【作者】 刘云；

【导师】 崔一平； 宗慎飞；

【作者基本信息】 东南大学 ， 光学工程， 2020， 硕士

【摘要】 免疫检测技术具有灵敏度高、特异性强等优点,是现代生命科学中重要的分析手段,在生物检测方面具有广泛的应用前景。但是,免疫检测中的非特异性吸附总是不可避免的。尽管人们已经开发出各种降低非特异性吸附的方法,但是假阳性事件一直是影响免疫检测结果可靠性的重要因素之一。光学超分辨技术突破了传统显微术衍射极限的限制,使得人们可以观察到更为精细的生物结构。单分子定位显微镜(SMLM)就是其中一种技术,这为降低免疫检测的假阳性带来了新的思路与技术支持。首先,本文提出了一种基于超分辨多色荧光共定位的免疫检测技术。该技术中,Alexa Fluor 488(A488)修饰的山羊抗兔IgG与Alexa Fluor 647(A647)修饰的山羊抗兔IgG在待检测抗原兔IgG存在的条件下形成了三明治夹心复合结构。利用SMLM检测A488与A647荧光位点的共定位系数,实现了对抗原的定量分析。该技术的检测限可低至0.1pg/m L,并且能在一定程度上降低假阳性事件带来的干扰。但由于SMLM成像时不同视野内荧光探针的数量不一致,导致不同区域内的荧光探针对于待测蛋白的捕获效率不同,容易造成分析误差。因此,本文进一步提出了基于金纳米阵列的超分辨多色荧光共定位免疫检测技术。利用有序金纳米阵列作为免疫反应的基底,有效降低了上述分析误差。该技术中,使用核酸适配体代替抗体,成功实现了假阳性结合位点的判断以及肿瘤细胞外泌体的检测。通过分析荧光共定位系数随外泌体浓度的变化曲线,得出该技术对于外泌体的检测限可低至10个外泌体/μL。这种基于超分辨成像的新型免疫检测技术具有灵敏度高、特异性好、普适性强等优点,在疾病标志物检测方面具有很好的应用前景。更多还原

【Abstract】 Immunoassay has high sensitivity and strong specificity,which is an important analytical method in modern life science and has broad application prospects in biological detection.However,non-specific adsorption in immunoassays is always inevitable.Although various methods have been developed to reduce non-specific adsorption,false positive events have always been one of the important factors affecting the reliability of immunoassay results.Super-resolution technology breaks through the diffraction limit of traditional fluorescence microscopy,allowing people to observe finer biological structures.Single-molecule localization microscopy(SMLM)is one of these technologies,which brings new ideas and technical support for reducing false positive events in immunoassays.Herein,at first,a new kind of immunoassay based on super-resolution multicolor fluorescence co-localization has been proposed.According to this technique,Alexa Fluor 488 modified goat anti-rabbit IgG(A488@Goat anti Rabbit IgG)and Alexa Fluor 647 modified goat anti-rabbit IgG(A647@Goat anti Rabbit IgG)formed a sandwich composite structure in the presence of rabbit IgG.Then,SMLM was used to detect the co-localization coefficients of A488 and A647 fluorescent sites to achieve quantitative analysis of the antigens.The detection limit of this technology can be as low as 0.1 pg / m L.More importantly,this method can reduce the interference caused by false positive events to a certain extent.However,due to the inconsistency of the number of fluorescent probes in different view fields during SMLM imaging,the fluorescent probes in different regions have different capture efficiencies for the antigens,which may easily cause analysis errors.Therefore,in order to solve this problem,we further proposed a super-resolution multicolor fluorescence co-localization immunoassay based on ordered gold nanoarrays.The ordered gold nanoarrays are used as the substrates in immune reactions,which effectively reduces the analysis errors mentioned above.In this technique,aptamers are used instead of antibodies.We successfully achieved the determination of false positive binding sites and the detection of tumor cell exosomes.The detection limit of this technique for exosomes can be as low as 10 particles/μL.This new type of immunoassay has the advantages of high sensitivity,good specificity and strong universality,and holds a good application prospect in the detection of disease markers.更多还原