CN107314992B - 一种fret超分子荧光传感体系及其在检测cd44蛋白中的应用 - Google Patents
一种fret超分子荧光传感体系及其在检测cd44蛋白中的应用 Download PDFInfo
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Abstract
本发明公开了一种FRET超分子荧光传感体系及其在检测CD44蛋白中的应用，该体系是将两亲性Eu(III)配合物和香豆素衍生物在水相中共组装形成结构稳定且平均粒径约为130nm的球形聚集体，然后基于荧光素标记的透明质酸与Eu(III)配合物的静电相互作用，构建的荧光共振能量转移传感平台。其中香豆素衍生物与荧光素两者间可进行有效的能量转移，荧光素的荧光被大幅敏化。向该传感体系中加入CD44蛋白后，上述荧光共振能量转移过程被阻断，荧光素的荧光猝灭同时香豆素衍生物的荧光恢复，据此实现了对CD44的选择性识别。本发明FRET超分子荧光传感体系制备方法简单，检测信号丰富，可用于CD44蛋白的检测。
Description
技术领域
本发明属于超分子传感材料技术领域，具体涉及一种FRET超分子荧光传感体系，以及该传感体系在检测CD44蛋白中的应用。
背景技术
CD44蛋白是一种广泛分布在不同类型细胞和组织中的跨膜糖蛋白，根据其mRNA的大小以及蛋白质的分子量，通常可将CD44蛋白大致分为两种形式：第一类在造血细胞中表达，其分子量为80～90KDa；第二类在上皮细胞中表达，分子量为130～160KDa。CD44蛋白与受体分子结合可对多种生理过程进行调节，其中在淋巴细胞归巢和激活、伤口愈合、细胞迁移等过程中发挥着重要的作用。研究发现，CD44蛋白与肿瘤的发生、恶化、转移、抗药有着密切联系，其在胰腺癌、乳腺癌、血液恶性肿瘤以及肝癌等肿瘤细胞中有着过度表达。例如，正常人体血清中CD44蛋白的含量约为2.7±1.1nM，而结肠癌患者血清中CD44蛋白的含量高达30.8±11nM。因而，在癌症的诊断和治疗中，CD44蛋白已被作为一种癌症的标识物。
众所周知，癌症已成为威胁人类生命健康的重大疾病之一。因此，做好肿瘤的早期诊断和治疗对降低癌症的死亡率有着至关重要的作用。目前，癌症的临床诊断主要依赖于成像技术和疑似患病组织的细胞形态学分析。前者包括：X射线、计算机断层扫描成像、内窥镜检查、磁共振成像以及超声等，由于这些技术成本高，操作繁琐复杂以及分析筛选过程耗时等原因难以被应用于癌症早期检测，并且低的灵敏度也限制了其区分良性和恶性肿瘤的能力。虽然细胞形态学分析可用于区分正常和病变的细胞或组织，但难以实现对癌症早期阶段的检测。因此，研发用于癌症早期检测的技术和仪器是非常必要的，同时也具有一定的难度。
发明内容
本发明所要解决的技术问题在于提供一种制备方法简单、生物相容性好的FRET超分子荧光传感体系，并为该传感体系提供一种新的应用。
解决上述技术问题所采用的FRET超分子荧光传感体系是：将香豆素衍生物与两亲性Eu(III)配合物溶于10mmol/L pH值为8.0的三(羟甲基)氨基甲烷缓冲溶液中，在50～60℃下保温1～3小时，自然冷却至常温，常温静置4～8小时，然后加入荧光素标记的透明质酸，即形成FRET超分子荧光传感体系。
上述香豆素衍生物的结构式为：
其制备方法为：将7-羟基-3-羧基香豆素、N-羟基琥珀酰亚胺和1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐溶于二甲基甲酰胺，在N2保护下冰浴搅拌30min后，加入十六胺，室温反应24h。反应结束后，将反应液滴加到去离子水中有沉淀析出，经抽滤后所得滤饼用甲醇洗涤，真空干燥得黄褐色固体即香豆素衍生物，其中7-羟基-3-羧基香豆素、N-羟基琥珀酰亚胺、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、十六胺的摩尔比为1:1.5:1.5:1。
上述两亲性Eu(III)配合物的结构式为：
