CN112876499B - 用于检测羧酸酯酶1的新型bodipy荧光探针及其制备方法与应用 - Google Patents
用于检测羧酸酯酶1的新型bodipy荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了用于检测羧酸酯酶1的新型BODIPY荧光探针及其制备方法与应用。本发明的小分子荧光探针是利用苯甲酰氯和氟硼二吡咯，通过有机合成反应制备得到。该新型BODIPY荧光探针是基于氟硼二吡咯的发光母核设计而成，在3位活泼甲基上通过克脑文盖尔缩合反应引入N‑乙基咔唑‑3‑甲醛基，延伸BODIPY荧光团的共轭结构，使发射波长红移到近红外区域。然后用苯甲酰氯取代8号位，形成酯键，对羧酸酯酶1具有识别性。本发明的新型BODIPY荧光探针具有较好的生物相容性，具有荧光量子产率高、摩尔吸收系数大、荧光谱峰窄，灵敏度高、光稳定性好等优异性能，能够实现对羧酸酯酶1(CES1)和农药残留的检测。
Description
技术领域
本发明属于生物分析检测领域，具体涉及用于检测羧酸酯酶1的新型BODIPY荧光探针及其制备方法与应用。
背景技术
羧酸酯酶1(CES1)是一种关键的广谱丝氨酸水解酶，它催化大量内源性和外源性物质的水解，如脂类、酰胺、硫酯和氨基甲酸酯。此外，羧酸酯酶1基于药物与酶活性位点的不可逆结合，被认为是重要的药物靶点和药物前体激活剂，参与药物代谢和农药解毒。CES1主要在肝脏表达，研究表明体内CES1水平异常可导致动脉粥样硬化、肥胖、间质细胞肿瘤、高脂血症、肝癌等疾病。因此，有必要开发专门的方法来监测生物样品中CES1的水平和活性。
食用含有大量高毒、剧毒农药残留引起的食物会导致人、畜急性中毒事故。长期食用农药残留超标的农副产品，虽然不会导致急性中毒，但可能引起人和动物的慢性中毒，导致疾病的发生，甚至影响到下一代。由于不合理使用农药，特别是除草剂，导致药害事故频繁，经常引起大面积减产甚至绝产，严重影响了农业生产，土壤中残留的除草剂是其中的一个重要原因。世界各国相继禁止了一些农药的使用，但由农药残留引起的食品安全事件仍然是一个巨大的挑战，因此开发快速有效的农药检测工具具有重要意义。
近年来，小分子荧光探针因其反应快、灵敏度高、重现性好、无创性等特点被广泛应用于生物分析和检测，并且是开发监测生理过程中CES1活性的良好平台。然而，目前报道的CES1探针很少，由于其发射波长较短、不可避免的背景荧光干扰、水溶性差、组织穿透性差、灵敏度低，在复杂生物系统中的应用受到很大限制。因此，有必要进一步开发更实用的荧光探针，高选择性地检测各种生物体系中的CES1活性，以及高通量筛选CES1抑制剂。
发明内容
发明目的：针对现有探针技术存在的发射波长较短、不可避免的背景荧光干扰、水溶性差、组织穿透性差、灵敏度低，在复杂生物系统中的应用受到很大限制问题。本发明提供了用于检测羧酸酯酶1的新型BODIPY荧光探针，其是一种基于氟硼二吡咯母核的小分子荧光探针，该探针不仅可以对羧酸酯酶1(CES1)进行检测，还可以用来检测食品中的农药残留。本发明还提供了检测羧酸酯酶1的新型BODIPY荧光探针的制备方法和应用。
技术方案：为实现上述发明目的，本发明采用如下技术方案：
用于检测羧酸酯酶1的新型BODIPY荧光探针，：以氟硼二吡咯作为发光母核，3号位上是N-乙基咔唑-3-甲醛基，8号位上是苯甲酸甲酯基，结构式如下：
进一步地，所述的用于检测羧酸酯酶1的新型BODIPY荧光探针的制备方法，包括如下步骤：
在化合物BOD-OH中加入二氯甲烷，化合物BOD-OH溶解后加入三乙胺，然后逐滴加入苯甲酰氯，反应混合物搅拌过夜，反应结束后旋蒸去除有机溶剂，然后纯化产物，得到用于检测羧酸酯酶1的新型BODIPY荧光探针CBZ-BOD；该制备方法的反应路线如下所示：
