CN116064032B - 一种高稳定绿色发光的纳米金簇材料及其在免疫层析检测黄曲霉素中的应用 - Google Patents
一种高稳定绿色发光的纳米金簇材料及其在免疫层析检测黄曲霉素中的应用 Download PDFInfo
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Abstract
本发明公开了一种高稳定绿色发光的纳米金簇材料及其在免疫层析检测黄曲霉素中的应用，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绿色发光的纳米金簇材料（Au8NC）使其表面羧基功能化，通过酰胺反应偶联黄曲霉素AFB1抗体达到标记抗体目的，用以免疫层析黄曲霉素。将其作为检测信号探针，制备成黄曲霉毒素B1免疫荧光检测试纸条，灵敏度高、特异性强、检测速度快，可实现黄曲霉毒素B1的现场快速检测。
Description
技术领域
本发明属于纳米材料化学和生物免疫检测的交叉领域，具体涉及一种检测黄曲霉毒素AFB1的检测探针及黄曲霉毒素的检测方法，尤其涉及一种高稳定绿色发光的纳米金簇材料及其在免疫层析检测黄曲霉素中的应用。
背景技术
黄曲霉毒素早在1993年即被世界卫生组织(WHO)的癌症研究机构划定为1类致癌物，其毒性和致癌力极高，比砒霜、氰化钾、三聚氰胺的毒性分别高出68倍、10倍、416倍，其致癌力分别是二甲基亚硝胺、奶油黄的75倍、900倍，诱发肝癌能力是苯并芘的4000倍，除了重金属、有害元素、农药残留以外，黄曲霉素(AFB1)污染已经成为食品安全的主要威胁。由于其具有痕量、剧毒、种类繁多、污染情形复杂等特点,造成人为控制的难度系数增加,同时也给实际检测带来很大难度,所以建立黄曲霉毒素高效、快速的检测方法对于其污染状况的研究及有效降低其危害具有重要意义。
当前报道的检测黄曲霉素的信号标记物大多为胶体金，发光量子点和荧光染料，然而胶体金标记在灵敏度和稳定性等方面不能满足一定分析要求，荧光染料易光漂白，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等优点，可有效消除背景荧光，提高分析的灵敏度和准确度。目前将金属团簇材料用于检测黄曲霉素的信号标记物未见相关报道。
发明内容
本发明的目的在于克服现有技术中信号标记物缺陷和不足,提供一种金属团簇材料，作为检测探针用于检测黄曲霉毒素。
为达到上述发明目的，本发明所述纳米金簇材料通过如下方法制备而成：
(1)将左炔诺孕酮室温搅拌溶解于二氯甲烷(DCM)和乙腈(CH3CN)的混合溶液中，加入三乙胺，室温搅拌反应；然后加入Me2SAuCl，继续搅拌，反应结束后将反应液置于室温避光缓慢挥发，析出晶体，经过滤，洗涤，室温晾干得到高稳定绿色发光的纳米金簇(Au8NC)。
(2)将聚(苯乙烯-co-顺丁烯二酸酐)(PSMA)和Au8NC分别溶于DCM，混合均匀，滴加至十二烷基磺酸钠(SDS)的水溶液中超声混匀，过夜搅拌离心收集得到PSMA包覆的金簇，然后在浓氨水的作用下水解，得到纳米金簇材料，简写为：PSMA-Au8NC-COOH。
所述步骤(2):PSMA和Au8NC质量比优选3:1，得到尺寸在40nm的均匀包覆的纳米金簇球。
利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化纳米金簇材料的-COOH,与小鼠单克隆抗体1偶联，PSMA-Au8NC-COOH与黄曲霉素单克隆抗体通过酰胺反应偶联得到免疫层析检测黄曲霉素探针，简写为：PSMA-Au8NC-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。
活化以及偶联最佳缓冲体系分别为pH 6.0的MES缓冲溶液和pH 9.6的CBS缓冲溶液。
所述PSMA包覆的金簇材料具有良好的分散性(图2和3所示)。在PSMA包覆荧光金簇并水解前后荧光发射中心没有改变(图4所示)。该荧光试纸条检测线上的荧光强度与AFB1浓度具有良好的线性关系，检测限低至0.3ng/mL(图5所示)。
本发明创新点及有益效果：采用的荧光标记示踪剂是利用高分子材料PSMA包裹一种高稳定绿色发光的纳米金簇，由此形成具有一定粒径的分散均匀的纳米级荧光微球。通过对微球表面进行化学修饰裸露大量羧基，可与抗体IgG分子上的氨基在交联剂EDC桥接下进行共价偶联，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，既保留了荧光簇原有的光谱特征，又在聚苯乙烯外壳保护作用下维持了荧光分子的稳定性，不易受各种理化因素影响导致易致淬灭。除此之外，荧光微球的高分子外壳表面因覆盖了大量共价基团，可结合的抗体分子数量和密度相比普通荧光素显著增加，由此对荧光信号产生级联放大作用而提高检测灵敏度，检测限低至0.3ng/mL,特异性强,从附图6可以看出，不同种类的黄曲霉素，与黄曲霉毒素B1具有更高的亲和力，与其他毒素无交叉，对AFB1具有更强的特异性。将其用于免疫层析试纸条检测黄曲霉毒素B1时无需任何其他试剂，10min即可获得结果，大大提高效率，可实现黄曲霉毒素B1的现场快速检测。可应用于理想缓冲溶液体系和实际样本的检测。为荧光试纸免疫检测的荧光标记材料提供了新的思路，开辟了新的途径。
