CN116496789B - 一种检测白细胞介素-6的合金团簇探针 - Google Patents
一种检测白细胞介素-6的合金团簇探针 Download PDFInfo
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Abstract
本发明公开了一种检测白细胞介素‑6的合金团簇探针，属于纳米材料化学和生物化学的交叉领域。本发明以苯乙烯‑马来酸酐聚合物包覆高稳定红色发光的合金团簇材料（Pt2Cu4NC）使其表面羧基功能化，通过酰胺反应偶联白细胞介素‑6抗体达到标记抗体目的，用于免疫层析检测白细胞介素‑6。将其制成免疫荧光检测试纸条，与白细胞介素‑6具有更高的亲和力，灵敏度高；与其他细胞因子无交叉，具有更强的特异性；免疫层析试纸条检测白细胞介素‑6时无需任何其他试剂，15 min即可获得结果，大大提高效率，有望应用于白细胞介素‑6的现场快速检测平台。
Description
技术领域
本发明属于纳米材料化学和生物化学交叉领域，具体涉及一种检测白细胞介素-6的检测探针及白细胞介素-6的荧光检测方法，尤其涉及一种高稳定红色发光的合金团簇探针及其在免疫层析检测白细胞介素-6中的应用。
背景技术
白细胞介素-6(Interleukin，IL-6)是单核细胞产生的细胞因子，具有促进免疫反应、炎症反应的作用。IL-6升高是慢性阻塞性肺疾病急性发作的生物学标志，尤其与吸烟相关的COPD急性发作有关。目前针对IL-6的检测方法主要为放免法和酶免法，要求操作人员具有较高的专业素质且耗时较长，亟需开发高灵敏快速检测方法。
当前报道的检测IL-6的信号标记物大多为胶体金、发光量子点和荧光染料。胶体金标记的灵敏度较低，荧光染料易光漂白且量子产率低。与传统的荧光标记物相比，金属团簇具有荧光寿命长、发射光谱窄、斯托克斯位移大和光稳定性强、量子产率高等优点，可有效消除背景荧光，提高分析灵敏度。因此，金属团簇探针作为检测IL-6的信号标记物具有很好的开发应用价值，目前未见相关报道。
发明内容
本发明的目的在于提供一种用于检测IL-6的高灵敏快速检测金属团簇探针，克服目前信号标记物缺陷。
为实现本发明的目的，本发明以苯乙烯-马来酸酐聚合物包覆合金团簇材料(Pt2Cu4NC)使其表面羧基功能化，通过酰胺反应偶联白细胞介素-6抗体达到标记抗体目的，用于免疫层析检测白细胞介素-6。
本发明具体技术方案为：
(1)将配体左炔诺孕酮溶解于二氯甲烷(DCM)中，室温搅拌溶解澄清；然后加入三乙胺，室温继续搅拌，再加入氯铂酸的甲醇溶液，室温搅拌反应；最后加入六氟磷酸四乙腈铜的二氯甲烷溶液，室温搅拌反应，反应结束以后将反应液置于室温下避光挥发，得到Pt2Cu4NC晶体后过滤洗涤，室温晾干。
(2)将苯乙烯-马来酸酐聚合物(PSMA)和Pt2Cu4NC分别溶于DCM混合均匀，滴加入十二烷基磺酸钠(SDS)的水溶液中超声混匀，过夜搅拌离心收集得到PSMA包覆的合金簇纳米材料，然后在浓氨水的作用下水解使PSMA上羧基裸露供以偶联抗体，简写为：PSMA-Pt2Cu4NC-COOH。
(3)用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化PSMA-Pt2Cu4NC-COOH荧光纳米球上的羧基，与IL-6小鼠单克隆抗体1偶联得到免疫层析检测IL-6探针，简写为：PSMA-Pt2Cu4NC-COOH-Antibody，得到的探针材料与目标物混合孵育进行免疫层析检测。
上述步骤(2)中PSMA和Pt2Cu4NC质量比选3:1，可以得到尺寸约40nm的纳米球。
所述PSMA包覆的合金团簇纳米材料具有良好的分散性(图2-3所示)。在PSMA包覆荧光合金簇并水解前后荧光发射中心保持不变(图4所示)。该荧光试纸条检测线上的荧光强度与IL-6浓度具有良好的线性关系和特异性(图5-6所示)，检测限低至42.66pg/mL。
本发明有益效果：本发明所述检测探针基于高稳定红色发光合金团簇纳米材料，由其制成的免疫荧光检测试纸条，与白细胞介素-6具有更高的亲和力，灵敏度高；与其他细胞因子无交叉，具有更强的特异性；免疫层析试纸条检测白细胞介素-6时无需任何其他试剂，15min即可获得结果，大大提高效率，检测操作快速便捷且灵敏度高，检测限低至42.66pg/mL，可应用于缓冲溶液体系和实际样本目标物检测。有望应用于白细胞介素-6的现场快速检测平台。
本发明原理：采用的荧光标记示踪剂是利用高分子材料PSMA包裹一种高稳定红色发光合金团簇的纳米材料，具有良好的分散性和稳定性。通过对微球表面进行化学反应裸露大量羧基，可与抗体IgG分子上的氨基进行共价偶联，形成稳定的荧光标记抗体，用于特异性结合和检测目标抗原。这种方法制备的荧光微球既保留了荧光团簇原有的光谱特征，又维持了荧光团簇的稳定性，荧光信号产生的级联放大作用可提高检测灵敏度，为荧光试纸免疫检测的荧光标记材料提供新的选择。
