【摘要】：獸藥殘留問題不僅嚴重威脅人們的身體健康,進入環(huán)境影響生態(tài),而且還嚴重影響我國的進出口貿(mào)易。近幾年我國在農(nóng)副產(chǎn)品貿(mào)易中因殘留物超標而導致的外貿(mào)經(jīng)濟損失每年高達數(shù)百億美元。食品生產(chǎn)過程中獸藥的非法、不合理使用是造成這種現(xiàn)狀的重要因素。因而,面對我國食品檢測量大,覆蓋面廣的問題,研發(fā)樣品前處理簡單、快速特異檢測方法是形成食品安全監(jiān)測能力的重要保障之一。本論文以獸藥殘留物己烯雌酚(DES)和雌二醇(E2)為靶標,抗體為識別分子,免疫納米顆粒為信號元件,針對免疫層析色譜(ICA)快速檢測技術的檢測通量較低的不足,探索了雙通道免疫層析試紙技術,實現(xiàn)了兩種目標物在一個免疫層析色譜上的同時檢測分析,且操作簡單,響應迅速,為多元快速檢測的ICA方法提供了技術支持;為了進一步提高檢測靈敏度,引入“turn-on”模式的生物傳感技術,制備了新型光譜可調(diào)的雙功能磁性納米顆粒,并在此基礎上建立了靈敏檢測雌二醇的電化學發(fā)光共振能量轉移傳感技術,實現(xiàn)了痕量殘留的檢測,為電化學發(fā)光共振能量轉移技術在食品安全領域的應用提供技術支撐。1.雙通道免疫層析技術研究首先合成了己烯雌酚半抗原,雌二醇半抗原,將其分別與OVA與BSA偶聯(lián)制備完全抗原,優(yōu)化后選取靶標抗原與BSA偶聯(lián)物固定到硝酸纖維素膜上的兩個檢測區(qū)域,以形成T1和T2兩條檢測線;兩種單克隆抗體與膠體金偶聯(lián)物吸附于結合墊;在樣品端口加入樣品后,由于毛細作用力,樣品流過結合墊和檢測線,從而實現(xiàn)二種物質(zhì)的同時檢測,其不需借助儀器,可實現(xiàn)裸眼檢測。優(yōu)化了抗原抗體濃度,以及封閉液的選擇,在最佳條件實現(xiàn)了對DES的快速檢測,分別在實際樣品如蝦組織、液態(tài)奶中檢測限達到25 ng/g,在奶粉中達到30ng/g,其結果可以在7-10分鐘判讀;E2在奶粉、液態(tài)奶及蝦肉組織的檢測限分別達到75ng/g、65ng/g和60ng/g,并且其結果可以3-4分鐘內(nèi)進行判斷。采用HPLC-MS/MS進行方法比對,通過測試從當?shù)厥袌鲑彽玫?7份牛奶和蝦組織樣本,通過統(tǒng)計學分析,證明這兩種方法之間結果無顯著性差異,具有良好的一致性。新型二聯(lián)免疫層析測試紙條的構建為多元快速檢測的ICA方法提供了技術支持,如果進一步擴展,就可以在同一條的免疫層析試紙實現(xiàn)多種殘留物的檢測,從而使其在食品安全等領域發(fā)揮更加重要的作用。2.雙功能復合納米顆�？煽睾铣裳芯糠謩e采用高溫熱分解法、溶劑熱法、共沉淀法制備了四氧化三鐵(Fe3O4),通過TEM結果可知,溶劑熱法合成的Fe3O4納米粒子粒徑均一但是較大,不利于下一步的Au殼的包覆;高溫熱分解法得到了單分散的油酸、油胺包被的Fe3O4,但是不能分散在水溶液中,限制了其進一步應用,后續(xù)通過相轉移手段,使其可分散在水溶液中,并在水相中完成了Au殼的包覆,通過調(diào)節(jié)巰基丙酸的量,成功實現(xiàn)Au殼的逐步生長,進而實現(xiàn)了復合納米Fe3O4@Au光譜調(diào)控550-575nm;共沉淀法合成的Fe3O4,雖然形貌不如前兩者,但其體積小、水溶性優(yōu),利于Au殼的包覆,成功制備了Fe3O4@Au,通過振動樣品磁強計(VSM)表征,Fe3O4:49.83emu/g;Fe3O4@Au:39.22emu/g,表明了磁性較好的保留,可以用于樣品前處理。3.多壁碳納米管-量子點電極電化學發(fā)光行為研究本章以硫化鎘(CdS)量子點為發(fā)光體,多壁碳納米管為信號放大材料,構建了新型CdS量子點電化學發(fā)光體系。合成水溶性CdS量子點,并進行了熒光光譜(PL)、紫外可見(UV-vis)、透射電鏡(TEM)表征。采用了CS-QD-MWCNTs-GCE的電極修飾方式,研究了量子點電化學發(fā)光機理,探討了多壁碳納米管(MWCNTs),殼聚糖(CS)對CdS量子點電化學發(fā)光的影響,考察了兩種碳納米管對ECL信號的影響,優(yōu)化了發(fā)光條件,pH以及量子點的含量,最終實現(xiàn)了多壁碳納米管對CdS的電化學發(fā)光(ECL)信號的放大,放大效果達10倍。這種簡單、高效的修飾方法為量子點電化學發(fā)光免疫檢測的研究奠定了基礎。4.電化學發(fā)光共振能量轉移免疫檢測技術研究以一種新型雙功能磁性納米復合物Fe3O4@Au作為ECL能量受體,CdS量子點修飾電極為能量供體,建立了一種“turn-on”模式的ECL共振能量轉移免疫分析方法。將Fe3O4@Au與雌二醇抗體偶聯(lián),通過間接競爭免疫的方法,構建了靈敏的雌二醇ECL-RET傳感檢測技術,其線性方程為A=472.2lgC+9523,相關系數(shù)(R2)為0.9936,線性范圍為2.0×10-10~2.0×10-4mg/mL,最低檢出限為3.2×10-12mg/mL,與二聯(lián)試紙條相比提高了3個數(shù)量級。雙功能納米顆粒Fe3O4@Au與量子點電化學發(fā)光相結合,實現(xiàn)了新型共振能量轉移的免疫檢測策略,為高靈敏檢測痕量污染物提供了一個富有應用前景的技術平臺。
[Abstract]:The problem of veterinary drug residues not only seriously threatens people's health, enters the environment and ecology, but also seriously affects China's import and export trade. Therefore, in the face of the problem of large detection and wide coverage of food in China, the development of sample pretreatment is simple, rapid and specific detection method is one of the important guarantees to form food safety monitoring ability. In this paper, veterinary drug residues diethylstilbestrol (DES) and estradiol (E2) as the target, antibodies as recognition molecules, immunity. Nanoparticles are signal components. Aiming at the low detection flux of immunochromatographic (ICA) rapid detection technology, a dual-channel immunochromatographic test paper technology is explored, which realizes the simultaneous detection and analysis of two target substances on an immunochromatographic (ICT) system with simple operation and rapid response. In order to further improve the detection sensitivity, the "turn-on" mode biosensor technology was introduced to prepare a new type of dual-functional magnetic nanoparticles with adjustable spectra. On this basis, a sensitive electrochemiluminescence resonance energy transfer sensing technology for the detection of estradiol was established, which realized the detection of trace residues and electrochemiluminescence. Resonance energy transfer technology provides technical support for the application of food safety. 