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)
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。
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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