| 题名 | 基于免疫层析技术的时间分辨荧光免疫分析仪研究 |
| 作者 | 任冰强 |
| 学位类别 | 硕士 |
| 答辩日期 | 2009 |
| 授予单位 | 中国科学院上海光学精密机械研究所 |
| 导师 | 黄惠杰 |
| 关键词 | 光学生物传感器 标记免疫学 时间分辨荧光免疫分析 免疫层析技术 免疫微球 |
| 其他题名 | Development of time-resolved fluorometer based on immunochromatography |
| 中文摘要 | 免疫层析技术是上世纪八十年代初发展起来的一种快速生物检测技术。由于免疫层析技术不须进行结合标记物与自由标记物的分离，因而操作简单、快速，非常适合现场检测之用。时间分辨荧光免疫分析是在传统荧光免疫分析的基础上创立的一种新型非放射性标记免疫分析技术。与传统的荧光免疫分析技术不同，它所用的标记物是具有独特荧光性能的镧系元素及其螯合物，利用这类标记物能发出长寿命与Stokes位移大的荧光的特点，同时采用时间分辨与光谱分辨技术可有效排除样品自然荧光的干扰，具有灵敏度高、特异性强、稳定性好等特点，在临床免疫检验和科学研究中的应用越来越广泛。 针对目前国内外尚无采用时间分辨技术的POCT（Point-of-Care Testing）分析仪的现状，本论文将时间分辨荧光免疫分析和免疫层析两种技术的优点相结合，研制了一种基于免疫层析技术的时间分辨荧光免疫分析仪。该分析仪既具有免疫层析技术操作简单与测量快速的特点，又具有时间分辨荧光技术检测灵敏度高与特异性强的特点，同时因为采用性能优异的紫外LED作激发光源而使整机体积小巧，可用作POCT分析仪，用于现场定量检测。 本论文的主要内容如下： 1.概括介绍了当前广泛应用于临床医学检测的几种标记免疫分析方法以及时间分辨荧光免疫分析仪的发展状况，重点探讨了临床医学检验的新趋势-POCT的特点及其对检验仪器的要求。 2.详细介绍了免疫层析技术、时间分辨荧光免疫分析和免疫微球技术的基本原理和相关知识，在此基础上建立了基于免疫层析技术的时间分辨荧光免疫分析仪的工作原理。 3.详细阐述了基于免疫层析技术的时间分辨荧光免疫分析仪的研制过程，介绍了各个部件的结构和设计方法，给出了主要的技术参数，并对测量软件进行了详细说明。针对时间分辨荧光的特性，提出了荧光信号检测过程的时间控制要求与实现方法。所研制的仪器采用紫外LED作为激发光源，主要由光学传感器、扫描平台及其驱动系统、数据采集与信号处理系统、微机控制与管理系统等几部分组成，采用扫描方式对免疫层析试纸条上荧光颗粒分布进行检测，依据特定算法可计算出目标被检物的浓度，计算结果可保存、拷贝或打印。 4.对基于免疫层析技术的时间分辨荧光免疫分析仪样机的性能进行了测试，并利用该仪器对空白层析膜的光学均匀性进行了分析。实验表明，使用内部填充有Eu3+螯合物的免疫微球标准样品溶液作为检测对象时，仪器的最小检测浓度可达到ng/ml量级，同一试纸条多次测量值的相对标准偏差小于3％，在5-103 ng/ml的浓度范围内具有良好的线性响应特性，与最小二乘法拟合出的直线具有很高的相关性，相关系数R2 ≥ 0.99。 5.总结了本论文取得的主要成果，列出了主要创新点，并讨论了进一步研究的内容。 |
| 英文摘要 | Immunochromatography was developed as a quick immunoassay technique in the 1980s. Immunochromatography can quickly determine the analytes without separating the combined marker and the free marker, so it is suitable for on-site test. Time-resolved fluoroimmunoassay (TRFIA) was applied in immunoassay firstly in the early 1980s. As a non-radioactive detection method, it is widely used in many scientific research fields at present. Different from the conventional fluorescein, TRFIA takes the lanthanide and its chelates as the fluorescent marker which has the particular fluorescent characteristic, such as long lifetime of fluorescence and exceptionally large Stokes shift, to eliminate the natural fluorescence of the sample by time-resolved technique and spectrum technique. TRFIA has the following advantages: high sensitivity, high specificity, high stability and high safety. At present there is no POCT instrument using TRFIA technique at home and abroad, so the time-resolved fluorometer based on immunochromatography described in this paper is developed on the basis of time-resolved fluorometers and it has the advantages of both the immunochromatography and TRFIA. This instrument can provide a rapid, quantitative measurement of analytes present in samples without any washing steps and complex operation and can be used to carry out point-of-care test (POCT). The primary contributions of this thesis are given in the following aspects: 1. An overview of the several tag immunoassay methods used widely in clinical detection and the development status of time-resolved fluorometer are given, and the trend of clinical detection - POCT and its impact on measuring instrument are discussed emphatically. 2. The principle and characteristics of immunochromatography, time-resolved fluoroimmunoassay and immune microsphere are introduced respectively in detail. On this basis, the principle of time-resolved fluoroimmunoassay based on immunochromatography is theorized. 3. The development process of the time-resolved fluorometer based on immunochromatography is described detailedly. The structure, design methods and main technical parameters of each part are given, and the analysis software is introduced in detail. For the characters of the time-resolved technique, the time control and implementation of the fluorescence detection is proposed. The instrument includes a specific optical sensor, a scanning stage, a signal processing system and a computer control system. It measures the distribution of Eu3+ chelates on the test trip surface in scanning mode, and the concentration of the target analyte in the sample can be obtained based on a specific algorithm. The final results are stored in the embedded computer and can be copied out or printed. 4. The performance of the instrument is tested and the uniformity of the surface of blank test strips using is evaluated by the instrument. The results show that, when Eu3 + chelates are used for marking substance, the sensitivity of the instrument is several ng/ml level, the relative standard deviation of the same test strip is less than 3% and response linear range is from several ng/ml to 103 ng/ml, in which correlation coefficient is 99.97%. 5. Achievements of this thesis are summarized, innovative aspects are listed, and some further improvements are proposed. |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.siom.ac.cn/handle/181231/16499]  |
| 专题 | 上海光学精密机械研究所_学位论文 |
| 推荐引用方式 GB/T 7714 | 任冰强. 基于免疫层析技术的时间分辨荧光免疫分析仪研究[D]. 中国科学院上海光学精密机械研究所. 2009. |
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