1. Two-channel immunochromatographic techniques were used to synthesize diethylstilbestrol hapten and estradiol hapten, and then coupled with OVA and BSA to prepare complete antigen respectively. After optimization, target antigen and BSA conjugate were selected and fixed on nitrocellulose membrane for two detection. Two detection lines, T1 and T2, were formed, two monoclonal antibodies and colloidal gold conjugates were adsorbed on the bonding pad, and after the sample was added at the sample port, the sample flowed through the bonding pad and the detection line due to capillary force, thus achieving the simultaneous detection of the two substances without the aid of an instrument. The concentration of antigen and antibody was optimized. The detection limits of DES in real samples such as shrimp tissues and liquid milk are 25 ng/g and 30 ng/g respectively, and the results can be interpreted in 7-10 minutes; the detection limits of E2 in milk powder, liquid milk and shrimp meat tissues are 75 ng/g, 65 ng/g and 60 ng/g respectively, and the results are satisfactory. The results can be judged in 3-4 minutes. The method of HPLC-MS/MS was used to compare 37 samples of milk and shrimp tissues purchased from the local market. Statistical analysis showed that there was no significant difference between the two methods and the results had good consistency. The ICA method provides technical support, if further expanded, it can be used in the same immunochromatographic test paper to detect a variety of residues, so that it can play a more important role in food safety and other fields. 2. The controllable synthesis of bifunctional composite nanoparticles were studied by high temperature thermal decomposition, solvothermal method and coprecipitation method, respectively. Fe3O4 was prepared. TEM results showed that the size of Fe 3O4 nanoparticles synthesized by solvothermal method was uniform but large, which was not conducive to the coating of Al shell. Monodispersed oleic acid and oleamine coated Fe 3O4 were obtained by high temperature pyrolysis method, but could not be dispersed in aqueous solution, which limited its further application. The Au shell can be dispersed in aqueous solution and coated in aqueous phase by transfer method. By adjusting the amount of Mercaptopropionic acid, the Au shell can be grown step by step, and then the composite nano-Fe3O4@Au spectrum can be controlled 550-575 nm. Fe3O4 @ Au was successfully prepared and characterized by vibrating sample magnetometer (VSM), Fe3O4:49.83emu/g; Fe3O4@Au:39.22emu/g, which indicated that the magnetic properties were well retained and could be used for sample pretreatment. A novel electrochemiluminescence system for CdS quantum dots was constructed. Water-soluble CdS quantum dots were synthesized and characterized by fluorescence spectroscopy (PL), ultraviolet-visible spectroscopy (UV-vis) and transmission electron microscopy (TEM). The effects of carbohydrate (CS) on the electrochemiluminescence (ECL) of CdS quantum dots were investigated. The luminescence conditions, pH and quantum dot content were optimized. Finally, the amplification effect of multi-walled carbon nanotubes (MWCNTs) on the electrochemiluminescence (ECL) of CdS quantum dots was 10 times. This simple and efficient modification method was quantum dot electrochemistry. 4. A novel bifunctional magnetic nanocomposite Fe3O4@Au was used as the ECL energy receptor and CdS quantum dot modified electrode as the energy donor to establish a "turn-on" mode of ECL resonance energy transfer immunoassay. 3O4@Au was coupled with estradiol antibody by indirect competitive immunization. A sensitive ECL-RET sensor for estradiol detection was constructed. The linear equation was A=472.2lgC+9523, the correlation coefficient (R2) was 0.9936, the linear range was 2.0 *10-10~2.0 *10-4mg/mL, and the minimum detection limit was 3.2 *10-12mg/mL, which was three orders of magnitude higher than that of the binary test strip. Bifunctional nanoparticles Fe3O4@Au combined with electrochemiluminescence of quantum dots (QDs) to achieve a novel resonance energy transfer immunoassay strategy, providing a promising platform for highly sensitive detection of trace pollutants.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：O657;R155.5

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