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-Au8NC-COOH的透射电镜图。
图3为本发明PSMA-Au8NC-COOH的动态光散射图。
图4为本发明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AFB1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真菌毒素的特异性选择图。
具体实施方式
下面通过实例对本发明做进一步的说明，以下百分比没有特别说明均为质量百分比：
实施例1：本发明高稳定绿色发光的纳米金簇及金簇羧基功能化的合成
(1)将配体左炔诺孕酮搅拌下溶解于二氯甲烷(DCM)和乙腈(CH3CN)的混合溶液中，加入三乙胺(Et3N),搅拌，然后加入Me2SAuCl继续室温搅拌反应，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，过滤，用乙腈洗涤，室温晾干即得到高稳定绿色发光的纳米金簇(Au8NC)。
(2)将苯乙烯-马来酸酐聚合物(PSMA)和Au8NC分别溶于DCM，混合均匀，滴加至十二烷基磺酸钠(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包覆的金簇，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-Au8NC-COOH。
实施例2：本发明荧光试纸条的制备
(1)取实施例1制得的PSMA-Au8NC-COOH利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化其-COOH,与小鼠单克隆抗体1偶联。
具体方法如下：取MES缓冲液(0.05mol/L,pH 6.0)加入荧光微球PSMA-Au8NC-COOH，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,pH 9.6)调节体系pH。加入偶联抗体1，置于摇床(250r/min),室温振摇反应2h。加入牛血清白蛋白(BSA)溶液至其终浓度为1％，置于摇床振摇封闭30min，离心弃上清，再用PBS缓冲液(0.05mol/L,pH 7.2,含0.5％ Tween-20)复溶，置于4℃保存。
(2)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。将各浓度目标抗原及抗体2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。将不同浓度的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，选择最佳抗原浓度组合用于试纸条的制备。
实施例3
实际应用检测举例：本发明试纸条可用于茶水中黄曲霉毒素B1的检测。茶叶泡水后，经磷酸盐缓冲液适当稀释后可直接加样检测，根据T线和C线的荧光强度情况判读结果。
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 (4)
1.纳米金簇材料，其特征在于，通过如下步骤制备而成：
（1）将左炔诺孕酮搅拌下溶解于二氯甲烷和乙腈的混合溶液中，加入三乙胺，搅拌，然后加入Me2SAuCl继续室温搅拌反应，反应结束后将反应液置于室温下避光缓慢挥发，析出晶体，经过滤，洗涤，室温晾干得到纳米金簇（Au8NC）；
（2）将苯乙烯-马来酸酐聚合物（PSMA）和Au8NC分别溶于二氯甲烷，混合均匀，滴加至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 包覆的金簇，然后在浓氨水的作用下水解，得到纳米金簇材料，简写为：PSMA-Au8NC-COOH。
2.如权利要求1所述的纳米金簇材料，其特征在于，步骤（2）PSMA和Au8NC质量比为3:1。
3.如权利要求1所述的纳米金簇材料的应用，其特征在于，以PSMA-Au8NC-COOH作为检测信号探针，将其用于免疫层析检测黄曲霉素。
4.如权利要求3所述的纳米金簇材料的应用，其特征在于，利用1-（3-二甲氨基丙基）-3-乙基碳二亚胺（EDC）活化PSMA-Au8NC-COOH，将活化后的 PSMA-Au8NC-COOH与黄曲霉素单克隆抗体偶联得到免疫层析检测黄曲霉素探针。
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