量子点生物传感膜快速同步定量检测肿瘤标志物的研究
发布时间：2019-03-16 09:44
【摘要】：恶性肿瘤是目前乃至未来病死率较高的病种之一,据世界卫生组织发布的《2014世界癌症报告》显示,2012年全球有820万人死于癌症。对肿瘤标志物的检测有利于实现癌症的早期诊断,从而提高治疗效率,降低死亡率。肿瘤标志物的检测方法多种多样,以免疫层析法最为受到关注。免疫层析技术因其简便、经济、低样品量、无需专业人员操作、可实现床旁快速检测等特点而迅速发展。但以胶体金为代表的免疫层析试纸条灵敏度不高,进行多组分分析时需要多个检测带,且以定性检测为主,无法满足对多种肿瘤标志物的早期同步定量检测需求。免疫层析法所采用的标记材料对免疫传感器的性能起决定性的作用。荧光量子点(QDs)具有荧光强度高、光化学稳定性好、吸收光谱连续、发射光谱窄而对等特性,可实现“一元激发,多元发射”。基于QDs的优势,针对目前免疫层析产品的不足,我们提出以多色QDs作荧光标记物,开发一种基于多色QDs标记的生物传感膜,以实现对多种肿瘤标志物的快速、同步、定量检测。本文以1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为偶联剂,制备546nm CdSe/ZnS QDs-抗甲胎蛋白单抗(QDs546-anti-AFP McAb)和620nm CdSe/ZnS QDs-抗癌胚抗原单抗(QDs620-anti-CEA McAb)偶联物。将两种偶联物按一定比例混合喷涂于结合垫上,鼠源甲胎蛋白单抗(anti-AFP McAb)和鼠源癌胚抗原单抗(anti-CEA McAb)混合物以及羊抗鼠IgG分别包被于硝酸纤维素膜(NC膜)上作为检测带(T带)和质控带(C带),在此基础上制作多色QDs生物传感膜。通过荧光阅读仪检测传感膜上T带和C带的荧光强度,计算出T带和C带的比值(T/C),对应相应的样品浓度绘制出标准曲线,即可达到定量检测的目的。在最优实验条件下,本课题组所开发的多色QDs生物传感膜对AFP和CEA的检测限分别为2 ng?ml和1 ng?ml,且无明显交叉反应,灵敏度、特异性高,样品恢复率好,批内稳定性好,批间差异小。本文将多色QDs与免疫层析技术相结合开发出的生物传感膜,NC膜上只需一条T带和一条C带便可实现对多种肿瘤标志物同步定量检测。其使用方便简单,无需专业人员操作,可在社区、家庭使用,并有利于将来检测仪器的小型化和智能化。
[Abstract]:Malignant tumor is one of the diseases with high mortality at present and even in the future. According to the World Cancer report 2014 published by the World Health Organization, 8.2 million people died of cancer in 2012. The detection of tumor markers is beneficial to the early diagnosis of cancer, so as to improve the therapeutic efficiency and reduce the mortality. There are many methods to detect tumor markers, so the method of epidemic chromatography is the most concerned. Because of its simplicity, economy, low sample size and no need of professional operation, immune chromatography technology can realize rapid detection on the bed side and so on. However, the immunochromatographic assay strip represented by colloidal gold is not sensitive and requires multiple detection bands for multi-component analysis, and qualitative detection is the main method, which can not meet the need of simultaneous and quantitative detection of multiple tumor markers in the early stage. The material used in immunochromatography plays a decisive role in the performance of the immunosensor. The fluorescence quantum dot (QDs) has the characteristics of high fluorescence intensity, good photochemical stability, continuous absorption spectrum, narrow emission spectrum and so on. It can realize "mono-excitation and multi-element emission". Based on the advantage of QDs and aiming at the deficiency of immunochromatographic products at present, we propose to develop a kind of biosensory membrane based on multicolor QDs as fluorescent marker to realize rapid, synchronous and quantitative detection of various tumor markers. In this paper, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) were used as coupling agents. The conjugates of 546nm CdSe/ZnS QDs- anti-alpha-fetoprotein monoclonal antibody (QDs546-anti-AFP McAb) and 620nm CdSe/ZnS QDs- anticancer embryo antigen monoclonal antibody (QDs620-anti-CEA McAb) were prepared. The two coupling materials are mixed and sprayed on the bonding pad in a certain proportion. The mixture of mouse-derived alpha-fetoprotein monoclonal antibody (anti-AFP McAb) and mouse-derived carcinoembryonic monoclonal antibody (anti-CEA McAb) and sheep anti-mouse IgG were coated on the nitrocellulose membrane (NC membrane) as a detection band (T-band) and a quality control band (C-band), respectively. On this basis, multicolor QDs biosensory membrane was prepared. The fluorescence intensity of T-band and C-band on the sensing membrane was measured by fluorescence reader, the ratio of T-band to C-band (T / C) was calculated, and the standard curve was drawn corresponding to the concentration of the sample, which could be used for the quantitative detection of T-band and C-band. Under the optimal experimental conditions, the detection limits of the multicolor QDs biosensor membrane for AFP and CEA were 2 ng?ml and 1 ng?ml, respectively, and there was no obvious cross-reaction, with high sensitivity, high specificity and good sample recovery rate. The intra-batch stability is good, and the difference between batches is small. In this paper, multi-color QDs combined with immuno-chromatography technique was combined to develop biological sensing membrane. Only one T-band and one C-band were needed on NC membrane to realize simultaneous quantitative detection of multiple tumor markers. It is easy to use, no professional operation, can be used in the community, home, and conducive to the future miniaturization and intelligence of testing instruments.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.4
