由无色变为蓝色(图6),这种荧光颜色变化的快速与明显性说明此探针可用于Hg2+的即时可视化检测。
实施例8探针ZY13试纸对Hg2+的可视化检测
用剪刀将滤纸剪成长为5cm,宽为1cm的纸条,用浓度为100μmol/L的探针ZY13母液浸泡,再取出,自然风干。然后将这些纸条放入不同浓度的Hg2+溶液(0mmol/L,0.1mmol/L,1mmol/L)中,置于紫外灯下(365nm)观察纸条的荧光颜色变化。如图7所示,按照Hg2+由低到高浓度的顺序,紫外灯下纸条的荧光颜色由白色逐渐变为浅蓝色和蓝色。因此,探针ZY13可以像pH试纸一样很方便地可视化检测水溶液中浓度低于毫摩尔每升的Hg2+,具有一定的实用性。
实施例9探针ZY13对活细胞中Hg2+的荧光成像
将培养好的HeLa细胞首先用磷酸盐缓冲溶液清洗3次,然后加入10μmol/L ZY13溶液(pH 7.4,H2O:EtOH:=4:1,v/v),在37℃恒温孵育30min,用磷酸缓冲溶液洗3次,将其放在倒置荧光显微镜下,观察细胞的荧光成像。再向上述细胞中加入Hg2+溶液(40μmol/L),37℃恒温孵育30min,用磷酸缓冲溶液清洗3次,观察细胞的荧光成像。相对于细胞内的ZY13荧光强度,每次都对细胞的明亮视野进行了成像,放大倍数为400倍。如图8所示,加入探针的Hela细胞几乎没有荧光(图8b),而加入Hg2+后,细胞有较强的蓝色荧光(图8d),因此,ZY13可实现在Hela活细胞中Hg2+的荧光成像,具有潜在的实际应用价值。
Claims (7)
2.一种如权利要求1所述的特异性识别汞离子的反应型苯并咪唑类荧光探针的制备方法,其特征在于包括如下步骤:
(A) 以醋酸为溶剂,将邻苯二胺和对苯二甲醛混合,回流,反应完毕后,向所得反应液加入70%~90%醋酸物质的量的NaHCO3粉末,调节溶液的pH,然后过滤,将固体成分用水清洗1遍,再过滤,所得固体通过柱层析法提纯,得到中间体,即2-(4-醛基苯基)苯并咪唑;
(B) 以无水四氢呋喃为溶剂,将中间体、乙硫醇和BF3·Et2O溶液混合,在低温条件下密封反应,在搅拌下将反应液倒入水中,随后用0.5 mol/L NaHCO3水溶液中和至pH为7,过滤,将所得固体通过柱层析法纯化,即得目标探针ZY13。
3.如权利要求2所述的制备方法,其特征在于:步骤(A)中所述醋酸的质量为邻苯二胺质量的100~150倍,邻苯二胺和对苯二甲醛的摩尔比为1:1~1:3,所述回流反应温度为100~120℃,反应时间为20~60分钟,所述调节溶液的pH为调节至pH 3~6,所述柱层析法提纯所用洗脱剂乙酸乙酯和二氯甲烷的体积比为5:1~3:1。
4.如权利要求2或3所述的制备方法,其特征在于:步骤(B)中所述四氢呋喃的质量为乙硫醇质量的40~60倍,中间体、乙硫醇和BF3·Et2O溶液的摩尔比为1:2:3~1:4:5,所述低温为 -10~10℃,所述密封反应时间为12~24小时,所述柱层析法纯化所用洗脱剂四氢呋喃和石油醚的体积比为1:2~1:6。
5.如权利要求1所述的特异性识别汞离子的反应型苯并咪唑类荧光探针在非疾病的诊断或治疗目的的Hg2+检测中的用途,其特征在于:所述用途均为通过检测所述探针的荧光强度而实现。
6.如权利要求5所述的用途,其特征在于:所述用途为用所述探针制成试纸,并可视化检测水溶液中的Hg2+,实现可视化检测水溶液中浓度低于毫摩尔每升级别的Hg2+。
7.如权利要求5所述的用途,其特征在于:所述用途为用所述探针实现活细胞中Hg2+的荧光成像。
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