附图说明
图1为本发明免疫层析检测示意图。
图2为本发明PSMA-Pt2Cu4NC-COOH的透射电镜图。
图3为本发明PSMA-Pt2Cu4NC-COOH的动态光散射图。
图4为本发明合金簇利用聚合物羧基功能化前后在365nm激发下的荧光发射图。
图5为本发明免疫荧光试纸条检测荧光强度与目标物IL-6浓度的标准曲线图。
图6为本发明免疫荧光试纸条检测不同细胞因子和蛋白的特异性选择图。
具体实施方式
下面通过实例对本发明做进一步的说明：
实施例1：免疫层析检测IL-6合金团簇探针的制备
(1)将配体左炔诺孕酮溶解于二氯甲烷(DCM)中，室温搅拌溶解澄清；然后加入三乙胺，室温继续搅拌，再加入氯铂酸的甲醇溶液，室温搅拌反应；最后加入六氟磷酸四乙腈铜的二氯甲烷溶液，室温搅拌2小时，反应结束以后将澄清溶液置于室温下避光挥发，得Pt2Cu4NC晶体后过滤洗涤，室温晾干。
(2)将苯乙烯-马来酸酐聚合物(PSMA)和Pt2Cu4NC分别溶于DCM混合均匀，滴加入十二烷基磺酸钠(SDS)的水溶液中超声混匀，用超声破碎仪使有机相和水相混合均匀，过夜搅拌离心收集得到PSMA包覆的合金团簇纳米材料，然后在浓氨水的作用下水解其酸酐基团成羧基供以偶联抗体，简写为：PSMA-Pt2Cu4NC-COOH。
(3)用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化PSMA-Pt2Cu4NC-COOH羧基,与IL-6小鼠单克隆抗体1偶联，得到免疫层析检测IL-6探针，简写为：PSMA-Pt2Cu4NC-COOH-Antibody。
具体方法如下：取MES缓冲液(0.05M,pH 5.0)加入PSMA-Pt2Cu4NC-COOH荧光纳米球，振荡混匀后，加入EDC溶液(2mg/mL)室温振荡活化；然后用碳酸盐缓冲液(0.05mol/L,pH9.6)调节体系pH，加入IL-6小鼠单克隆抗体1置于摇床(250r/min),室温振摇反应2h。加入牛血清白蛋白(BSA)溶液至其终质量浓度为1％，置于摇床振摇封闭2h，离心弃上清，再用PBS缓冲液(0.05mol/L,pH 7.2,含质量百分比0.5％ Tween-20)复溶，置于4℃保存。
实施例2：本发明荧光试纸条的制备
将目标抗原用磷酸盐缓冲液(0.01mol/L,pH 7.5)溶解，配制成不同浓度的溶液。将IL-6小鼠单克隆抗体2和羊抗鼠二抗分别喷涂于NC膜的检测线(T线)和控制线(C线)，喷样量1μg/cm，室温干燥。将NC膜和结合垫交叉组合，分别按样品垫、NC膜、检测垫和吸水垫的组装顺序贴在底板上，相邻两膜间重叠0.2cm。将试纸条裁剪成宽4mm的规格，根据不同浓度目标物检测时的T线和C线的荧光强度情况，实现白细胞介素-6的定性检测。
实施例3：荧光试纸条实际应用检测
将待检测样品与探针在96孔板微孔中混匀反应10分钟，插入制备好的试纸条，3分钟后在紫外灯下判读检测结果。T线荧光强度与样品中所含白细胞介素-6量成正比，检测限低至42.66pg/mL。
以上实施例仅用于说明本发明的内容，除此之外，本发明还有其它实施方式。但是，凡采用等同替换或等效变形方式形成的技术方案均落在本发明的保护范围内。
Claims (3)
1.一种检测白细胞介素-6的合金团簇纳米材料，其特征在于，通过如下步骤制备而成：
（1）将配体左炔诺孕酮溶解于二氯甲烷溶液中，加入三乙胺，搅拌，加入H2PtCl6的甲醇溶液搅匀，然后加入六氟磷酸四乙腈铜的二氯甲烷溶液，持续搅拌，反应结束以后将反应液置于室温下避光缓慢挥发，析出晶体Pt2Cu4NC，过滤，室温晾干；
（2）将苯乙烯-马来酸酐聚合物（PSMA）和Pt2Cu4NC分别溶于二氯甲烷混合均匀，滴加入十二烷基磺酸钠的水溶液中超声混匀，过夜搅拌离心收集得到 PSMA 包覆的合金簇纳米材料，然后在浓氨水的作用下水解，得到的产物简写为：PSMA-Pt2Cu4NC-COOH；
（3）用1-(3-二甲氨基丙基)-3-乙基碳二亚胺活化PSMA-Pt2Cu4NC-COOH荧光纳米球上的羧基，与IL-6小鼠单克隆抗体偶联得到检测白细胞介素-6的合金团簇纳米材料。
2.如权利要求1所述的检测白细胞介素-6的合金团簇纳米材料，其特征在于，步骤（2）中PSMA和Pt2Cu4NC质量比为3:1。
3.如权利要求1或2所述的检测白细胞介素-6的合金团簇纳米材料的应用，其特征在于，将其制成荧光免疫层析试纸条。
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