本文選題：納米材料 + 廣譜性抗體　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：食品中真菌毒素嚴(yán)重威脅人類健康,其檢測(cè)技術(shù)研究一直是分析化學(xué)領(lǐng)域研究的熱點(diǎn)。但由于樣品基質(zhì)復(fù)雜,待測(cè)物大多是痕量或超痕量存在,現(xiàn)有前處理技術(shù)操作繁瑣,凈化效率低,特異性差,消耗大量有機(jī)溶劑,不能滿足實(shí)際檢測(cè)的要求,開(kāi)發(fā)簡(jiǎn)單、快速、高效、綠色環(huán)保的樣品前處理技術(shù)顯得尤為必要。本文將高親和力、高特異性的生物識(shí)別材料與分離高效、吸附性能好、結(jié)構(gòu)穩(wěn)定的磁性納米顆粒等載體有機(jī)結(jié)合,研究開(kāi)發(fā)高效分離凈化樣品前處理技術(shù),并將其應(yīng)用于谷物等基質(zhì)中黃曲霉毒素檢測(cè)。成功制備了免疫殼聚糖磁性微球,并對(duì)其進(jìn)行了表征和活性鑒定。通過(guò)一步水熱法合成了殼聚糖四氧化三鐵核-殼形納米顆粒(Fe_30_4@CTS),用XRD、SEM、TEM、FTIR、EDS和TGA對(duì)其進(jìn)行了表征,結(jié)果顯示所制備的Fe_30_4@CTS晶形良好,屬于核殼形結(jié)構(gòu),CTS成功包裹于Fe_30_4表面。采用EDC/sulfo-NHS法將黃曲霉毒素單克隆抗體(AFs-mAb)與Fe_30_4@CTS納米顆粒偶聯(lián)(Fe_30_4@CTS@AF_s-mAb),偶聯(lián)率大于90%,對(duì)AF_s-mAb的最大偶聯(lián)載量是23.5 mg/g。該微球?qū)?AFB_1、AFB_2、AFG_1、AFG_2、AFM_1AFM_2 的最大吸附量分別為 337、351、306、113、462 和 389 ng/mL�；谥苽涞腇e_30_4@CTS@AF_s-mAb微球,建立了分離凈化玉米、花生、橄欖油等樣品基質(zhì)中黃曲霉毒素(AFB_1、AFB_2、AFG_1、AFG_2、AFM_1和AFM_2)的前處理方法。樣品經(jīng)60%甲醇水提取,加入Fe_30_4@CTS@AF_s-mAb微球吸附后,棄去上清,再加入甲醇解吸,整個(gè)吸附解吸過(guò)程可在1 min內(nèi)完成,比傳統(tǒng)的SPE方法時(shí)間縮短了至少60倍。凈化樣品用UPLC-MS/MS檢測(cè),黃曲霉毒素檢測(cè)限在0.003-0.007 μg/kg之間,添加回收率范圍63.0%-112.0%,相對(duì)標(biāo)準(zhǔn)偏差小于15.6%。對(duì)AOAC方法標(biāo)定的3個(gè)標(biāo)準(zhǔn)參考樣本進(jìn)行了 Fe_3O_4@CTS@AF_s-mAb微球分離凈化處理測(cè)定,檢測(cè)結(jié)果與標(biāo)準(zhǔn)參考值一致。成功制備了 AF_s-mAb修飾還原氧化石墨烯薄膜,并將其用于兔血清中黃曲霉毒素的分離凈化。采用減壓抽濾法制備氧化石墨烯(GO)薄膜,經(jīng)高溫處理得到還原氧化石墨烯(rGO)薄膜,在rGO薄膜上通過(guò)π-π共輒作用修飾1-芘丁酸(PBA),再通過(guò)EDC/sulfo-NHS法偶聯(lián)鏈霉親和素(SA),最后與生物素化的AF_s-mAb偶聯(lián)(rGO@PBA@SA@B-AFs-mAb),偶聯(lián)率為99.5%。兔血清樣品經(jīng)酶解后,取100 μL滴加于rGO@PBA@SA@B-AF_s-mAb表面,凈化樣品用UPLC-MS/MS檢測(cè),黃曲霉毒素的檢測(cè)限在0.05-0.17 μg/L之間,添加回收率范圍55.1%-75.3%,相對(duì)標(biāo)準(zhǔn)偏差小于9.4%。研究建立了同時(shí)分離凈化玉米、大米、花生、啤酒、花生油、大豆油、醬油、豆醬共8種糧油制品中黃曲霉毒素的免疫親和色譜技術(shù)。以CNBr-Sepharose 4B為載體,偶聯(lián)AF_s-mAb,偶聯(lián)率大于90%,四種黃曲霉毒素最大吸附量在45-293 ng/mLgel之間。樣品用60%甲醇水提取,上樣后用去離子水洗滌,純甲醇洗脫,凈化樣品用UPLC-MS/MS檢測(cè),黃曲霉毒素檢測(cè)限在0.003-0.005μg/kg之間,添加回收率范圍60.2%-119.8%,相對(duì)標(biāo)準(zhǔn)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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS207.5

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本文編號(hào)：2057340