【作者】 刘丽；

【导师】 吴永江；

【作者基本信息】 浙江大学 ， 药学， 2020， 硕士

【摘要】 赭曲霉毒素A（OTA）是一类由青霉素属和曲霉素属的某些真菌在适宜条件下产生的次级有毒代谢产物,主要污染农作物及其制品。研究表明,OTA具有很强的肾毒性、肝毒性、免疫毒性、致癌性,对人和动物的健康造成巨大的威胁。目前,常规的检测分析方法虽然可实现OTA的灵敏检测,但这些技术昂贵、耗时且需要高精密仪器、复杂的样品制备以及专业人员操作,难以满足日常快速检测的需求。因此,建立新型、快速、灵敏的OTA检测方法,对保障食品、药材的安全以及人类健康有重大意义。荧光传感器因其具有灵敏度高、选择性好、荧光可调等优势成为检测领域的研究热点,而纳米材料具备独特的光、电性能,生物相容性好、水溶性较佳等优点成为荧光传感器的辅助手段。基于此,本课题以OTA为研究对象,利用纳米材料的不同特性以及适配体的特异性构型转换,构建了多种基于纳米材料和适配体的荧光传感器,实现了食品和中药材中OTA的检测,为快速检测OTA提供了新思路。本课题主要的研究内容和成果如下:1.基于碳纳米粒子（CNPs）氧化物和标记型适配体的荧光传感器测定OTA的研究。本研究以FAM修饰的适配体作为供体,水溶性CNPs氧化物作为受体,充分利用CNPs氧化物对适配体的单链核苷酸以及适配体与OTA结合后形成的折叠结构的敏感性差异,从而构建基于CNPs氧化物和标记型适配体的荧光传感器用于OTA的检测。利用紫外可见光谱（UV）对CNPs氧化物及其与适配体混合进行表征,并优化了CNPs氧化物的浓度及混合时间等实验条件。所构建的传感器对OTA检测的线性范围为10-80 ng mL^-1,检测限（LOD）为3.681 ng mL^-1,选择性较好,并成功用于红酒样品中的OTA检测,回收率为98.9%-106.11%。2.基于卟啉和量子点的非标记适配体荧光传感器测定OTA的研究。本研究以水溶性CdTe量子点（QDs）为荧光探针,四-（4-甲基吡啶基）卟啉（TMPyP）为淬灭剂,利用无修饰的适配体结合OTA后的构型转换形成反向平行的G-四链体,使得TMPyP与反向平行G-四链体结合引起荧光恢复,从而构建适配体荧光传感器用于OTA的检测。利用CD光谱对适配体与OTA形成的G-四链体进行表征,优化了TMPyP的浓度、适配体与OTA的孵育时间等实验条件。在最优的实验条件下,应用该荧光传感器对OTA进行检测,线性范围为0.2-20 ng mL^-1,LOD为0.16 ng mL^-1,选择性好。采用黄芪药材进行加标回收实验,回收率为98.9-102.2%之间,表明该传感器可用于实际样品中OTA的检测。3.基于自组装卟啉和ZnCdSe QD的“Turn off-on”型荧光传感器检测OTA的研究。本研究以自组装锌卟啉作为淬灭剂,水溶性ZnCdSe QDs为荧光探针,构建了“Turn off-on”型荧光传感器用于OTA的检测。利用绿色环保的十二烷基二甲基甜菜碱为软模板对四吡啶基锌卟啉（ZnTPyP）进行自组装,所形成的自组装ZnTPyP（SA-ZnTPyP）的水溶性显著提高,光学性能提升。在最佳实验条件下,荧光传感器表现出良好的检测性能,包括较宽的线性范围(0.5-80 ng mL^-1),较低的LOD(0.33 ng mL^-1),良好的稳定性,高选择性,较短的检测时间。本方法可以较好的应用于实际样品如牛奶和咖啡的检测中,并利用超高效液相色谱–串联质谱联用（UHPLC–MS/MS）验证了结果的准确性。更多还原

