CN113502157A - 一种组合型荧光纳米探针及其制备方法 - Google Patents
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Abstract
本发明涉及一种组合型荧光探针，所述荧光探针包含Cy7染料以及IR786S染料。针对现有的荧光纳米探针特异性和灵敏性不高，检测的细菌浓度有限，以及无法实现广谱细菌检测的问题，本申请的荧光探针在检测广谱细菌时具有高特异性和高灵敏性。
Description
技术领域
本发明荧光探针技术领域，具体涉及一种针对细菌感染诊断的组合型荧光探针。
背景技术
病原微生物诱导的ROS自由基在感染介导的疾病发生和生物体内抗性中起着重要作用。活性氧自由基(O2-,·OH,H2O2,ClO-等)来源于细胞内NADPH氧化酶和线粒体呼吸链，广泛参与感染相关的炎症反应。因此，活性氧是鉴别感染的重要指标之一。
目前研究细菌感染和检测活性自由基异常变化的常规方法主要依赖于组织学或生化等终点分析方法。这些方法在实时监测自由基动态变化与感染病理演变的关系方面存在局限性，不足以准确诊断感染性疾病，以及为早期治疗和干预提供依据。因此，有必要开发一种更简单有效的自由基实时检测方法。
近年来，荧光成像在体内外检测自由基方面显示出良好的特异性和敏感性，并能有效地解释自由基应激与细菌感染的相关机制。但目前很少有特异性次氯酸根应答的荧光探针分子，用于感染组织的诊断。
发明内容
为了解决上述现有技术存在的问题，本发明的目的在于针对现有的荧光纳米探针特异性和灵敏性不高，检测的细菌浓度有限，以及无法实现广谱细菌检测的问题，提供一种兼具广谱细菌检测荧光纳米探针的制备方法，制得的荧光纳米探针在检测广谱细菌时具有高特异性和高灵敏性。
本申请第一方面提供一种组合型荧光探针，其特征在于，所述荧光探针包含Cy7染料以及IR786S染料。
优选的，所述荧光探针中Cy7染料与IR786S染料的摩尔比为1:(0.1-10)。
进一步优选的，所述荧光探针中Cy7染料与IR786S染料的摩尔比为1:1。
进一步优选的，所述Cy7染料和IR786S染料吸附在表面含有多巴胺或者其衍生物的纳米材料表面。
进一步优选的，所述Cy7染料和IR786S染料吸附在包裹有聚多巴胺的纳米材料表面。
本申请第二方面提供上述组合型荧光探针的制备方法，其特征在于，将表面含有多巴胺或其衍生物的纳米材料，与含Cy7的溶液共混吸附若干小时，离心之后，再与含IR786S的溶液共混若干小时后，离心冷冻干燥。
优选的，含Cy7的溶液和含IR786S的溶液的浓度均为1mg/ml。
所述的纳米材料为本领域常见纳米材料，例如可为金属有机框架纳米材料或者介孔二氧化硅纳米材料。
本申请第三方面提供上述组合型荧光探针的用途，其特征在于，所述荧光探针用于制备检测次氯酸根离子的试剂。
本申请第四方面提供上述组合型荧光探针的用途，其特征在于，所述荧光用于制备检测细菌感染的试剂。
本发明的有益效果在于：针对现有的荧光纳米探针特异性和灵敏性不高，检测的细菌浓度有限，以及无法实现广谱细菌检测的问题，提供一种兼具广谱细菌检测荧光纳米探针的制备方法，制得的荧光纳米探针在检测广谱细菌时具有高特异性和高灵敏性。
附图说明
图1是Cy7和IR786s的荧光曲线，图1中上方为Cy7的荧光曲线，下方为IR786s的荧光曲线；
图2是IR786s的1H-NMR核磁谱图；
图3是荧光探针ClO-特异性应答测试结果；
图4中(A)是感染后细胞模型的测试结果；图4中(B)是细胞感染后荧光探针检测的机理图；
图5是感染老鼠模型的诊断结果。
具体实施方式
下面结合具体实例，进一步阐述本发明，需理解这些实例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1荧光探针的制备
(1)纳米材料的制备
