CN106939015B - 一种丙二醛荧光探针及其制备方法和应用 - Google Patents
一种丙二醛荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种丙二醛荧光探针及其制备方法和应用。该探针结构式如下:该探针分子合成方法简单,实现了水溶液中丙二醛的检测,还可通过共聚焦显微镜对分别共同孵育了荧光探针FDP和丙二醛的人宫颈癌细胞或洋葱组织进行荧光成像,观察绿色通道荧光信号的变化以达到检测哺乳动物细胞或植物组织环境中丙二醛的目的。
Description
技术领域
本发明涉及一种丙二醛荧光探针及其制备方法和应用,属于分析化学技术领域。
背景技术
丙二醛(MDA)是机体内的氧自由基攻击生物膜中的多不饱和脂肪酸形成的脂质过氧化物,它的产生还能加剧膜的损伤。由于丙二醛的生物活性很高,被认作为是一种能诱导有机体突变和导致动脉粥样硬化的有毒物质。在人体体内,异常水平的丙二醛被证实与白血病、糖尿病、癌症、心血管疾病、气喘、动脉粥样硬化等疾病有关。在植物衰老生理和抗性生理研究中,丙二醛含量是一个常用指标,测试丙二醛的含量可以反映机体内脂质过氧化的程度,反映出细胞损伤的程度。因此,在植物衰老和抗性生理研究中,丙二醛含量是一个非常重要的指标。另外,还可以通过丙二醛含量的测定,间接评估植物膜系统受损程度以及植物的抗逆性。因此,发明一种方便快速检测生理环境中丙二醛含量的方法,具有重要的现实意义。
传统的测定方法是利用过氧化降解产物中的丙二醛与硫代巴比妥酸(TBA)在高温和酸性条件下缩合,形成的红色产物三甲川(3,5,5-三甲基恶唑2,4-二酮)于532nm处有最大吸收峰来进行测定的。该方法需要对生物样本进行破坏性的处理,无法对生理环境中的丙二醛进行无损检测。近年来,小分子有机荧光探针受到科学界的广泛关注,它与特定目标分析物发生作用后,荧光信号会发生变化,以达到检测目的。利用荧光探针进行检测的荧光分析法具有特异选择性、高灵敏度、响应时间快等特征,并且对细胞内目标分子进行非侵入性的实时在线成像检测,形象具体地观察到信号变化。因此,研究一种简便、快速的丙二醛含量检测方法是十分必要的。
发明内容
本发明通过分子设计,合成出了一种丙二醛荧光探针,简称FDP;并进一步提供了探针FDP的制备方法和应用。
本发明的技术方案如下:
一种丙二醛荧光探针,它具有如下式(Ⅰ)所示的结构:
所述荧光探针激发波长为373nm,随着丙二醛含量的逐渐增加,其在554nm处的荧光峰值逐渐增强。
所述的丙二醛荧光探针能抗甲醛、乙醛、丙醛、丁醛、异戊醛、2-乙基丙烯醛、2-乙基丁醛、3-甲基-2-丁烯醛、乙醛酸、乙酰丙酮、丙酮、硝酸根、亚硝酸根、一氧化氮、超氧根、单线态氧、双氧水、次氯酸、硫化氢、L-半胱氨酸和还原谷胱甘肽的干扰。
上述的丙二醛荧光探针的制备方法,包括如下步骤:
1)将0.2mmol的4-氯-7-氯磺酰基-2,1,3-苯并恶二唑和0.2mmol的3-丙胺基三苯基膦溴化盐置于圆底烧瓶中,滴加二氯甲烷至溶解,随后加入一滴三乙胺,然后氮气保护下,常温搅拌反应1小时,待反应完全后除去反应液中的二氯甲烷得到粗产品,将粗产品分离提纯得化合物3;
2)取0.16mmol化合物3置于圆底烧瓶中,滴加乙醇至反应物完全溶解,随后用滴管滴加5滴80%水合肼,在常温和搅拌条件下搅拌反应2小时,反应完毕后,通过旋转蒸发仪减压蒸馏除去反应液中的溶剂得到粗产品,并用硅胶层析柱纯化得到纯净目标探针化合物,简记为FDP。
所述步骤(1)中化合物3的分离提纯方法为:以体积比为2:1的乙酸乙酯与石油醚为洗脱剂,用柱层析色谱进行纯化分离。
所述步骤(2)中硅胶柱层析纯化所用洗脱剂体积比为乙酸乙酯:乙醇=100:1。
上述的荧光探针的合成路线如下:
