CN109283330A - 一种基于拉曼光谱法的快速检测癌细胞的方法 - Google Patents
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Abstract
本发明属于生物技术领域,公开了一种基于拉曼光谱法的快速检测癌细胞的方法,包括如下步骤:步骤1:在磁性微球表面标记能够识别癌细胞的抗体或核酸适配体;步骤2:在步骤1得到的磁性微球表面通过抗体或核酸适配体富集癌细胞;步骤3:将能够特异性识别癌细胞的荧光纳米金探针与步骤2得到的磁性微球的靶标物结合;步骤4:将磁性微球表面的带有抗体或核酸适配体‑靶标物‑荧光纳米金探针的三明治结构分离;步骤5:通过拉曼光谱法检测荧光纳米金探针的表面形貌和吸收光谱得到癌细胞浓度的检测结果。该方法可实现痕量检测,检测精度高。
Description
技术领域
本发明涉及生物技术领域,特别是一种基于拉曼光谱法的快速检测癌细胞的方法。
背景技术
恶性肿瘤是一种以细胞异常增生为基本特征的人类疾病。伴随着肿瘤的发生发展,其大分子物质(包括核酸、蛋白和多糖等)可以由癌细胞释放进入血液循环,此类游离(Circulating)大分子作为生物标志物(Biomarkers)在肿瘤诊断、预后评估及病情随访中均具有广泛的临床价值。
但是现有的基于磁珠抗体富集技术的检测方法普遍存在检测精度低的问题,无法实现痕量检测。
发明内容
为了解决上述的缺点和不足,本发明提供了一种检测精度高的基于电化学方法和磁珠抗体富集技术的快速检测靶标物的方法。
其具体方案为:一种基于拉曼光谱法的快速检测癌细胞的方法,包括如下步骤:
步骤1:在磁性微球表面标记能够识别癌细胞的抗体或核酸适配体;
步骤2:在步骤1得到的磁性微球表面通过抗体或核酸适配体富集癌细胞;
步骤3:将能够特异性识别癌细胞的荧光纳米金探针与步骤2得到的磁性微球的靶标物结合;
步骤4:将磁性微球表面的带有抗体或核酸适配体-靶标物-荧光纳米金探针的三明治结构分离;
步骤5:通过拉曼光谱法检测荧光纳米金探针的表面形貌和吸收光谱得到癌细胞浓度的检测结果。
在上述的基于拉曼光谱法的快速检测癌细胞的方法中,步骤1的磁性微球为经过羧基化的磁性微球。
在上述的基于拉曼光谱法的快速检测癌细胞的方法中,所述的荧光纳米金探针是组装有荧光物质、能够识别癌细胞的DNA标记物的纳米金颗粒。
在上述的基于拉曼光谱法的快速检测癌细胞的方法中,所述的荧光物质为罗丹明B或罗丹明6G。
在上述的基于拉曼光谱法的快速检测癌细胞的方法中,所述的步骤2中抗体或核酸适配体与靶标物的偶联通过碳化二亚胺进行偶联。
在上述的基于拉曼光谱法的快速检测癌细胞的方法中,所述的磁性微球的粒径为25nm。
本发明的有益效果在于:
本发明通过纳米金探针和磁珠抗体富集技术进行结合,可以对靶标物实现痕量检测,检测精度高。
具体实施方式
下面结合具体实施方式,对本发明的技术方案作进一步的详细说明,但不构成对本发明的任何限制。
为了更加清楚的对本发明进行说明,列举如下实施例来说明本发明的优越性。
实施例1
EpCAM抗体与Magpearl Streptavidin偶联
按照5:1(bio-anti-salmonella抗体与Magpearl Streptavidin)的浓度比例将bio-anti-salmonella抗体和Magpearl Streptavidin混合20分钟,中间间隔几分钟上下颠倒晃动离心管使之充分结合;反应完时间后,将离心管靠近磁架,小心的吸去上清液,加入原体积的PBS溶液重悬;重复此步骤3次,以洗去游离的bio-anti-salmonella抗体分子;最终用等体积的PBS重悬,放4℃保存。
3.2.3胶体金的制备