其根据公布号为CN 105277523 A中公开的方法制备而成。
上述荧光素标记的透明质酸的结构式为：
其中透明质酸的数均分子量为4000～10000，荧光素的标记度为0.5％～1％。
上述FRET超分子荧光传感体系中，香豆素衍生物的浓度为2～12μmol/L、两亲性Eu(III)配合物的浓度为100μmol/L、荧光素标记的透明质酸的浓度为2～6mg/L，优选香豆素衍生物的浓度为6μmol/L、两亲性Eu(III)配合物的浓度为100μmol/L、荧光素标记的透明质酸的浓度为4.5mg/L。
本发明FRET超分子荧光传感体系在检测CD44蛋白中的用途，具体检测方法为：
1、向FRET超分子荧光传感体系中加入CD44蛋白标准样品，利用荧光光谱仪测量不同浓度CD44蛋白对应体系的荧光发射光谱，并绘制I448/I524值随CD44蛋白浓度变化的标准曲线。
2、按照步骤1的方法测量待测样品的荧光强度，结合标准曲线的线性方程即可高灵敏度识别CD44蛋白并确定待测样品中CD44蛋白的浓度。
本发明将两亲性Eu(III)配合物和香豆素衍生物在水相中共组装形成结构稳定且平均粒径约为130nm的球形聚集体，基于荧光素标记的透明质酸与Eu(III)配合物的静电相互作用，构建了荧光共振能量转移传感平台，即FRET超分子荧光传感体系。起初该传感体系中香豆素衍生物与荧光素两者间可进行有效的能量转移，荧光素的荧光被大幅敏化。向体系中加入可与荧光素标记的透明质酸特异性结合的CD44蛋白后，上述荧光共振能量转移过程被阻断，荧光素的荧光猝灭同时香豆素衍生物的荧光恢复，据此实现了对CD44蛋白的高选择性、高灵敏度识别。与现有技术相比，本发明具有的有益效果如下：
1、本发明基于荧光共振能量转移(FRET)原理和超分子动态组成化学概念设计构建了一种新型的FRET超分子荧光传感体系，该传感体系制备过程简单，检出信号丰富、设计思路新颖。
2、本发明构建FRET超分子荧光传感体系所使用的小分子化合物结构简单、易于合成；荧光素修饰的透明质酸提高了该传感体系的生物相容性；同时具有多个受体分子的自组装体可大幅提高传感体系的选择性和灵敏度。
3、本发明利用CD44蛋白与透明质酸的特异性作用，实现了对传感体系荧光共振能量转移过程的调控，致使传感体系的光物理信号发生改变，最终实现对CD44蛋白的简单、快速、高选择性、高灵敏度检测。
4、本发明可通过多重荧光信号有效的削弱环境pH、光源扰动、探针自身浓度以及背景荧光等外部因素对测试结果的干扰，实现了对分析物更精准的检测。
附图说明
图1是EuL3+/CA二元体系的透射电子显微镜图。
图2是EuL3+/CA二元体系的粒径分布图。
图3是EuL3+/CA二元体系中添加荧光素标记的透明质酸前后的荧光光谱图。
图4是FRET超分子荧光传感体系随CD44蛋白浓度变化的荧光光谱图。
图5是FRET超分子荧光传感体系的相对荧光强度随CD44蛋白浓度变化的线性曲线图。
图6是FRET超分子荧光传感体系在不同氨基酸和蛋白质体系中的相对荧光强度对比图。
具体实施方式
下面结合附图和实施例对本发明进一步详细说明，但本发明的保护范围不仅限于这些实施例。
实施例1
将香豆素衍生物(CA)、两亲性Eu(III)配合物(EuL3+)溶于3mL 10mmol/LpH＝8.0的Tris缓冲溶液中，在60℃下保温2小时，自然冷却至常温，常温静置5小时，EuL3+和CA在缓冲溶液中共组装形成了EuL3+/CA二元体系。分别采用动态光散射仪、透射电子显微电镜对形成的EuL3+/CA二元体系进行表征，结果见图1、图2。由图可见，在EuL3+/CA二元体系中，EuL3+和CA共组装形成直径约为130nm的球形聚集体。然后向EuL3+/CA二元体系中加入荧光素标记的透明质酸(HA-FA，其中透明质酸的数均分子量为8600，荧光素的标记度为0.64％)，即形成FRET超分子荧光传感体系(EuL3+/CA+HA-FA)。所得FRET超分子荧光传感体系中CA的浓度为6μmol/L、EuL3+的浓度为100μmol/L、HA-FA的浓度为4.5mg/L。