本发明丙二醛荧光探针的用途:通过荧光光谱仪测试在水溶液中由丙二醛引起荧光探针FDP的荧光光谱变化来检测水环境中的丙二醛;或者通过共聚焦显微镜对分别共同孵育了荧光探针FDP和丙二醛的人宫颈癌细胞或洋葱组织进行荧光成像,观察绿色通道荧光信号的变化以达到检测哺乳动物细胞或植物组织环境中的丙二醛的目的。
本发明的优点:(1)探针合成简单,并且产率较高;(2)本发明实现了水溶液中的丙二醛的检测;(3)本发明实现了哺乳动物细胞中的丙二醛的检测;(4)本发明实现了植物细胞中的丙二醛的检测。
附图说明
图1是实施例1中化合物3的1H NMR图谱;
图2是实施例1中化合物3的13C NMR图谱;
图3是实施例1中化合物FDP的1H NMR图谱;
图4是实施例1中化合物FDP的13C NMR图谱;
图5是实施例1中探针FDP随不同量丙二醛的加入荧光谱图的变化情况;图中,丙二醛浓度依次为:0、10、30、50、70、100、150、200、250、300、350、450、550、650、750、900、1050、1200μmol/L的荧光光谱;
图6是实施例3中探针FDP与丙二醛随时间变化的554nm处荧光强度值变化图;
图7是肉眼观察到探针FDP(10μmol/L)在PBS缓冲溶液与丙二醛在便携式紫外灯365nm光源照射条件下的变化。
图8是实施例4中探针FDP对不同干扰分析物的选择性柱状荧光数据图;图中,1,空白;2,甲醛;3,乙醛;4,丙醛;5,丁醛;6,异戊醛;7,2-乙基丙烯醛;8,2-乙基丁醛;9,3-甲基-2-丁烯醛;10,乙醛酸;11,乙酰丙酮;12,丙酮;13,硝酸根;14,亚硝酸根;15,一氧化氮;16,超氧根;17,单线态氧;18,双氧水;19,次氯酸;20,硫化氢;21,L-半胱氨酸;22,还原谷胱甘肽。
图9是实施例5中探针FDP与HeLa细胞中丙二醛响应的荧光成像图;图中,(a-c)是加入探针FDP孵育20分钟后的成像,(d-f)是先加入探针FDP孵育20分钟,随后加入丙二醛继续培育30分钟后的成像。(a,d)明场成像;(b,e)绿色通道成像;(c,f)明场和绿色通道成像叠加图;激发波长为405nm;标尺为20微米。
图10是实施例6中探针FDP与洋葱组织中丙二醛响应的荧光成像图;图中,(a-c)是加入探针FDP孵育30分钟后的成像,(d-f)是先加入β-丙氨酰-L-组氨酸孵育60分钟,随后加入探针FDP继续培育30分钟后的成像。(a,d)明场成像;(b,e)绿色通道成像;(c,f)明场和绿色通道成像叠加图;激发波长为405nm;标尺为100微米。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1
化合物3的合成:
将取50mg 4-氯-7-氯磺酰基-2,1,3-苯并恶二唑(1)(0.2mmol)和80mg 3-丙胺基三苯基膦溴化盐(2)(0.2mmol)至于圆底烧瓶中,加入二氯甲烷使之完全溶解,随后滴加入一滴三乙胺,在氮气保护和常温下搅拌1小时,反应完毕通过旋转蒸发仪减压蒸馏将溶剂(二氯甲烷)除去得到粗产品。以体积比为2:1的乙酸乙酯与石油醚为洗脱剂,用硅胶(200-300目)层析柱进行纯化,得到79mg淡黄色固体3(产率为:63%)。1H NMR(400MHz,CD3OD),δ(ppm):1.81-1.87(m,2H),3.15-3.16(2H),3.39-3.47(2H),7.64-7.66(d,J=7.6Hz,1H),7.73-7.75(7H),7.77-7.79(4H),7.87(s,1H),7.88-7.91(3H),8.01-8.03(d,J=7.6Hz,1H);13C NMR(100MHz,CD3OD):δ(ppm):20.23,24.64,44.02,119.14,120.00,128.03,129.40,131.45,131.59,131.72,134.79,134.89,135.72,136.43,146.64,150.39。