500mL的圆底烧瓶用超纯水清洗干净后,加入400mL的超纯水,再加入1%的氯金酸溶液4mL,磁力搅拌加热至沸腾,加入12mL提前配好的1%柠檬酸三钠溶液,继续搅拌加热,直至溶液颜色由最初的淡黄色变为均匀透亮的酒红色,再继续搅拌加热10分钟后,停止加热,继续搅拌至溶液冷却至室温后移至4℃保存备用。
3.2.4制备AuNP-DNA发卡探针
1)取10mL胶体金溶液,9600rpm离心10分钟,弃去上清液,用1mL超纯水重悬,加入100uL去离子水溶解的DNA(巯基修饰的DNA),充分混匀,4℃过夜反应;
2)加入10%牛血清蛋白至终浓度为1%,震荡混匀,封闭30分钟;
3)加入1.5M的NaCl溶液和1%的SDS溶液,分别至终浓度0.15M和0.01%,4℃老化24小时;
4)在4℃下,12,000rpm,离心20分钟,小心的用移液枪弃去上清。用1mL含20mMNa3PO4、1%BSA,0.25%Tween-20和10%蔗糖的重悬液重悬,重复此步骤2-3次,洗去多余的核酸,最后用100μL的重悬液重悬;
1、传感界面的制备
采用多晶光滑金电极作为传感界面。在捕获探针固定前,电极首先用0.3和0.05μm氧化铝抛光粉抛光至镜面并依次用超纯水、乙醇、超纯水超声清洗;然后于piranha溶液中浸泡清洗20min,水冲洗干净后于氮气中吹干。
2、肿瘤细胞的检测
将巯基标记的GR-5捕获探针(HS-ssDNA-GR5)通过Au-S键固定到金电极表面,4℃反应12小时。然后将电极放入PBS中搅洗,随后浸入1mM MCH中10min,放入PBS中搅拌洗涤,MCH用于阻止DNA在金电极表面的非特异性吸附以提高互补序列间的杂交效率;也可有效防止AuNPs在金电极表面的非特异性吸附。
用血清培养基培养肿瘤细胞,收集,离心然后梯度稀释加入到PBS中,形成不同细胞数量:10个/ml,50个/ml,100个/ml,500个/ml,1000个/ml。各加入EpCAM-磁珠,颠倒混匀10min。EpCAM抗体-磁珠将肿瘤细胞富集后,加入DNA修饰的AuNPs溶液中,反应30min,经过磁力架富集洗涤多余的DNA修饰的AuNPs后,PBS重悬,将所得电极在37℃放入重悬液中浸泡30min,并冲洗干净,重复该过程2次,所得电极用氮气吹干以备拉曼检测。
以上所述的仅为本发明的较佳实施例,凡在本发明的精神和原则范围内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种基于拉曼光谱法的快速检测癌细胞的方法,其特征在于,包括如下步骤:
步骤1:在磁性微球表面标记能够识别癌细胞的抗体或核酸适配体;
步骤2:在步骤1得到的磁性微球表面通过抗体或核酸适配体富集癌细胞;
步骤3:将能够特异性识别癌细胞的荧光纳米金探针与步骤2得到的磁性微球的靶标物结合;
步骤4:将磁性微球表面的带有抗体或核酸适配体-靶标物-荧光纳米金探针的三明治结构分离;
步骤5:通过拉曼光谱法检测荧光纳米金探针的表面形貌和吸收光谱得到癌细胞浓度的检测结果。
2.根据权利要求1所述的基于拉曼光谱法的快速检测癌细胞的方法,其特征在于,步骤1的磁性微球为经过羧基化的磁性微球。
3.根据权利要求2所述的基于拉曼光谱法的快速检测癌细胞的方法,其特征在于,所述的荧光纳米金探针是组装有荧光物质、能够识别癌细胞的DNA标记物的纳米金颗粒。
4.根据权利要求3所述的基于拉曼光谱法的快速检测癌细胞的方法,其特征在于,所述的荧光物质为罗丹明B或罗丹明6G。
5.根据权利要求1所述的基于拉曼光谱法的快速检测癌细胞的方法,其特征在于,所述的步骤2中抗体或核酸适配体与靶标物的偶联通过碳化二亚胺进行偶联。
6.根据权利要求1所述的基于拉曼光谱法的快速检测癌细胞的方法,其特征在于,所述的磁性微球的粒径为25nm。
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