CN110551498A - 一种检测Hg2+的香豆素类荧光探针的制备及应用 - Google Patents
一种检测Hg2+的香豆素类荧光探针的制备及应用 Download PDFInfo
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Abstract
本发明涉及一种香豆素类荧光探针制备方法及其应用,化合物名称为:7‑(二乙基氨基)‑3‑(1,3‑二噻烷‑2‑基)‑2H‑吡喃‑2‑酮。本发明所述的化合物分子质量小,结构简单,对Hg2+具有十分灵敏的效果。表现出极好的选择性和灵敏度。适用于Hg2+的裸眼检测。它的检测限为5.0nM,与最严格的Hg2+测量标准相同,具有原位检测的潜力。这种既定方法在分析环境和海产品样品中的应用提供了令人满意的结果。因此,本发明所述的化合物为Hg2+检测提供了一种很有前途的方法,并激发了感应其他重金属离子和过渡金属离子的发展。在各种水介质中,如水,土壤和海产品,进一步显示出测量和管理HTM离子的潜力,具有广泛的应用前景。
Description
本发明涉及一种香豆素类衍生物荧光分子探针的制法与在检测Hg2+中的应用。
背景技术
近年来,重金属和过渡金属(HTM)离子受到了相当的关注,因为它们对环境以及生命***均造成有害影响。在这些HTM离子中,汞是一种众所周知的剧毒物质,即使在非常低的浓度下也会对人类的生命和环境产生有害影响。据报道,从自然和工业来源释放的Hg2+很容易进入生物膜,并通过食物链在人体内积累,从而严重损害人类健康,如水俣病,耳聋和认知障碍。目前用于检测HTM离子的分析方法包括电化学技术,电感耦合等离子体质谱法,比色法,气相色谱法和荧光探针。这些技术常见的缺点包括需要复杂的操作过程或昂贵的设备,以及不适合进行原位分析。随着近年来HTM检测技术的发展,荧光探针由于其高选择性,高灵敏度,简单的仪器,尤其适用于现场和实时分析而具有更多的优势。因此,设计具有实际应用的Hg2+选择性和原位荧光探针仍然是科学家面临的一个挑战。
我们通过制备香豆素类衍生物得到一种有效灵敏荧光分子探针,一系列实验表明,该荧光分子具有良好的性能,具有潜在的应用价值。
发明内容
本发明的目的在于提供一种新型的香豆素类并对Hg2+进行裸眼检测的生物荧光探针的制备方法及在实际中的应用。
本发明的技术方案如下:
一种由喹啉环胺类衍生物荧光分子探针,其特征是它有如下结构:
一种制备上述香豆素类衍生物的方法,它包括如下步骤:
步骤1.将4-二乙基氨基水杨醛加入到乙醇中,使其充分溶解,再加入丙二酸二乙酯和哌啶,物质的量之比4-二乙基氨基水杨醛∶丙二酸二乙酯∶哌啶=1∶2∶1,将反应混合物回流10h,通过TLC监测,减压除去溶剂,然后加入浓盐酸和冰醋酸进行水解,并在1()()℃下搅拌6h,溶液冷却至室温并倒入冰水中,加入50%NaOH溶液调节pH至7.0-8.0,再加入二氯甲烷,有机层用水洗涤三次,用无水亚硫酸钠干燥,减压浓缩,该粗品经硅胶柱层析纯化,洗脱剂为乙酸乙酯与二氯甲烷之比为1∶8,得到第一步产物;
步骤2.在0℃下将DMF逐滴添加至三氯氧磷,DMF与三氯氧磷之比为6:5,并搅拌30min以产生红色溶液,再将第一步产物溶于DMF中,逐滴加入到红色溶液中,将混合物在60℃搅拌过夜,然后倒入冰水中,加入50%NaOH溶液以调节pH至固体析出,通过过滤获得粗产物并用冷乙醇洗涤,然后用乙醇和二氯甲烷重结晶,得到第二步产物DAC-CHO;
步骤3.将步骤2中得到的产物与1,3-丙二硫醇溶解于二氯甲烷溶液中,再加入三氟化硼醚合物,并在室温下搅拌24h,然后真空蒸发除去溶剂,残留物用冷石油醚洗涤三次,得到的固体在乙酸乙酯和石油醚的混合液中重结晶,其体积比是乙酸乙酯∶石油醚=1∶3,得到目标化合物DAC-Hg。
本发明的优点是:本发明所述的化合物对Hg2+具有十分灵敏的效果,表现出极好的选择性和灵敏度,适用于Hg2+的裸眼检测与在各种水介质中的实际应用,如水,土壤和海产品,进一步显示出测量和管理HTM离子的潜力,具有广泛的应用前景。
具体实施方式
通过以下实施例进一步详细说明本发明,但应注意本发明的范围并不受这些实施例的任何限制。
实施例一:7-(二乙基氨基)-3-(1,3-二噻烷-2-基)-2H-吡喃-2-酮的制备
