【摘要】：獸藥殘留問題不僅嚴(yán)重威脅人們的身體健康,進(jìn)入環(huán)境影響生態(tài),而且還嚴(yán)重影響我國(guó)的進(jìn)出口貿(mào)易。近幾年我國(guó)在農(nóng)副產(chǎn)品貿(mào)易中因殘留物超標(biāo)而導(dǎo)致的外貿(mào)經(jīng)濟(jì)損失每年高達(dá)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(duì)我國(guó)食品檢測(cè)量大,覆蓋面廣的問題,研發(fā)樣品前處理簡(jiǎn)單、快速特異檢測(cè)方法是形成食品安全監(jiān)測(cè)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(biāo),抗體為識(shí)別分子,免疫納米顆粒為信號(hào)元件,針對(duì)免疫層析色譜(ICA)快速檢測(cè)技術(shù)的檢測(cè)通量較低的不足,探索了雙通道免疫層析試紙技術(shù),實(shí)現(xiàn)了兩種目標(biāo)物在一個(gè)免疫層析色譜上的同時(shí)檢測(cè)分析,且操作簡(jiǎn)單,響應(yīng)迅速,為多元快速檢測(cè)的ICA方法提供了技術(shù)支持;為了進(jìn)一步提高檢測(cè)靈敏度,引入“turn-on”模式的生物傳感技術(shù),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(chǔ)上建立了靈敏檢測(cè)雌二醇的電化學(xué)發(fā)光共振能量轉(zhuǎn)移傳感技術(shù),實(shí)現(xiàn)了痕量殘留的檢測(cè),為電化學(xué)發(fā)光共振能量轉(zhuǎn)移技術(shù)在食品安全領(lǐng)域的應(yīng)用提供技術(shù)支撐。1.雙通道免疫層析技術(shù)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(biāo)抗原與BSA偶聯(lián)物固定到硝酸纖維素膜上的兩個(gè)檢測(cè)區(qū)域,以形成T1和T2兩條檢測(cè)線;兩種單克隆抗體與膠體金偶聯(lián)物吸附于結(jié)合墊;在樣品端口加入樣品后,由于毛細(xì)作用力,樣品流過結(jié)合墊和檢測(cè)線,從而實(shí)現(xiàn)二種物質(zhì)的同時(shí)檢測(cè),其不需借助儀器,可實(shí)現(xiàn)裸眼檢測(cè)。優(yōu)化了抗原抗體濃度,以及封閉液的選擇,在最佳條件實(shí)現(xiàn)了對(duì)DES的快速檢測(cè),分別在實(shí)際樣品如蝦組織、液態(tài)奶中檢測(cè)限達(dá)到25 ng/g,在奶粉中達(dá)到30ng/g,其結(jié)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(cè)限分別達(dá)到75ng/g、65ng/g和60ng/g,并且其結(jié)果可以3-4分鐘內(nèi)進(jìn)行判斷。采用HPLC-MS/MS進(jìn)行方法比對(duì),通過測(cè)試從當(dāng)?shù)厥袌?chǎng)購(gòu)得的37份牛奶和蝦組織樣本,通過統(tǒng)計(jì)學(xué)分析,證明這兩種方法之間結(jié)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(cè)試紙條的構(gòu)建為多元快速檢測(cè)的ICA方法提供了技術(shù)支持,如果進(jìn)一步擴(kuò)展,就可以在同一條的免疫層析試紙實(shí)現(xiàn)多種殘留物的檢測(cè),從而使其在食品安全等領(lǐng)域發(fā)揮更加重要的作用。2.雙功能復(fù)合納米顆粒可控合成研究分別采用高溫?zé)岱纸夥�、溶劑熱法、共沉淀法制備了四氧化三鐵(Fe3O4),通過TEM結(jié)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?zé)岱纸夥ǖ玫搅藛畏稚⒌挠退帷⒂桶钒坏腇e3O4,但是不能分散在水溶液中,限制了其進(jìn)一步應(yīng)用,后續(xù)通過相轉(zhuǎn)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(shí)現(xiàn)Au殼的逐步生長(zhǎng),進(jìn)而實(shí)現(xiàn)了復(fù)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(dòng)樣品磁強(qiáng)計(jì)(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(diǎn)電極電化學(xué)發(fā)光行為研究本章以硫化鎘(CdS)量子點(diǎn)為發(fā)光體,多壁碳納米管為信號(hào)放大材料,構(gòu)建了新型CdS量子點(diǎn)電化學(xué)發(fā)光體系。合成水溶性CdS量子點(diǎn),并進(jìn)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(diǎn)電化學(xué)發(fā)光機(jī)理,探討了多壁碳納米管(MWCNTs),殼聚糖(CS)對(duì)CdS量子點(diǎn)電化學(xué)發(fā)光的影響,考察了兩種碳納米管對(duì)ECL信號(hào)的影響,優(yōu)化了發(fā)光條件,pH以及量子點(diǎn)的含量,最終實(shí)現(xiàn)了多壁碳納米管對(duì)CdS的電化學(xué)發(fā)光(ECL)信號(hào)的放大,放大效果達(dá)10倍。這種簡(jiǎn)單、高效的修飾方法為量子點(diǎn)電化學(xué)發(fā)光免疫檢測(cè)的研究奠定了基礎(chǔ)。4.電化學(xué)發(fā)光共振能量轉(zhuǎn)移免疫檢測(cè)技術(shù)研究以一種新型雙功能磁性納米復(fù)合物Fe3O4@Au作為ECL能量受體,CdS量子點(diǎn)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(zhuǎn)移免疫分析方法。將Fe3O4@Au與雌二醇抗體偶聯(lián),通過間接競(jìng)爭(zhēng)免疫的方法,構(gòu)建了靈敏的雌二醇ECL-RET傳感檢測(cè)技術(shù),其線性方程為A=472.2lgC+9523,相關(guān)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(gè)數(shù)量級(jí)。雙功能納米顆粒Fe3O4@Au與量子點(diǎn)電化學(xué)發(fā)光相結(jié)合,實(shí)現(xiàn)了新型共振能量轉(zhuǎn)移的免疫檢測(cè)策略,為高靈敏檢測(cè)痕量污染物提供了一個(gè)富有應(yīng)用前景的技術(shù)平臺(tái)。
[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(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：O657;R155.5