本文编号：2441144
[Abstract]:Malignant tumor is one of the diseases with high mortality at present and even in the future. According to the World Cancer report 2014 published by the World Health Organization, 8.2 million people died of cancer in 2012. The detection of tumor markers is beneficial to the early diagnosis of cancer, so as to improve the therapeutic efficiency and reduce the mortality. There are many methods to detect tumor markers, so the method of epidemic chromatography is the most concerned. Because of its simplicity, economy, low sample size and no need of professional operation, immune chromatography technology can realize rapid detection on the bed side and so on. However, the immunochromatographic assay strip represented by colloidal gold is not sensitive and requires multiple detection bands for multi-component analysis, and qualitative detection is the main method, which can not meet the need of simultaneous and quantitative detection of multiple tumor markers in the early stage. The material used in immunochromatography plays a decisive role in the performance of the immunosensor. The fluorescence quantum dot (QDs) has the characteristics of high fluorescence intensity, good photochemical stability, continuous absorption spectrum, narrow emission spectrum and so on. It can realize "mono-excitation and multi-element emission". Based on the advantage of QDs and aiming at the deficiency of immunochromatographic products at present, we propose to develop a kind of biosensory membrane based on multicolor QDs as fluorescent marker to realize rapid, synchronous and quantitative detection of various tumor markers. In this paper, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) were used as coupling agents. The conjugates of 546nm CdSe/ZnS QDs- anti-alpha-fetoprotein monoclonal antibody (QDs546-anti-AFP McAb) and 620nm CdSe/ZnS QDs- anticancer embryo antigen monoclonal antibody (QDs620-anti-CEA McAb) were prepared. The two coupling materials are mixed and sprayed on the bonding pad in a certain proportion. The mixture of mouse-derived alpha-fetoprotein monoclonal antibody (anti-AFP McAb) and mouse-derived carcinoembryonic monoclonal antibody (anti-CEA McAb) and sheep anti-mouse IgG were coated on the nitrocellulose membrane (NC membrane) as a detection band (T-band) and a quality control band (C-band), respectively. On this basis, multicolor QDs biosensory membrane was prepared. The fluorescence intensity of T-band and C-band on the sensing membrane was measured by fluorescence reader, the ratio of T-band to C-band (T / C) was calculated, and the standard curve was drawn corresponding to the concentration of the sample, which could be used for the quantitative detection of T-band and C-band. Under the optimal experimental conditions, the detection limits of the multicolor QDs biosensor membrane for AFP and CEA were 2 ng?ml and 1 ng?ml, respectively, and there was no obvious cross-reaction, with high sensitivity, high specificity and good sample recovery rate. The intra-batch stability is good, and the difference between batches is small. In this paper, multi-color QDs combined with immuno-chromatography technique was combined to develop biological sensing membrane. Only one T-band and one C-band were needed on NC membrane to realize simultaneous quantitative detection of multiple tumor markers. It is easy to use, no professional operation, can be used in the community, home, and conducive to the future miniaturization and intelligence of testing instruments.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.4
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