【Abstract】 Ochratoxin A（OTA）is a toxic metabolite derived mainly from Aspergillus ochraceus and Penicillium verrucosum,which are widely presented as contaminants in a variety of products,including crops and their products.OTA is found to cause severe toxic effects,such as teratogenic,embryotoxic,genotoxic,neurotoxic,immunosuppressive,carcinogenic,and nephrotoxic to humans.Current analysis of OTA is conducted by various methods,although these methods have both high accuracy and sensitivity,they still have some shortcomings,such as time consuming,tedious sample pretreatment and expensive instruments.Therefore,it would be highly desirable to establish a rapid,convenient,low-cost,and sensitive method for OTA detection.Recently,fluorescent analysis methods have become hotspots in the detection field due to their advantages of high sensitivity,good selectivity,and adjustable fluorescence.Nanomaterials have unique optical and electrical properties,good biocompatibility,and better water solubility,which have become auxiliary methods for fluorescent sensors.Based on this,this project developed nanomaterials and aptamer-based fluorescent sensor for the determination of OTA in food and traditional Chinese medicine,which is expected to provide a novel tool for the rapid and sensitive detection of OTA.The main research works are as follows:1.Development of labled fluorescent aptasensor for detection of OTA based on Carbon nanoparticles（CNPs）oxide.FAM-modified aptamers were used as donors,and water-soluble CNPs oxides were used as acceptors.Utilizing folded structure formed by the aptamers and OTA could not combine with CNPs oxide,we constructed a fluorescent sensor based on CNPs oxide and labeled aptamer for OTA detection.The ultraviolet-visible spectrum（UV）was used to characterize CNPs oxides and their mixture with aptamers.The experimental conditions such as CNPs oxide concentration and mixing time were optimized.Under optimized conditions,the linear range of the fluorescent sensor for OTA detection was 10-80 ng mL^-1,and the detection limit（LOD）was 3.681ng mL^-1.The fluorescent sensor has good selectivity and has been used for OTA detection in wine samples with a recovery rate of 98.9%-106.11%.2.Development of label-free fluorescent aptasensor for OTA of detection based on porphyrin and CdTe Quantum Dots.In this study,water-soluble CdTe QDs were used as fluorescent probes,and（N-methyl-4-pyridy）porphyrin（TMPyP）was used as a quencher.A novel label-free fluorescent aptasensor utilizing the interaction between OTA-triggered antiparallel G-quadruplex and TMPyP for the rapid and sensitive determination of OTA was established.The Circular Dichroism（CD）spectrum was used to characterize the G-quadruplex formed by the aptamer and OTA,and the experimental conditions such as the concentration of TMPyP and the incubation time of the aptamer and OTA were optimized.Under optimized conditions,the LOD of the designed aptasensor was 0.16 ng mL^-1,with a linear range of 0.2 to 20 ng mL^-1 and good selectivity.Furthermore,this aptasensor was applied in Astragalus membranaceus samples with a recovery rate of 98.9-102.2%,indicating that the sensor can be used to detect OTA in actual samples.3.Development of“Turn off-on”fluorescent sensor for OTA detection based on ZnCdSe QDs and self-assembled zinc porphyrin.In this study,a self-assembled zinc porphyrin was used as a quencher,and a water-soluble ZnCdSe QDs was used as fluorescent probes.A“Turn off-on”fluorescent sensor was established for the detection of OTA.Using environmentally friendly dodecyl dimethyl betaine as“soft template”,zinc5,10,15,20-tetra（4-pyridyl）-21H-23H-porphine（ZnTPyP）was self-assembled into nanorods through a green process,which has significantly improved their water solubility and optical performance.Under the optimized experimental conditions,the newly developed fluorescent sensor showed a linear response in concentration range 0.5–80 ng mL^-1,with a LOD of 0.33 ng mL^-1,good stability,high selective,and short detection time.The excellent applicability of the system has been verifed in milk and coffee samples,with relative recoveries between 98.33%and 103.70%.The results obtained were further verified by Ultra-high performance liquid chromatography–tandem mass spectrometry（UHPLC–MS/MS）.更多还原