量子点免疫层析试纸条同步定检测
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作者签名:
导师签名: 日期: 年 月 日
摘要
摘要
乙型肝炎、丙型肝炎和艾滋病是三种常见的血液传染病,严重威胁着人类健 康,在临床输血和献血过程中,国家卫生部规定医疗和卫生机构必须对这三种传 染病进行强制性检测。目前,针对这三种血液传染病的临床诊断方法有很多,但 各有缺点:如检测灵敏度较低,且不能同步定量检测;需要特殊检测仪器,且检 测耗时较长等。针对这些检测方法的不足之处,本研究将量子点(Quantum dot) 技术和免疫层析技术相结合,研制一种量子点免疫层析试纸条,从而实现乙型肝 炎、丙型肝炎和艾滋病的同步定量检测。
论文题目 量子点免疫层析试纸条同步定量检测
血液传染病三种指标的研究
学科专业 学号 作者姓名 指导教师
生物化学与分子生物学 201121090112 侯飞 马义才 副教授
分类号 UDC 注1
密级
学位论文
量子点免疫层析试纸条同步定量检测 血液传染病三种指标的研究
(题名和副题名)
侯飞
(作者姓名)
指导教师
通过将量子点技术和免疫层析技术相结合,以量子点作为荧光标记物,从而
I
摘要
取代胶体金,研制一种量子点免疫层析试纸条,非常适合血液及其他样品的同步 定量分析,可以实现乙型表面抗原、丙肝抗体和艾滋病抗体进行同步定量检测, 从而使得血液传染病的检测更为简单、快速和经济,并建立一种特异性强、灵敏 度高和稳定性好的膜相同步定量检测方法。 关键词:量子点,免疫层析,血液传染病,同步定量检测
In this study, EDC/NHS are used as cross-linkers to activate carboxyl QDs. Activated QDs with emission wavelengths at 530nm, 576nm and 620nm are coupled to hepatitis B surface antibody , HCV antigen and HIV antigen respectively .Quantum dots coupling compound are added to a glass fiber membrane to obttain the conjugate pad. Hepatitis B surface antigen polyclonal antibody, HCV core antigen and HIV antigen are coated onto the NC membrane as testing line, sheep anti mouse IgG as control line. Then the sample pad, conjugate pad, NC membrane and absorbent pad are pasted on the PVC backing plate in sequence to make up a quantum dots based ICTS.
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作者签名:
日期: 年 月 日
论文使用授权
本学位论文作者完全了解电子科技大学有关保留、使用学位论文 的规定,有权保留并向国家有关部门或机构送交论文的复印件和磁盘, 允许论文被查阅和借阅。本人授权电子科技大学可以将学位论文的全 部或部分内容编入有关数据库进行检索,可以采用影印、缩印或扫描 等复制手段保存、汇编学位论文。
马义才
副教授
电子科技大学
成都
(姓名、职称、单位名称)
申请学位级别 硕士 学科专业 生物化学与分子生物学
提交论文日期 2014.04 论文答辩日期
2014.05
学位授予单位和日期 电子科技大学 2014 年 06 月 日
答辩委员会主席
评阅人
注 1:注明《国际十进分类法 UDC》的类号。
Quantum dot-based immunochromatography test strip for Synchronous and quantitative detection of
Hepatitis B, hepatitis C and AIDS
A Master Thesis Submitted to University of Electronic Science and Technology of China
Major: Biochemistry and Molecular Biology
本文以EDC/NHS为反应活化剂,分别制备530nm CdSe/ZnS QD-抗HBsAg单 抗、576nm CdSe/ZnS QD-HCV Ag和620nm CdSe/ZnS QD-HIV Ag偶联物,并将三 种量子点偶联物喷涂于结合垫上,把抗HBsAg多抗、HCV Ag和HIV Ag包被于NC 膜上作为检测线,质控线包被羊抗鼠IgG,然后将样品垫、结合垫、NC膜、吸水 垫按顺序组装在PVC底板上,制备成量子点免疫层析试纸条。通过荧光阅读仪阅 读量子点试纸条的检测带和质控带的荧光值,根据不同浓度的HBsAg、HCV抗体 和HIV抗体的检测带(T)和质控带(C)荧光值的不同,可以得出T/C的比值,此 比值对应HBsAg、HCV抗体和HIV抗体浓度做标准曲线,即可实现乙型肝炎、丙 型肝炎和艾滋病的同步定量检测。
Author:
Hou fei
Advisor:
Professor Ma Yi cai
School : School of Life Science and Technology
独创性声明
本人声明所呈交的学位论文是本人在导师指导下进行的研究工作 及取得的研究成果。据我所知,除了文中特别加以标注和致谢的地方 外,论文中不包含其他人已经发表或撰写过的研究成果,也不包含为 获得电子科技大学或其它教育机构的学位或证书而使用过的材料。与 我一同工作的同志对本研究所做的任何贡献均已在论文中作了明确的 说明并表示谢意。
Fluorescence intensity of test line and control line on a single strip are both measured by a fluorescent reader. Different concentration of HBsAg, HCV antibody and HIV antibody captured on the test line have different fluorescence intensity of QDs both on test line and control line, and the results are recorded by the ratio of the fluorescence intensities of test line and control line (T/C ratio). Synchronous quantitative detection of HBV, HCV ang AIDS are realized by the standard curve drawed according to the T/C ratio.
