【作者】 马文蔚；

【导师】 胥传来；

【作者基本信息】 江南大学 ， 食品科学， 2012， 硕士

【摘要】 特异性强、灵敏度高的重金属、毒素的快速检测方法,对于食品安全有着重大的意义。通过指数富集配体系统进化技术（SELEX）筛选得到的核酸适体,在毒素、重金属的检测等很多领域有重要作用。本论文选取了代表性的生物毒素（赭曲霉毒素）和重金属离子(Hg²⁺)作为检测对象,以核酸适配体为基础,构建了检测Hg²⁺的核磁共振方法,以及胶体金和量子点试纸条检测赭曲霉毒素（OTA）的方法。本文以这几个方面为主要内容:1、制备了核酸适配体功能化的胶体金试纸条,采用竞争法检测OTA,主要优化了金标核酸适配体的偶联比、重悬液体系、硝酸纤维素膜和结合垫上点样量等条件。在金:核酸=1:10,0.01M pH7.4 PB（含0.5%PEG,1%蔗糖,0.1%Tween20,0.02% MgSO4,0.05% （NH4）2SO4）的溶液体系下,采用CN140硝酸纤维素膜,试纸条能够在15min内实现对OTA的定性及半定量测。肉眼检测限2.5ng/mL,标准曲线得到的线性范围0.2-2.5ng/mL,检测限0.18ng/mL。2、构建了核酸适配体的量子点荧光试纸条,采用竞争法,量子点与适配体偶联后,优化重悬液体系、硝酸纤维素膜和结合垫上点样量等条件。发现在0.01M pH7.4 PB（含1.5%PVP,1%蔗糖,0.1%Tween20、0.02% MgSO4,0.05% （NH4）2SO4）体系中,采用M135硝酸纤维素膜,试纸条能够在15min内实现对OTA的定性及半定量测,在最优条件下,肉眼检测限5ng/mL,标准曲线得到的线性范围2.2-10ng/mL,检测限1.9ng/mL。3、优化磁粒子标记适配体的偶联比以后,探针与Hg²⁺反应,形成T-Hg²⁺-T错配结构,磁粒子发生聚集,水中质子状态变化。利用核磁共振成像仪器,得到T2加权图像,T2造影图像。实验结果中,随着Hg²⁺浓度的提高,水中的质子环境改变越多,MRI图像强度减弱,即T2加权图像强度变暗,T2弛豫时间逐渐减小,线性范围0.25pg/mL-10pg/mL,检测限0.15pg/mL。更多还原

【Abstract】 Specificity, high sensitivity and rapid detection of heavy metals and toxins have great significance for food safety. Aptamer can get through systematic evolution of ligands by exponential enrichment （SELEX）. Aptamer have played an important pole in many areas of the toxin and heavy metals’detection. Based on aptamer, we have build the NMR method to detect Hg²⁺ ion, as well as colloidal gold and quantum dot strip for the detection of ochratoxin A method.In this paper, these aspects as the main content:1. Aptamer of OTA marked gold nanoparticles, prepared by this complex, we developed a quick and on-site sensor for detection of OTA in the competion format. The best ratio between gold nanoparticles and DNA is 1:10 , the best buffer is 0.01M pH7.4 PB（0.5%PEG,1%sucrose,0.1%Tween20,0.02% MgSO4,0.05% （NH4）2SO4）and take the CN140 nitrocellulose membrane. The test strip took the qualitative and semi-quantitative measurement of OTA within 15min .We got a good linear relationship between OTA and the response signal. The limit of detection with naked eyes is 2.5ng/mL, and according to the standard curve, the linear range is 0.2-2.5ng/mL and LOD is 0.18ng/mL.2. Aptamer of OTA marked quantum dots, prepared by this complex, we developed a quick and on-site sensor for detection of OTA in the competion format. The best buffer is 0.01M pH7.4 PB（1.5%PVP,1%sucrose,0.1%Tween20、0.02% MgSO4,0.05% （NH4）2SO4） and take the M135 nitrocellulose membrane. The test strip took the qualitative and semi-quantitative measurement of OTA within 15min. The limit of detection with naked eyes is 5ng/mL, and according to the standard curve, the linear range is 2.2-10ng/mL and LOD is 1.9ng/mL.3. We conjugated the T-rich nucleic acid to magnetic nanoparticles which would form the T- Hg²⁺-T structure with the accession of Hg²⁺. Then magnetic nanoparticles began to aggregate, hence lead to a change in the distribution of water proton. Using the magnetic resonance imaging apparatus, we got T2–weighted images and T2 contrast image. The more Hg²⁺ concentration increased, the more water proton environment changed. Accordingly T2 contrast image became dimmed and T2 relaxation time gradually decreased. We got a good linear relationship between the analyte concentration and the response signal. The linear range is 0.25-10pg/mL, and the limit of detection is 0.15pg/mL.更多还原