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐國(guó)寶,董紹俊;電化學(xué)發(fā)光及其應(yīng)用[J];分析化學(xué);2001年01期

2 張?chǎng)┢G,闕肖冬,馬興剛,錢麗娜,潘維平,郭玉芹;電化學(xué)發(fā)光在分析科學(xué)中的應(yīng)用[J];中國(guó)衛(wèi)生工程學(xué);2002年01期

3 安鏡如;林金明;;新試劑5-(對(duì)-苯胺偶氮)-2,3-二氫-1,4-酞嗪二酮的電化學(xué)發(fā)光研究[J];分析化學(xué);1991年03期

4 呂家根,章竹君,鄭鵠志;電化學(xué)發(fā)光新體系及其在原位、在線、實(shí)時(shí)監(jiān)測(cè)家兔血液銅代謝過程中的應(yīng)用[J];化學(xué)學(xué)報(bào);2002年07期

5 袁濤;劉中原;胡連哲;徐國(guó)寶;;電化學(xué)和電化學(xué)發(fā)光核酸適體傳感器[J];分析化學(xué);2011年07期

6 林金明,安鏡如;新試劑6-(2-羥基-4-二乙基氨苯偶氮)-2,3-二氫-1.4-酞嗪二酮的電化學(xué)發(fā)光研究[J];福州大學(xué)學(xué)報(bào)(自然科學(xué)版);1991年01期

7 王鵬,張文艷,周泓,朱果逸;免疫電化學(xué)發(fā)光[J];分析化學(xué);1998年07期

8 張成孝,漆紅蘭;電化學(xué)發(fā)光分析研究進(jìn)展[J];世界科技研究與發(fā)展;2004年04期

9 童碧海;梅群波;李志文;董永平;張千峰;;系列2-苯基喹啉類銥配合物的合成及電化學(xué)發(fā)光性能研究[J];化學(xué)學(xué)報(bào);2012年23期

10 席強(qiáng);王捷;陳鈺;劉仲明;;量子點(diǎn)電化學(xué)發(fā)光及其在生物分析中的應(yīng)用[J];化學(xué)研究;2014年02期

相關(guān)會(huì)議論文 前10條

1 呂家根;;一種新的與微型化自發(fā)電池整合的電化學(xué)發(fā)光檢測(cè)芯片研究[A];第二屆全國(guó)微全分析系統(tǒng)學(xué)術(shù)會(huì)議論文摘要集[C];2004年

2 漆紅蘭;高紅方;趙瑩;張成孝;;有機(jī)物及有機(jī)納米顆粒電化學(xué)和電化學(xué)發(fā)光行為研究[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第04分會(huì)：納米生物傳感新方法[C];2014年

3 余林頗;黃澤柱;劉揚(yáng);周明;;水溶性環(huán)金屬銥配合物的電化學(xué)發(fā)光[A];中國(guó)化學(xué)會(huì)第27屆學(xué)術(shù)年會(huì)第09分會(huì)場(chǎng)摘要集[C];2010年

4 楊秀榮;邱海波;嚴(yán)吉林;趙曉翠;汪爾康;;微芯片毛細(xì)管電泳電化學(xué)/電化學(xué)發(fā)光同時(shí)檢測(cè)藥物分子[A];第二屆全國(guó)微全分析系統(tǒng)學(xué)術(shù)會(huì)議論文摘要集[C];2004年

5 董曼曼;黨倩;黃銀;漆紅蘭;張成孝;;電化學(xué)發(fā)光生物傳感器測(cè)定酪蛋白激酶Ⅱ[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第04分會(huì)：納米生物傳感新方法[C];2014年

6 陳金花;林振宇;陳國(guó)南;;電化學(xué)發(fā)光猝滅法測(cè)定桑葉中黃酮類成分[A];第八屆全國(guó)發(fā)光分析暨動(dòng)力學(xué)分析學(xué)術(shù)研討會(huì)論文集[C];2005年

7 胡連哲;韓雙;李海娟;徐國(guó)寶;;電化學(xué)發(fā)光分析新材料及新應(yīng)用[A];中國(guó)化學(xué)會(huì)第27屆學(xué)術(shù)年會(huì)第09分會(huì)場(chǎng)摘要集[C];2010年

8 張成孝;張靜;漆紅蘭;;納米磁性粒子電化學(xué)發(fā)光生物傳感新方法[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第04分會(huì)：納米生物傳感新方法[C];2014年

9 周小明;邢達(dá);;基于磁珠和納米粒子的電化學(xué)發(fā)光擴(kuò)增方法及其在高靈敏的基因檢測(cè)中的應(yīng)用[A];第十一次中國(guó)生物物理學(xué)術(shù)大會(huì)暨第九屆全國(guó)會(huì)員代表大會(huì)摘要集[C];2009年

10 尹學(xué)博;辛有英;劉東元;唐春霞;趙s，

本文編號(hào)：2213911