本课题研究结果:(1)量子点免疫层析试纸条和胶体金试纸条以及 ELISA 法 同时检测临床标本 58 份,其中 HBsAg 阳性 38 份,阴性 20 份,用量子点试纸条 和胶体金试纸条以及 ELISA 法分别检测,比较结果显示:量子点试纸条的符合率 为 98.1%,灵敏度为 100%,特异性为 95%,检测下限浓度为 2.6ng∕ml,检测用时 为 10 分钟。相对 ELISA 法,大大节省了检测时间,与金标法相比,灵敏度得到 提高。(2)量子点试纸条同步检测 HBsAg 和 HIVAb 时,HBsAg 阴性符合率为 94.2%,HIVAb 阴性符合率为 89.6%;HBsAg 阳性符合率为 87.2%,HIVAb 阳性 符合率为 83.7%;HBsAg 检测灵敏度为 95%,HIVAb 检测的灵敏度为 90.1%;同 步检测 HBsAg 和 HIVAb 时,HBsAg 和 HIVAb 的变异系数分别为 12%和 14.5%, 均小于 15%。(3)量子点免疫层析试纸条同步定量检测 HBsAg、HCV Ab 和 HIV Ab 探索实验显示:同步检测 HBsAg、HCV Ab 和 HIV Ab 时,对这三种指标的定 量检测下限浓度分别为 2.6 ng∕ml、15 ng∕ml 和 10 ng∕ml。(4)量子点免疫层析试纸 条在 4-30℃保存 1 个月,其稳定性、特异性和灵敏度基本不变。
The research results of this article: (1) 58 clinical samples (HBsAg positive 38, HBsAg negative 20) were simultaneously detected with the quantum dots based ICTS and the colloidal gold based test strip and ELISA kit respectively. The results show that the accordance rates, sensitivity , specificity of quantum dots based ICTS were
II
ABSTRACT
ABSTRACT
Hepatitis B, hepatitis C and AIDS are three common infectious diseases that seriously threaten human health. During clinical blood transfusion and blood donation, the Medical and Sanitary Institution must follow the provisions of the Ministry of Health to carry on mandatory examination on these three kinds of infectious diseases. At present, there are lots of clinical and experimental methods for detecting HBV、HCV and HIV, but each has shortcomings. For example, low detection sensitivity, cannot achieve synchronous quantitative detection require special testing instruments or timeconsuming, etc. Considering the deficiency of these existing test methods, we developed a quantum dot (QD) based immunochromatography test strip (ICTS) by combining quantum dot labeling technology with immunochromatography to realize synchronous quantitative detection of hepatitis B, hepatitis C and AIDS.
III
ABSTRACT
98.1%,100%, 95%, the minimum detection limit and detection time is 2.6ng∕ml,10 minutes. It is faster than ELSIA, and has high level of sensitivity compared with colloidal gold based test strip. (2) When HbsAg and HIVAb were were simultaneously detected with the quantum dots based ICTS, the negative accordance rates for HBsAg and HIVAb were 94.2%, 89.6%; the positive accordance rates for HBsAg and HIVAb were 87.2%, 83.7%; the detection sensitivity for HBsAg and HIVAb were 95%, 90.1%; the detection results of HBsAg and HIVAb show that the cofficient variation (CV) were 12%, 14.5%, both less than 15%. (3) The exploration experiment shows that when use a quantum dots based ICTS to detect HBsAg and HCV Ab and HIVAb synchronously and quantitatively, the minimum detection limit of these three indicators were 2.6 ng/ml, 15 ng/ml and 10 ng/ml. (4) The stability, specificity and sensitivity of the quantum dots based ICTS which stored at 4℃ did not change obviously after one month.