将5mmol,965mg 4-二乙基氨基水杨醛加入到20.0mL的乙醇中,使其充分溶解,再加入10mmol,1.56mL的丙二酸二乙酯和5mmol,494μL的哌啶,物质的量之比4-二乙基氨基水杨醛∶丙二酸二乙酯∶哌啶=1∶2∶1,将反应混合物回流10h,通过TLC监测,减压除去溶剂,然后加入10.0mL浓盐酸和10.0mL冰醋酸进行水解,并在100℃下搅拌6h,溶液冷却至室温并倒入50mL冰水中,加入50%NaOH溶液调节pH至7.0-8.0,再加入二氯甲烷,有机层用水洗涤三次,用无水亚硫酸钠干燥,减压浓缩,该粗品经硅胶柱层析纯化,洗脱剂为乙酸乙酯与二氯甲烷之比为1∶8,得到第一步产物;在0℃下将11.7mmol,902μL的DMF逐滴添加至9.75mmol,907μL三氯氧磷,DMF与三氯氧磷之比为6∶5,并搅拌30min以产生红色溶液,再取846.3mg上一步的产物溶于5.0mL的DMF中,逐滴加入到红色溶液中,将混合物在60℃搅拌过夜,然后倒入50mL冰水中。加入50%NaOH溶液以调节pH至固体析出,通过过滤获得粗产物并用冷乙醇洗涤,然后用乙醇和二氯甲烷重结晶,得到第二步产物;取2.0mmol,490.0mg上一步的产物与2.0mmol,201μL的1,3-丙二硫醇溶解于253μL的二氯甲烷溶液中,再加入三氟化硼醚合物,并在室温下搅拌24h,然后真空蒸发除去溶剂,残留物用冷石油醚洗涤三次,得到的固体在乙酸乙酯和石油醚的混合液中重结晶,其体积比是乙酸乙酯∶石油醚=1∶3,得到目标化合物0.55g,产率82%。1H NMR(600MHz,DMSO-d6)δ/ppm 7.96(s,1H),7.56(d,J=8.9Hz,1H),6.72(dd,J=8.8,1.9Hz,1H),6.55(s,1H),5.35(s,1H),3.43(q,J=7.1Hz,4H),3.11(t,J=13.3Hz,2H),2.91(t,J=3.SHz,1H),2.88(t,J=3.3Hz,1H),2.14-2.10(m,1H),1.75-1.68(m,1H),1.12(t,J=7.2Hz,6H).13C NMR(151MHz,DMSO-d6)δ/ppm 160.23,156.05,142.57,130.20,44.76,43.29,31.50,25.22,12.69.HR-MS(ESI-TOF m/z):Calculated for[C17H22NO2S2]+336.1092,Found 336.1078.
该荧光分子化合物的性质与应用实验,将实施例一中制备的荧光分子探针进行实施例二到八的试验,具体数据和分析如下:
实施例二:
图1:目标化合物检测Hg2+的机理研究
实施例三:
图2:在PBS缓冲液中,该荧光分子探针以及与Hg2+反应后紫外/可见吸收光谱图
对PBS缓冲液(10mM,10%DMSO,pH7.4)中游离的DAC-Hg(10μM)的发射光谱进行了25次采集。激发波长为400nm,光电倍增管电压设定在500V,狭缝宽度设定为5.0nm,在岛津UV-2550仪器上进行检测。
结果显示,游离探针DAC-Hg在403nm处显示峰,而DAC-CHO和DACHg+Hg2+均在444nm处显示峰。随后测定的DAC-Hg的荧光量子产率为0.93,检测产物DAC-CHO为0.27。
实施例四:
图3:DAC-Hg的荧光强度及其对pH环境的响应
将10μM荧光分子探针溶解在PBS(pH 7.4,10mM,10%DMSO)溶液中,将其置于不同pH值下测定该探针的荧光强度变化,pH设定范围为2-12。激发波长为400nm,光电倍增管电压设定在500V,狭缝宽度设定为5.0nm。
这两种物质在较宽的pH范围内均表现出稳定性,这对实际应用是有利的。
实施例五:
图4:在PBS溶液中,该荧光分子探针随时间响应的荧光光谱图
将10μM荧光分子探针溶解在PBS(pH 7.4,10mM,10%DMSO)溶液中,分别在不同的时间下检测其性能,时间设定范围为0-24h。激发波长为400nm,光电倍增管电压设定在500V,狭缝宽度设定为5.0nm。
结果显示该反应在2小时内完成,并保持在12h以上,这说明该荧光分子探针检测***的稳定性。
实施例六:
