基于黄曲霉毒素广谱性抗体的高效分离净化样品前处理技术研究

发布时间：2018-06-23 13:56

本文选题：纳米材料 + 广谱性抗体　；参考：《中国农业大学》2017年博士论文

【摘要】：食品中真菌毒素严重威胁人类健康,其检测技术研究一直是分析化学领域研究的热点。但由于样品基质复杂,待测物大多是痕量或超痕量存在,现有前处理技术操作繁琐,净化效率低,特异性差,消耗大量有机溶剂,不能满足实际检测的要求,开发简单、快速、高效、绿色环保的样品前处理技术显得尤为必要。本文将高亲和力、高特异性的生物识别材料与分离高效、吸附性能好、结构稳定的磁性纳米颗粒等载体有机结合,研究开发高效分离净化样品前处理技术,并将其应用于谷物等基质中黄曲霉毒素检测。成功制备了免疫壳聚糖磁性微球,并对其进行了表征和活性鉴定。通过一步水热法合成了壳聚糖四氧化三铁核-壳形纳米颗粒(Fe_30_4@CTS),用XRD、SEM、TEM、FTIR、EDS和TGA对其进行了表征,结果显示所制备的Fe_30_4@CTS晶形良好,属于核壳形结构,CTS成功包裹于Fe_30_4表面。采用EDC/sulfo-NHS法将黄曲霉毒素单克隆抗体(AFs-mAb)与Fe_30_4@CTS纳米颗粒偶联(Fe_30_4@CTS@AF_s-mAb),偶联率大于90%,对AF_s-mAb的最大偶联载量是23.5 mg/g。该微球对 AFB_1、AFB_2、AFG_1、AFG_2、AFM_1AFM_2 的最大吸附量分别为 337、351、306、113、462 和 389 ng/mL。基于制备的Fe_30_4@CTS@AF_s-mAb微球,建立了分离净化玉米、花生、橄榄油等样品基质中黄曲霉毒素(AFB_1、AFB_2、AFG_1、AFG_2、AFM_1和AFM_2)的前处理方法。样品经60%甲醇水提取,加入Fe_30_4@CTS@AF_s-mAb微球吸附后,弃去上清,再加入甲醇解吸,整个吸附解吸过程可在1 min内完成,比传统的SPE方法时间缩短了至少60倍。净化样品用UPLC-MS/MS检测,黄曲霉毒素检测限在0.003-0.007 μg/kg之间,添加回收率范围63.0%-112.0%,相对标准偏差小于15.6%。对AOAC方法标定的3个标准参考样本进行了 Fe_3O_4@CTS@AF_s-mAb微球分离净化处理测定,检测结果与标准参考值一致。成功制备了 AF_s-mAb修饰还原氧化石墨烯薄膜,并将其用于兔血清中黄曲霉毒素的分离净化。采用减压抽滤法制备氧化石墨烯(GO)薄膜,经高温处理得到还原氧化石墨烯(rGO)薄膜,在rGO薄膜上通过π-π共辄作用修饰1-芘丁酸(PBA),再通过EDC/sulfo-NHS法偶联链霉亲和素(SA),最后与生物素化的AF_s-mAb偶联(rGO@PBA@SA@B-AFs-mAb),偶联率为99.5%。兔血清样品经酶解后,取100 μL滴加于rGO@PBA@SA@B-AF_s-mAb表面,净化样品用UPLC-MS/MS检测,黄曲霉毒素的检测限在0.05-0.17 μg/L之间,添加回收率范围55.1%-75.3%,相对标准偏差小于9.4%。研究建立了同时分离净化玉米、大米、花生、啤酒、花生油、大豆油、酱油、豆酱共8种粮油制品中黄曲霉毒素的免疫亲和色谱技术。以CNBr-Sepharose 4B为载体,偶联AF_s-mAb,偶联率大于90%,四种黄曲霉毒素最大吸附量在45-293 ng/mLgel之间。样品用60%甲醇水提取,上样后用去离子水洗涤,纯甲醇洗脱,净化样品用UPLC-MS/MS检测,黄曲霉毒素检测限在0.003-0.005μg/kg之间,添加回收率范围60.2%-119.8%,相对标准偏差小于15.7%。
[Abstract]:Mycotoxin in food is a serious threat to human health, and its detection technology has been a hot spot in analytical chemistry. However, due to the complexity of the sample matrix and the presence of trace or ultra-trace samples, the existing pretreatment techniques are complicated in operation, low in purification efficiency, poor in specificity and consumption of a large amount of organic solvents, which can not meet the requirements of actual detection and can not be developed easily and quickly. High efficiency, green sample pretreatment technology is particularly necessary. In this paper, the high affinity, high specificity biometric materials were combined with the carriers with high separation efficiency, good adsorption performance and stable structure to study and develop the high efficiency separation and purification sample pretreatment technology. It was applied to the detection of aflatoxin in cereals and other substrates. The immuno-chitosan magnetic microspheres were successfully prepared and characterized. Fe30304CTS (Fe30304CTS) was synthesized by one step hydrothermal method and characterized by means of XRDX SEMSEMTEMTEMEREDS and TGA. The results show that the prepared Fe304CTS has a good crystal shape and belongs to the core-shell structure CTS which is successfully encapsulated on the surface of Fe304. The aflatoxin monoclonal antibody (AFs-mAb) was coupled