化合物FDP的合成:
取50mg化合物3(0.16mmol)至于圆底烧瓶中,加入乙醇使反应物完全溶解,随后用滴管滴加5滴80%水合肼,在常温和搅拌条件下搅拌反应2小时。反应完毕后,通过旋转蒸发仪减压蒸馏将溶剂(乙醇)除去得到粗产品。最后以体积比为100:1的乙酸乙酯与乙醇为洗脱剂,用硅胶(200-300目)层析柱进行纯化,得到54mg白色固体FDP(产率为55%)。1H NMR(400MHz,CD3OD),δ(ppm):1.78-1.86(m,2H),3.14-3.17(d,J=6.2Hz,2H),3.38-3.45(2H),7.73-7.75(5H),7.76-7.77(4H),7.78(s,3H),7.88-7.89(1H),7.90-7.92(2H),7.93(s,1H);13C NMR(100MHz,CD3OD):δ(ppm):20.69,24.38,43.71,99.35,111.29,119.20,120.06,124.90,131.45,131.56,131.68,133.90,134.73,134.82,136.40,140.46,144.48,144.92,146.95,147.59,150.18。
实施例2
探针FDP与不同浓度丙二醛反应的荧光光谱变化
取实施例1制备的探针FDP溶于乙醇中,制成浓度为1mM的探针母液,将220mg 1,1,3,3-四乙氧基丙烷溶于80mL浓度为10mM的盐酸水溶液中,在50度下加热1小时,冷却至室温后再用盐酸水溶液稀释至100mL,得到浓度为100mM的丙二醛母液。从探针母液中取出30μL加入到5mL的离心管当中,加入不同当量(0-120eq)的丙二醛母液(所述当量是指丙二醛母液中丙二醛的物质的量相对于探针母液中探针的物质的量的倍数),用不同体积的PBS水溶液(浓度25mmol/L,pH 7.4)稀释至3mL,配置成探针浓度为10μmol/L,含1%乙醇的测试溶液。用荧光光谱仪测试探针与不同当量丙二醛反应液的荧光光谱变化(激发波长为373nm),荧光光谱变化情况如图5由图可见,随着丙二醛加入当量的逐渐增加,探针FDP溶液在554nm处的荧光峰值逐渐增强。当荧光强度达到最大值时,比探针空白液的荧光强度增强约774倍。如图6所示,还可以通过肉眼可以观察到测试液在便携式紫外灯照射下(356nm光源)荧光明显增强。实验结果说明探针FDP可以通过荧光光谱仪检测水溶液中的丙二醛,并且用肉眼观察前后荧光发生的变化。
实施例3
探针FDP与丙二醛随时间变化的荧光变化
从实施例2中荧光探针母液中取出30μL加入到5mL的离心管当中,加入30μL浓度为100mmol/L的丙二醛母液,再用2.94mL PBS缓冲溶液(浓度25mM,pH 7.4)稀释至3mL,配制成探针浓度为10μmol/L,含1%乙醇的测试液。用373nm的激发波长,测试其随时间变化的荧光光谱。由图7可见,随着时间增加,554nm处的荧光强度逐渐变大,并且在15分钟左右达到最大值。
实施例4
探针FDP对不同干扰分析物的选择性和竞争性研究
从实施例2中荧光探针母液中取出30μL加入到5mL的离心管当中,分别加入2.0mmol的干扰物,比如甲醛、乙醛、丙醛、丁醛、异戊醛、2-乙基丙烯醛、2-乙基丁醛、3-甲基-2-丁烯醛、乙醛酸、乙酰丙酮、丙酮、硝酸根、亚硝酸根、一氧化氮、超氧根、单线态氧、双氧水、次氯酸、硫化氢、L-半胱氨酸、还原谷胱甘肽,测试其选择性;之后,再分别在每个测试管加入1.0μmol/L的丙二醛,测试其竞争性。用不同体积的PBS水溶液(浓度25mM,pH 7.4)稀释至3mL,配置成探针浓度为10μmol/L,含1%乙醇的测试溶液。由图8可知,加入各种干扰物,包括醛酮类物质、活性氮、活性氧、硫醇等,测试液荧光强度几乎没有变化。然而,加入丙二醛的测试液的荧光强度发生了显著增强。实验结果说明探针FDP对丙二醛检测具有良好的选择性。