图5:在PBS溶液中,FT-IR光谱法验证实验
将10μM荧光分子探针溶解在PBS(pH 7.4,10mM,10%DMSO)溶液中,37℃孵育激后进行测定。激发波长为400nm,光电倍增管电压设定在500V,狭缝宽度设定为5.0nm。
结果显示,在~1722cm-1和3125cm-1处的吸收信号分别对应于DAC-Hg中-C=C-C=O,-CH2-CH2-CH2-的伸缩振动(受香豆素结构影响)。Hg2+的添加引起在2972cm-1,1683cm-1,1635cm-1,1581cm-1,1523cm-1处出现典型的羰基吸收峰,在3125cm-1出现-C=C-C=O的消失。DAC-Hg+Hg2+的光谱图与DAC-CHO几乎相同,说明DAC-Hg与Hg2+的脱保护反应成功。
实施例七:
图6:在PBS溶液中,该荧光分子探针在不同氨基酸和阴离子中对Hg2+的选择性
将100μM荧光分子探针溶解在PBS(pH 7.4,10mM,10%DMSO)溶液中,分别在不同的氨基酸和阴离子下检测其性能,不同阴离子的浓度为1.0mM。激发波长为400nm,光电倍增管电压设定在500V,狭缝宽度设定为5.0nm。
从图中可以得出结论,只有Hg2+离子才能导致显着的荧光猝灭。所有这些结果表明DAC-Hg能够通过以高选择性显示“关闭”效应而不受其他金属离子或氨基酸的显著干扰。
实施例八:
图7:EDTA添加的不同顺序对Hg2+于DAC-Hg不可逆机理的影响
将10μM荧光分子探针溶解在PBS(pH 7.4,10mM,10%DMSO)溶液中,改变EDTA的添加顺序检测检测该荧光分子探针的荧光强度。激发波长为400nm,光电倍增管电压设定在500V,狭缝宽度设定为5.0nm。
从图中可以看出,包括DAC-Hg和Hg2+在内的检测***完全孵育后,加入高浓度EDTA(200μM)不会对检测结果产生干扰。但是,当Hg2+首先与EDTA或与EDTA和DAC-Hg的混合物反应时,检测可能被阻止。因此,DAC-Hg意味着不可逆转的检测机制,不受已经螯合的Hg2+的干扰。
实施例九:
图8:本发明目标化合物的氢谱与碳谱解析
结果表明,在实际应用中,本发明中的荧光分子探针具有高选择性和灵敏性,可用于裸眼检测Hg2+。该荧光分子探针对Hg2+的“关闭”感应超过其他金属离子。它的检测限为5.0nM,与最严格的Hg2+测量标准相同,具有原位检测的潜力。这种既定方法在分析环境和海产品样品中的应用提供了令人满意的结果。因此,DAC-Hg为Hg2+检测提供了一种很有前途的方法,并激发了感应其他重金属离子和过渡金属离子的发展。
Claims (3)
1.一种检测Hg2+的香豆素类荧光探针,其特征在于,所述荧光探针由如下结构式构成:
2.根据权利要求1所述的香豆素类衍生物,包括如下步骤制备:
步骤1.将4-二乙基氨基水杨醛加入到乙醇中,使其充分溶解,再加入丙二酸二乙酯和哌啶,物质的量之比4-二乙基氨基水杨醛∶丙二酸二乙酯∶哌啶=1∶2∶1,将反应混合物回流10h,通过TLC监测,减压除去溶剂,然后加入浓盐酸和冰醋酸进行水解,并在100℃下搅拌6h,溶液冷却至室温并倒入冰水中,加入50%NaOH溶液调节pH至7.0-8.0,再加入二氯甲烷,有机层用水洗涤三次,用无水亚硫酸钠干燥,减压浓缩,该粗品经硅胶柱层析纯化,洗脱剂为乙酸乙酯与二氯甲烷之比为1∶8,得到第一步产物;
步骤2.在0℃下将DMF逐滴添加至三氯氧磷,DMF与三氯氧磷之比为6∶5,并搅拌30min以产生红色溶液,再将第一步产物溶于DMF中,逐滴加入到红色溶液中,将混合物在60℃搅拌过夜,然后倒入冰水中,加入50% NaOH溶液以调节pH至固体析出,通过过滤获得粗产物并用冷乙醇洗涤,然后用乙醇和二氯甲烷重结晶,得到第二步产物;
步骤3.将步骤2中得到的产物与1,3-丙二硫醇溶解于二氯甲烷溶液中,再加入三氟化硼醚合物,并在室温下搅拌24h,然后真空蒸发除去溶剂,残留物用冷石油醚洗涤三次,得到的固体在乙酸乙酯和石油醚的混合液中重结晶,其体积比是乙酸乙酯∶石油醚=1∶3,得到目标化合物。
3.根据权利要求1和2所述的荧光分子的光谱特征与实际应用。
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