with Fe304CTS nanoparticles by EDC / sulfo-NHS method. The coupling rate of AFs-mAb was more than 90%. The maximum coupling capacity of AFs-mAb was 23.5 mg / g. The maximum adsorption capacity of the microsphere for AFB1AFB _ 2 / AFG _ S _ 1 and AFG _ 2T _ 2AFM _ 2 is 337351306113462 and 389 ng / mL, respectively. The maximum adsorption capacity of the microsphere is 337351306113462 and 389ng / mL, respectively. Based on the microspheres of Fe304CTSAFS-mAb, a pretreatment method for separating and purifying aflatoxin (AFB1) from corn, peanut, olive oil and other sample substrates was established. The sample was extracted with 60% methanol water, and then adsorbed by Festud 304CTS @ AFs-mAb microspheres, then the supernatant was discarded and methanol desorbed. The whole adsorption and desorption process could be completed within 1 min, which was 60 times shorter than that of the traditional SPE method. The detection limit of aflatoxin was 0.003-0.007 渭 g/kg, the recovery range was 63.0 ~ 112.0, and the relative standard deviation was less than 15.60.The purified sample was detected by UPLC-MS / MS, and the detection limit of aflatoxin was 0.003-0.007 渭 g/kg. Three standard reference samples calibrated by AOAC method were separated and purified by Fe3O\ +\ {4CTSAFs-mAb\} microspheres, and the results were in agreement with the standard reference values. Aflatoxin in rabbit serum was successfully purified by AFs-S mAb modified reduced graphene thin film. Graphene oxide (go) thin films were prepared by vacuum filtration, and reduced graphene oxide (rGO) films were obtained by high temperature treatment. The 1-pyrene butyric acid (PBA) was modified by 蟺 -co-action on rGo thin film, and then the SA was coupled by EDC / sulfo-NHS method. Finally, the biotinylated AFs-mAb (rGOPBAOR-BAB-AFs-mAb) was coupled with biotinylated SAB-AFs-mAb. The coupling rate was 99.555%. After enzymatic hydrolysis, 100 渭 L of rabbit serum samples were added to the surface of rGOOIBALR PBAORSAAORTABOR-BAFS-mAb. The purified samples were detected by UPLC-MS-MS. The detection limit of aflatoxin was 0.05-0.17 渭 g / L, the recovery range was 55.1 -75.3 and the relative standard deviation was less than 9.4%. An immunoaffinity chromatography technique for simultaneous separation and purification of aflatoxin from corn, rice, peanut, beer, peanut oil, soybean oil, soy sauce and soy sauce was established. Using CNBr-Sepharose 4B as the carrier, AFs-mAbcoupling was carried out, and the coupling rate was greater than 90. The maximum adsorption capacity of four aflatoxins was between 45-293 ng / mLgel. The samples were extracted with 60% methanol water, washed with deionized water and eluted with pure methanol. The purified samples were detected by UPLC-MS / MS. The detection limit of aflatoxin was 0.003-0.005 渭 g/kg. The recovery range was 60.2-119.8.The relative standard deviation was less than 15.7.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS207.5

【参考文献】