进一步地，所述的反应结束后旋蒸去除有机溶剂具体为：用柱层析法进一步纯化化合物，得到检测羧酸酯酶1的新型BODIPY荧光探针CBZ-BOD。
进一步地，在通N2的条件下，在化合物1中加入甲苯溶解后加入N-乙基咔唑-3-甲醛，然后加入对甲苯磺酸和哌啶；回流，反应混合物搅拌过夜；反应结束后旋蒸去除有机溶剂，然后用柱层析法纯化产物，得到化合物BOD-OH；其反应路线如下所示：
进一步地，所述的反应结束后旋蒸去除有机溶剂具体为：用柱层析法进一步纯化化合物，得到化合物BOD-OH。
进一步地，所述的用于检测羧酸酯酶1的新型BODIPY荧光探针在与MOF材料结合的应用，用于检测羧酸酯酶1的新型BODIPY荧光探针与ZIF-8的结合。
进一步地，所述的用于检测羧酸酯酶1的新型BODIPY荧光探针在检测羧酸脂酶1(CES1)中的应用，利用探针中的酯键被CES1催化水解并转化为羟基，导致荧光强度降低，从而检测羧酸酯酶1(CES1)。
进一步地，所述的用于检测羧酸酯酶1的新型BODIPY荧光探针在检测农药残留中的应用，CES1作为检测农药的生物指示剂，因为它的活性位点可以不可逆地与有机磷酸酯类农药结合，农药会抑制CES1，导致探针荧光的变化，从而检测农药残留。
有益效果：与现有技术相比，本发明具有如下优点：本发明的新型BODIPY荧光探针包含氟硼二吡咯基团和酯键，具有较好的生物相容性，具有荧光量子产率高、摩尔吸收系数大、荧光谱峰窄，灵敏度高、光稳定性好等优异性能；在BODIPY的8位引入酯键，使得探针具有对羧酸酯酶1(CES1)的识别功能，在3位活泼甲基上通过克脑文盖尔缩合反应引入N-乙基咔唑-3-甲醛基，延伸BODIPY荧光团的共轭结构，使发射波长红移到近红外区域。由于有机磷酸酯类农药与CES1活性位点的不可逆结合，农药会抑制CES1，导致探针荧光的变化，从而检测有机磷酸酯类农药残留。发明的新型BODIPY荧光探针制备方法，制备工艺简单、原料易得、成本低，易于规模化生产。
附图说明
图1为新型的BODIPY荧光探针的合成路线；
图2为新型的BODIPY荧光探针的最大吸收波长和最大发射波长的测定实验结果；
图3为新型的BODIPY荧光探针对羧酸酯酶1(CES1)的选择性测试结果；
图4为新型的BODIPY荧光探针的动力学实验结果；
图5为新型的BODIPY荧光探针的pH稳定性实验结果；
图6为新型的BODIPY荧光探针对羧酸酯酶1(CES1)浓度滴定实验结果；
图7为新型的BODIPY荧光探针对农药残留的检测结果；
图8为新型的BODIPY荧光探针与ZIF-8结合后的扫描电镜(SEM)图。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
本发明中使用的实验方法如无特殊说明，均为常规方法。实验所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
检测羧酸酯酶1的新型BODIPY荧光探针的制备
其具体制备合成路线如图1所示：
中间体化合物1的合成
称量对羟基苯甲醛(1.00g)于圆底烧瓶中，用二氯甲烷(150mL)溶解后加入2,4-二甲基吡咯(1.69mL)，开始搅拌，搅拌速度为500rpm。加入6滴三氟乙酸作为催化剂催化反应，反应12h后加入四氯苯醌(2.01g)作为氧化剂，反应5h。利用恒压滴液漏斗逐滴加入三乙胺(10mL)，半小时后再逐滴加入三氟化硼乙醚(10mL)。反应过夜。反应结束后用水进行萃取，取有机层，用无水硫酸钠干燥，然后旋蒸除去溶剂，用柱层析法(PE：EA＝2：1)进一步纯化化合物，得到化合物1
中间体化合物BOD-OH的合成
