CN113004220A - 一种酯酶检测荧光探针、制备方法及应用 - Google Patents
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Abstract
Description
技术领域
本发明涉及一种荧光探针、制备方法及应用,更具体地说涉及一种酯酶检测荧光探针、制备方法及应用。
背景技术
酯酶是生物体中一类发挥着重要作用的酶,它可以催化酯水解成相应的醇和酸,具有很高的底物特异性。酯酶可以调节各种代谢功能,包括基因表达,酯代谢,物质转运和排毒。另外,酯酶也是重要的药物靶标和前药激活剂。研究结构表明,当生物体内的酯酶浓度水平不能维持在正常的生理水平范围内时,将会导致生物体的多种疾病,例如:沃尔曼病,肥胖症,动脉粥样硬化,癌症,高脂血症和肝脂肪变性。由此可见,监测并维持细胞内正常的酯酶浓度水平对很多细胞功能是至关重要的。
目前为止,已开发了多种检测酯酶的方法,包括比色法,分光光度法,平板测定法和色谱法,但是,这些方法大多操作复杂,并且无法实现有效灵敏的实时检测。而荧光探针方法的检测反应迅速,选择性高,灵敏度高,操作简便,可以实现酯酶的实时无损成像。因此,该方法在生物成像中具有广泛的应用。
近年来,一些能特异性检测酯酶的小分子荧光探针被报道,但是也出现了水溶性差、灵敏度低,不适宜细胞成像等缺陷,这些缺陷很大程度上影响了探针的应用。因此,开发出能克服这些缺陷的能够高效检测酯酶的新型荧光探针是非常必要的。
发明内容
本发明所要解决的技术问题是:克服现有技术存在的不足,提供一种酯酶检测荧光探针,该探针具有高选择性和高灵敏度。
此外,本发明还提供该荧光探针的制备方法以及该探针在酯酶检测中的实际应用。
本发明的技术构思和原理如下:该基于苯并噻唑衍生物探针可选择性与酯酶反应并产生强烈荧光,且在0-0.2U/mL酯酶浓度范围内,所产生的荧光强度与酯酶浓度有着较好的线性关系。本发明人首次制备酯酶检测荧光探针即相关苯并噻唑衍生物并首次将其用于酯酶的选择性检测,从而解决现有技术中存在的问题。
本发明解决其技术问题的技术方案如下:
本发明的酯酶检测荧光探针,其具有如式结构HBT-EA、HBT-MA-EA或HBT-Py-EA所示的苯并噻唑衍生物:
本发明上述的酯酶检测荧光探针的制备方法,其包括以下步骤:
将HBT、HBT-MA或HBT-Py分别与溴甲基乙酸酯、碳酸铯在无水N,N-二甲基甲酰胺中反应生成如式结构HBT-EA、HBT-MA-EA或HBT-Py-EA所示的苯并噻唑衍生物即为酯酶检测荧光探针。
本发明上述的酯酶检测荧光探针的制备方法,其进一步的技术方案是所述的反应在N2保护且避光条件下,20-30℃温度下反应8-12h。
本发明上述的酯酶检测荧光探针的制备方法,其再进一步的技术方案是所述的反应后进行纯化:将去离子水缓慢加入反应液中,在此过程中逐渐产生沉淀;然后收集沉淀过滤,真空干燥,得到粗产物;最后,将粗产物通过硅胶柱层析进一步纯化得到酯酶检测荧光探针。
本发明上述的探针可以在检测酯酶中进行应用,主要用于环境中酯酶含量的检测、生物样本中酯酶荧光显影、含量检测。
本发明具有以下有益效果:
1)本发明荧光探针在pH=7.4的缓冲溶液中几乎没有荧光,与酯酶反应后分别释放出强烈的绿色和红色荧光。
2)采用本发明荧光探针后,检测灵敏度高,对酯酶的检测限可达到1.22×10-4U/mL。
3)本发明荧光探针仅与酯酶发生荧光反应,对其它常见离子,氨基酸和酶均无反应,具有很好的选择性和特异性。具有适宜的荧光发射波长(分别为527nm,542nm,614nm)。
4)本发明荧光探针制备工艺简单易行,易于规模化生产。
附图说明
图1为本发明实施例2荧光探针对酯酶的选择特异性