5ml 2-甲基咪唑(50mM)甲醇溶液和5mL含Zn(NO3)2·6H2O(50mM)的甲醇混合在一起,然后让混合物在40℃下反应24小时,通过离心收集ZIF-8金属有机框架材料，并用水冲洗三次以去除未反应的离子和2-甲基咪唑。
上述反应得到的ZIF-8在含50mM盐酸多巴胺的pH＝8.5TRIS盐酸溶液中，搅拌半小时，得到聚多巴胺包裹的ZIF-8@PDA，离心并用乙醇洗涤三次做进一步使用。
(2)荧光探针的制备
将上述制备得到的纳米材料50mg，与1mg/ml的Cy7(CAS号943298-08-6，分子式C35H41KN2O8S2)在乙醇中共混吸附12小时，离心之后，再与1mg/ml IR786S在乙醇中共混12个小时后，离心冷冻干燥。
实施例2荧光探针ClO-特异性应答测试
将实施例1制备的纳米荧光材料，分别用5μM ClO-,1mM H2O2,1mM·OH,0.02μM黄嘌呤氧化酶和25μM黄嘌呤(产生超氧阴离子O2-)刺激，之后收集其荧光曲线以及在790nm的吸光度，通过与原始对照组的吸光度比值确定探针的特异性，具体结果如图3所示，从图3中可以看出(图3左侧最上方曲线为5μMClO-刺激后的荧光曲线)，纳米荧光探针针对ClO-的特异性和响应型均最好。
实施例3细菌感染细胞模型的建立及其测试结果
为了建立细菌感染的细胞模型，将人脐静脉内皮细胞(2×104)在盖玻片上培养过夜，然后加入10μL金黄色葡萄球菌溶液(1×104CFU/mL)。培养1h后，用PBS洗涤人脐静脉内皮细胞3次，用ClO-探针(GMS10135.1，GENMED，USA)检测内源性ClO-，用激光扫描共聚焦显微镜(CLSM，leicatcs-SP8)拍摄图像，具体结果如图4所示，图4中显示细菌刺激细胞后细胞ClO-表达上调，相应图像中出现大量绿色荧光部分。
实施例4感染老鼠模型建立及其诊断结果
来自上海实验动物公司的BALB/c小鼠(6周龄，雄性)，作为动物模型。在小鼠背部剃光鼠毛，罗露出皮肤并消毒，同时使用环形皮肤采样器产生直径10mm的缺损。随后，创面注射含Xen29生物发光细菌的PBS 100μL，细菌浓度为1×106cfu/ml。然后，注射纳米材料，观察是否有荧光信号产生，具体结果如图5所示，从图5中可以看出随着细菌量增多，荧光信号增强。
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
Claims (9)
1.一种组合型荧光探针，其特征在于，所述荧光探针包含Cy7染料以及IR786S染料。
2.根据权利要求1所述的一种组合型荧光探针，其特征在于，所述荧光探针中Cy7染料与IR786S染料的摩尔比为1:(0.1-10)。
3.根据权利要求1所述的一种组合型荧光探针，其特征在于，所述荧光探针中Cy7染料与IR786S染料的摩尔比为1:1。
4.根据权利要求1所述的一种组合型荧光探针，其特征在于，所述Cy7染料和IR786S染料吸附在表面含有多巴胺或者其衍生物的纳米材料表面。
5.根据权利要求4所述的一种组合型荧光探针，其特征在于，所述Cy7染料和IR786S染料吸附在包裹有聚多巴胺的纳米材料表面。
6.一种权利要求1-5任一项所述的组合型荧光探针的制备方法，其特征在于，将表面含有多巴胺或者其衍生物的纳米材料50mg，与含Cy7的溶液共混吸附若干小时，离心之后，再与含IR786S的溶液共混若干小时后，离心冷冻干燥。
7.根据权利要求6所述的荧光探针的制备方法，其特征在于，含Cy7的溶液和含IR786S的溶液的浓度均为1mg/ml。
8.权利要求1-5任一项所述组合型荧光探针的用途，其特征在于，所述荧光探针用于制备检测次氯酸根离子的试剂。
9.权利要求1-5任一项所述组合型荧光探针的用途，其特征在于，所述荧光用于制备检测细菌感染的试剂。
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