实施例5
探针FDP与HeLa细胞中的丙二醛荧光成像
从实施例2中荧光探针母液中取出5μL加入到育有HeLa细胞的培养皿(含1mL PBS培养基)中,探针浓度为5μmol/L,孵育20分钟,作为控制组;在其中一组控制组样品中分别加入250μmol/L的丙二醛,继续孵育30分钟,作为实验组。随后分别用共聚焦显微镜对控制组和实验组进行荧光成像,使用激发波长为405nm的光源激发,收集530-580nm范围的荧光,结果如图9所示。在控制组的荧光成像中,只能观察到非常微弱的荧光;然而,在实验组中,可以观察到明显的绿色荧光,荧光显著增强。实验结果说明探针FDP可以通过共聚焦显微镜检测细胞环境中的丙二醛,具有潜在的实际应用价值。
实施例6
探针FDP与洋葱组织中的丙二醛荧光成像
从实施例2中荧光探针母液中取出10μL加入到育有洋葱切片组织的培养皿(含1mLPBS培养基)中,探针浓度为10μmol/L,孵育30分钟,作为实验组;在其中一组控制组样品中预先加入2mmol/L的β-丙氨酰-L-组氨酸(α-β不饱和醛清除剂),并孵育60分钟,然后加入10μL探针溶液继续孵育30分钟,作为控制组。随后分别用共聚焦显微镜对控制组和实验组进行荧光成像,使用激发波长为405nm的光源激发,收集530-580nm范围的荧光,结果如图10所示。在实验组的荧光成像中,可以观察到很强的绿色荧光;然而,在控制组中,荧光显著减弱。实验结果说明探针FDP可以通过共聚焦显微镜检测植物组织中的内源性丙二醛,对研究植物的抗性具有潜在的实际应用价值。
Claims (5)
1.一种丙二醛荧光探针,其特征在于,它具有如下式(Ⅰ)所示的结构:
。
2.一种权利要求1所述的丙二醛荧光探针的制备方法,其特征在于,包括如下步骤:
1)将0.2mmol的4-氯-7-氯磺酰基-2,1,3-苯并恶二唑和0.2mmol的3-丙胺基三苯基膦溴化盐置于圆底烧瓶中,滴加二氯甲烷至溶解,随后加入一滴三乙胺,然后氮气保护下,常温搅拌反应1小时,待反应完全后除去反应液中的二氯甲烷得到粗产品,将粗产品分离提纯得化合物3;所述化合物3的结构式如下所示:
;
2)取0.16mmol化合物3置于圆底烧瓶中,滴加乙醇至反应物完全溶解,随后用滴管滴加5滴80%水合肼,在常温和搅拌条件下搅拌反应2小时,反应完毕后,通过旋转蒸发仪减压蒸馏除去反应液中的溶剂得到粗产品,并用硅胶层析柱纯化得到纯净目标探针化合物,简记为FDP。
3.根据权利要求2所述的制备方法,其特征在于,所述步骤(1)中化合物3的分离提纯方法为:以体积比为2:1的乙酸乙酯与石油醚为洗脱剂,用柱层析色谱进行纯化分离。
4.根据权利要求2所述的制备方法,其特征在于,所述步骤(2)中硅胶柱层析纯化所用洗脱剂体积比为乙酸乙酯:乙醇=100:1。
5.一种权利要求1所述的丙二醛荧光探针的应用,其特征在于,用于检测水环境、动物细胞中或植物细胞中的丙二醛。
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CN112266366B (zh) * | 2020-11-13 | 2022-03-18 | 河南省农业科学院农业质量标准与检测技术研究所 | 基于硝基呋咱衍生物的用于甲醛检测的荧光分子及其制备方法和应用 |
CN114668841B (zh) * | 2022-03-14 | 2022-12-09 | 湖南大学 | 一种可激活的纳米光敏剂及其制备方法和应用 |
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