图2为本发明实施例3荧光探针与酯酶反应的荧光增量图
图3为本发明实施例3荧光探针对酯酶浓度的荧光强度工作曲线
图4为本发明实施例3荧光探针对0-0.2U/mL酯酶的荧光强度线性关系图
图5为本发明实施例3荧光探针对细胞内源性酯酶的荧光成像图
图6为实施例1中HBT-EA纯品1H-NMR图
图7为实施例1中HBT-EA纯品高分辨质谱图
图8为实施例1中HBT-MA-EA纯品1H-NMR图
图9为实施例1中HBT-MA-EA纯品高分辨质谱图
图10为实施例1中HBT-Py-EA纯品1H-NMR图
图11为实施例1中HBT-Py-EA纯品高分辨质谱图
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。本发明的探针制备反应方程式如下:
实施例1制备酯酶检测荧光探针
将640mg HBT和610mg溴甲基乙酸酯以及860mg碳酸铯加入到无水25mL无水DMF中;在N2保护,避光,20-30℃下反应12h;反应结束后,先将去离子水缓慢加入反应液中,在此过程中逐渐产生白色沉淀。然后收集沉淀过滤,真空干燥,得到粗产物。最后,将粗产物通过硅胶柱层析进一步纯化得到白色固体750mg,即为酯酶检测探针HBT-EA纯品(1H-NMR图和高分辨质谱图见图6、图7)。所得荧光探针HBT-EA纯品实测分子量为313.08。
将1.17g HBT-MA和1.00g溴甲基乙酸酯以及1.42g碳酸铯加入到无水50mL无水DMF中;在N2保护,避光,20-30℃下反应12h;反应结束后,先将去离子水缓慢加入反应液中,在此过程中逐渐产生白色沉淀。然后收集沉淀过滤,真空干燥,得到粗产物。最后,将粗产物通过硅胶柱层析进一步纯化得到白色固体880mg,即为酯酶检测探针HBT-MA-EA纯品(1H-NMR图和高分辨质谱图见图8、图9)。所得荧光探针HBT-MA-EA纯品实测分子量为341.07。
将1.17g HBT-Py和1.00g溴甲基乙酸酯以及1.42g碳酸铯加入到无水50mL无水DMF中;在N2保护,避光,20-30℃下反应12h;反应结束后,将反应液用H2O(20mL)和CH2Cl2(20mL)萃取3次,取有机相溶液进行减压浓缩,得到黄色固体粗产物。最后,粗产物通过硅胶柱层析进一步纯化得到黄色固体750mg,即为酯酶检测探针HBT-Py-EA纯品(1H-NMR图和高分辨质谱图见图10、图11)。所得荧光探针HBT-Py-EA纯品实测分子量为558.04。
实施例2酯酶检测荧光探针与各种离子、氨基酸、酶反应的光谱性质
分别称取3.1mg,3.4mg,5.6mg实施例1制得酯酶检测荧光探针,配成浓度为1mM的10mL DMSO溶液,作为母液。
荧光光谱测试:将30μL上述母液加入到一定量的10mM PBS缓冲溶液(pH 7.4)中,然后分别加入各种离子:K+,Na+,CO3 2-,SO3 2-,Cl-,ClO-,S2-,氨基酸和肽:Cys,Hcy,GSH,酶:人红细胞中的碳酸酐酶Ⅰ,过氧化物酶,牛乳中的黄嘌呤氧化酶,无机焦磷酸化酶,乙酰胆碱酯酶,使分析物终浓度为10μM或20U/mL,荧光探针终浓度为10μM。分别在350nm(HBT-EA),375nm(HBT-MA-EA),400nm(HBT-Py-EA)激发光波长下即时测试其荧光发射光谱。激发与发射的狭缝宽度分别为3/5nm(HBT-EA),5/5nm(HBT-MA-EA),10/10nm(HBT-Py-EA)。所得荧光图如图1所示。
以上结果表明:
(1)实施例1制得荧光探针本身在溶液呈无色且几乎没有荧光,但随着酯酶的加入,探针HBT-EA在527nm处绿色荧光逐渐增强,探针HBT-MA-EA在542nm处绿色荧光逐渐增强,探针HBT-Py-EA在614nm处红色荧光逐渐增强。