称取化合物1(0.21mg)中加入甲苯(15mL)溶解后加入N-乙基咔唑-3-甲醛(0.69mg)和对甲苯磺酸(0.14mg)，最后加入哌啶(0.65mL)。通N2，回流，反应混合物温度保持在110℃，搅拌速度为500rpm，搅拌12h。反应结束后旋蒸去除有机溶剂，然后柱层析法(PE：EA＝3：1)进一步纯化化合物，得到化合物BOD-OH。
目标化合物CBZ-BOD的合成
称取化合物BOD-OH(0.08g)于圆底烧瓶中，加入二氯甲烷(15mL)溶解后加入三乙胺(0.03mL)，在24℃下搅拌，搅拌速度为500rpm。然后逐滴加入苯甲酰氯(0.03mL)，反应时间为12h。反应结束后旋蒸去除溶剂，用柱层析法(PE：EA＝3：1)纯化产物，得到一种紫黑色固体，即为检测羧酸酯酶1的新型BODIPY荧光探针。
其具体合成路线如图1所示。
实施例2
检测羧酸酯酶1的新型BODIPY荧光探针最大吸收波长和最大发射波长的测定实验
以DMSO为溶剂配制20mM的探针母液，稀释得到浓度为2mM的探针母液，离心管中配制2.500mL PBS(100mM,pH＝7.4)、2.475mL乙腈、0.025mL探针的混合液。混合溶液中探针的浓度为10μM，并设置对照。将样品置于紫外分光光度计(UV-6100a)进行测定，以确定探针的最大吸收波长。在获得探针的最大吸收波长后，将样品放在在荧光分光光度计(F97Pro，上海棱光)中测定最大发射波长。结果如图2所示，得最大吸收波长为585nm，最大发射波长为624nm。
实施例3
检测羧酸酯酶1的新型BODIPY荧光探针对羧酸酯酶1(CES1)的选择性测试
为了测定探针对羧酸酯酶1的选择性，将探针添加到在不同的酶溶液中。配制总体积为100μL的混合溶液，包含98μL PBS缓冲溶液，1μL探针母液(2mM)和1μL CES1(1mg/mL)。将混合液在37℃孵育60min，加入100μL的乙腈来淬灭反应。探针和CES1的最终浓度分别为10μM和5μg/mL。在条件不变的情况下，用溶菌酶、胰蛋白酶、木瓜蛋白酶、胰凝乳蛋白酶、乙酰胆碱酯酶、血清蛋白酶、溶壁酶、脂肪酶、胃蛋白酶、蜗牛酶、蛋白酶K和牛血清蛋白替代CES1，得到不同的酶溶液，用酶标仪(Synergy H1)测定荧光强度。如图3所示，羧酸脂酶1溶液的荧光强度低于其他酶溶液的荧光强度，探针中的酯键会被CES1催化水解并转化为羟基，导致荧光强度降低。这些结果表明本发明中的探针可以选择性地检测CES1。
实施例4
检测羧酸酯酶1的新型BODIPY荧光探针动力学实验
依次加入100μL PBS，2μL探针(2mM)，100μL乙腈，2μL羧酸酯酶1(CES1)，得到400μL的混合溶液，并设置对照。用酶标仪测定荧光强度，进行CES1催化水解探针的动力学测试。测定结果如图4所示。随着孵育时间的增加，探针的荧光强度逐渐减小，40min后荧光强度趋于稳定。
实施例5
检测羧酸酯酶1的新型BODIPY荧光探针pH稳定性实验
为了验证探针是否能在一个较为广泛的pH范围内稳定存在，并将其应用于生理环境中，分别准备了pH为3、4、5、6、7、8、9、10的PBS缓冲溶液，在99μL不同pH值的PBS缓冲溶液中加入1μL探针(2mM),100μL乙腈，得到200μL的混合溶液，用酶标仪测定荧光强度，如图5所示，探针在pH值为3到10范围内，荧光强度几乎没有变化，表明探针的稳定性良好；同样在98μL不同pH值的PBS缓冲溶液中加入1μL探针(2mM),1μL CES1(1mg/mL)，孵育半小时后，加入100μL乙腈，用酶标仪测定荧光强度，如图5所示，在加入羧酸酯酶1后探针的荧光强度下降幅度几乎一致，表明在CES1存在下探针水解后产物在pH值为3到10范围内稳定性也十分优异。