利用荧光光谱仪对EuL3+/CA二元体系中加入HA-FA前后的荧光性能进行测试，结果见图3。由图可知，EuL3+/CA二元体系中未加入HA-FA时，能量给体CA在448nm处的荧光强度为5.09×105；当向体系中加入HA-FA后，CA和荧光素(FA)两者间发生荧光共振能量转移，致使CA的荧光强度被猝灭42％，FA的特征荧光被敏化了113倍。对照实验结果表明，向仅含有EuL3+的Tris缓冲溶液中加入HA-FA后(EuL3++HA-FA)，在420nm到610nm波长范围内几乎观察不到FA的特征荧光。
实施例2
实施例1的FRET超分子荧光传感体系在检测CD44蛋白中的用途，具体检测方法如下：
1、向FRET超分子荧光传感体系中加入CD44蛋白标准样品，使体系中CD44蛋白的浓度分别为0、3.2×10-6、6.4×10-6、9.6×10-6、1.28×10-5、1.96×10-5g/mL，采用荧光光谱仪在最大激发波长为405nm、发射波长为450nm测量不同浓度CD44蛋白对应体系的荧光发射光谱(见图4)，并绘制I448/I524值随CD44蛋白浓度变化的标准曲线，见图5。
由图4可见，随体系中CD44蛋白浓度的增大，FA在524nm处的荧光强度(I524)逐渐减弱，同时CA在448nm处的荧光强度(I448)逐渐增强，该结果说明FRET超分子荧光传感体系对CD44蛋白的检测灵敏度很高。由图5可见，I448/I524值与CD44蛋白浓度呈线性关系，线性方程为：
y＝0.00519x+0.410
式中y为I448/I524值，x为CD44蛋白浓度，相关系数R为0.997，由相关系数可见，I448/I524值与CD44蛋白浓度的线性关系很好。
2、按照步骤1的方法用荧光光谱仪测量待测样品的荧光强度，根据待测样品I448/I524的值，结合标准曲线的线性方程即可高灵敏度识别CD44蛋白并确定待测样品中CD44蛋白的浓度。
为了证明本发明的有益效果，发明人采用实施例2的方法分别测量浓度为1.96×10-5g/mL的精氨酸、赖氨酸、谷氨酸、天冬氨酸、丙氨酸、丝氨酸、单磷酸腺苷、细胞色素C、溶菌酶、胰蛋白酶对应体系在最大激发波长为405nm、发射波长为450nm处的荧光发射光谱，并计算不同体系的相对荧光强度(相对荧光强度＝(F0-F)/F，其中F＝I524/I448)，结果见图6。由图6可见，在这些氨基酸以及蛋白质的体系中，CD44蛋白与透明质酸的特殊作用能够使传感体系的光物理行为发生明显的变化，而其他氨基酸及蛋白质对体系的光物理行为影响不大，说明本发明FRET超分子荧光传感体系能够实现对CD44蛋白的高选择性识别。
Claims (2)
1.一种FRET超分子荧光传感体系，其特征在于：将香豆素衍生物与两亲性Eu(III)配合物溶于10mmol/L pH值为8.0的三(羟甲基)氨基甲烷缓冲溶液中，在50～60℃下保温1～3小时，自然冷却至常温，常温静置4～8小时，然后加入荧光素标记的透明质酸，即形成FRET超分子荧光传感体系；所述FRET超分子荧光传感体系中，香豆素衍生物的浓度为6μmol/L、两亲性Eu(III)配合物的浓度为100μmol/L、荧光素标记的透明质酸的浓度为4.5mg/L；
上述香豆素衍生物的结构式为：
上述两亲性Eu(III)配合物的结构式为：
上述荧光素标记的透明质酸的结构式为：
其中透明质酸的数均分子量为4000～10000，荧光素的标记度为0.5％～1％。
2.一种权利要求1所述的FRET超分子荧光传感体系在检测CD44蛋白中的用途，具体方法如下：
(1)向FRET超分子荧光传感体系中加入CD44蛋白标准样品，利用荧光光谱仪测量不同浓度CD44蛋白对应体系的荧光发射光谱，并绘制I448/I524值随CD44蛋白浓度变化的标准曲线；
(2)按照步骤(1)的方法测量待测样品的荧光强度，结合标准曲线的线性方程即可高灵敏度识别CD44蛋白并确定待测样品中CD44蛋白的浓度。
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