(2)实施例1制得荧光探针对酯酶具有高度的选择性和特异性,并且在上述条件下,能够从K+,Na+,CO3 2-,SO3 2-,Cl-,ClO-,S2-等常见离子,Cys,Hcy,GSH等常见氨基酸和肽以及人红细胞中的碳酸酐酶Ⅰ,过氧化物酶,牛乳中的黄嘌呤氧化酶,无机焦磷酸化酶,乙酰胆碱酯酶等常见酶中区分出酯酶。
实施例3酯酶检测荧光探针与酯酶反应产物的光谱性质
将30μL实施例2中的母液加入到一定量的10mM PBS缓冲溶液(pH 7.4)中,然后加入不同当量的酯酶,使荧光探针的终浓度为10μM,酯酶终浓度分别为0U/mL、0.02U/mL、0.04U/mL、0.06U/mL、0.08U/mL、0.10U/mL、0.12U/mL、0.14U/mL、0.16U/mL、0.18U/mL、0.20U/mL。酯酶加入后,室温孵育20min,测量其荧光发射光谱。荧光发射光谱测定时分别以350nm(HBT-EA),375nm(HBT-MA-EA),400nm(HBT-Py-EA)激发波长;激发与发射的狭缝宽度分别为3/5nm(HBT-EA),5/5nm(HBT-MA-EA),10/10nm(HBT-Py-EA)。所得荧光强度增量图见图2;分别以527nm(HBT-EA),542nm(HBT-MA-EA),614nm(HBT-Py-EA)的数据制作工作曲线,结果见图3。
该实验结果表明,反应后荧光强度随次酯酶浓度的增加而增加;反应后荧光强度与0-0.20U/mL范围内的酯酶浓度呈较好的线性关系,可以用于酯酶含量的定量分析检测,527nm(HBT-EA)处荧光强度与0-0.16U/mL范围内的酯酶浓度线性关系曲线;542nm(HBT-MA-EA)处荧光强度与0-0.20U/mL范围内的酯酶浓度线性关系曲线;614nm(HBT-Py-EA)以与0-0.20U/mL范围内的酯酶浓度线性关系曲线见图4。
实施例4酯酶检测荧光探针对细胞内源性酯酶的荧光成像。
取探针母液20μL加入到1mL的培养基中,此时探针的浓度为20μM,37℃条件下培养HeLa细胞3h;然后用荧光倒置显微镜对HeLa细胞进行细胞成像实验。加入之后浓度为20μM的双(4-硝基苯基)磷酸酯(一种酯酶抑制剂),37℃条件下培养HeLa细胞1h;接着加入相同剂量荧光探针,培养3h;然后用荧光倒置显微镜对HeLa细胞进行细胞成像实验。结果如图5所示,图(a-c)表示探针在明场下HeLa细胞的形态;图(d-f)表示探针在HeLa细胞荧光成像图;图(g-i)表示探针和抑制剂在HeLa细胞中的明场图;图(j-l)表示探针和抑制剂在HeLa细胞中的荧光成像图;证明探针对细胞内源性酯酶具有良好的成像能力。
Claims (5)
2.一种如权利要求1所述的酯酶检测荧光探针的制备方法,其特征在于包括以下步骤:
将HBT、HBT-MA或HBT-Py分别与溴甲基乙酸酯、碳酸铯在无水N,N-二甲基甲酰胺中反应生成如式结构HBT-EA、HBT-MA-EA或HBT-Py-EA所示的苯并噻唑衍生物即为酯酶检测荧光探针。
3.根据权利要求2所述的酯酶检测荧光探针的制备方法,其特征在于,所述的反应在N2保护且避光条件下,20-30℃温度下反应8-12h。
4.根据权利要求3所述的酯酶检测荧光探针的制备方法,其特征在于,所述的反应后进行纯化:将去离子水缓慢加入反应液中,在此过程中逐渐产生沉淀;然后收集沉淀过滤,真空干燥,得到粗产物;最后,将粗产物通过硅胶柱层析进一步纯化得到酯酶检测荧光探针。
5.一种如权利要求1所述的探针在检测酯酶中的应用。
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