实施例6
检测羧酸酯酶1的新型BODIPY荧光探针对羧酸酯酶1(CES1)浓度滴定实验
绘制CES1浓度滴定的标准曲线，定量检测CES1。方法为：将2μL的探针(2mM)溶于PBS中，调整CES1的量，得到200μL的混合液。将混合液在37℃下孵育60min，加入200μL乙腈使反应失活，得到总体积为400μL，CES1浓度分别为0、1、2、3、4、5、6、7、8、9、10、15μg/mL的混合溶液。用酶标仪测定荧光强度。结果如图6所示，探针在624nm处的荧光强度随着CES1浓度的逐渐增加呈下降趋势。此外，探针的荧光强度与酶浓度在0～5μg/mL之间具有良好的线性关系。结果表明，本发明探针可以定量地测定CES1。
实施例7
检测羧酸酯酶1的新型BODIPY荧光探针对农药残留的检测
将2μL的探针(2mM)溶于198μL的PBS中，CES1的量为2μL，调整毒死蜱的量，得到毒死蜱浓度为0，1，2，3，4，5，6，7，8μM的400μL的混合溶液，孵育30min后用酶标仪测定荧光强度。测定结果如图7所示，探针在624nm处的荧光强度随着毒死蜱浓度的逐渐增加呈线性上升趋势，结果表明，本发明探针可以检测毒死蜱农药残留。
实施例8
检测羧酸酯酶1的新型BODIPY荧光探针与ZIF-8载体的结合。
将2-甲基咪唑(0.657g)在圆底烧瓶中5mL去离子水中溶解，加入CBZ-BOD(0.015g)超声溶解。用5ml去离子水配制新鲜醋酸锌二水合物(0.088g)溶液，加入混合液中。最后的混合溶液静置12h后，离心6min(10000rpm)。收集沉淀，用20mL乙醇洗涤，再次离心。经多次洗涤，离心后上清为无色，沉淀产物经真空干燥即得。采用扫描电子显微镜(SEM)进行表征，结果如图8所示，呈现出了典型的ZIF-8形貌。
Claims (8)
1.用于检测羧酸酯酶1的BODIPY荧光探针，其特征在于：以氟硼二吡咯作为发光母核，3号位上是N-乙基咔唑-3-甲醛基，8号位上是苯甲酸甲酯基，结构式如下：
2.权利要求1所述的用于检测羧酸酯酶1的BODIPY荧光探针的制备方法，其特征在于，包括如下步骤：
在化合物BOD-OH中加入二氯甲烷，化合物BOD-OH溶解后加入三乙胺，然后逐滴加入苯甲酰氯，反应结束后旋蒸去除有机溶剂，然后纯化产物，得到用于检测羧酸酯酶1的新型BODIPY荧光探针CBZ-BOD；该制备方法的反应路线如下所示：
3.根据权利要求2所述的用于检测羧酸酯酶1的BODIPY荧光探针的制备方法，其特征在于，所述的反应结束后旋蒸去除有机溶剂具体为：用柱层析法进一步纯化化合物，得到检测羧酸酯酶1的新型BODIPY荧光探针CBZ-BOD。
4.权利要求1所述的用于检测羧酸酯酶1的BODIPY荧光探针的制备方法，其特征在于，在通N2的条件下，在化合物1中加入甲苯溶解后加入N-乙基咔唑-3-甲醛，然后加入对甲苯磺酸和哌啶；回流，反应结束后旋蒸去除有机溶剂，然后用柱层析法纯化产物，得到化合物BOD-OH；其反应路线如下所示：
5.根据权利要求4所述的用于检测羧酸酯酶1的BODIPY荧光探针的制备方法，其特征在于，所述的反应结束后旋蒸去除有机溶剂具体为：用柱层析法进一步纯化化合物，得到化合物BOD-OH。
6.权利要求1所述的用于检测羧酸酯酶1的BODIPY荧光探针在与MOF材料结合的应用。
7.权利要求1所述的用于检测羧酸酯酶1的BODIPY荧光探针在检测羧酸脂酶1中的以非疾病诊断和治疗为目的的应用。
8.权利要求1所述的用于检测羧酸酯酶1的BODIPY荧光探针在检测农药残留中的应用。
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