本文選題：鎘 + X射線吸收精細(xì)結(jié)構(gòu)光譜��； 參考：《華中農(nóng)業(yè)大學(xué)》2011年碩士論文

【摘要】：鎘(Cadmium, Cd)是最常見的污染食品和飲料的重金屬元素之一。Cd可以通過環(huán)境污染,生物濃縮和含Cd化肥的使用而導(dǎo)致食品和水體的污染。我國水稻、蔬菜等農(nóng)作物Cd的檢出率較高,超標(biāo)現(xiàn)象嚴(yán)重。它具有較強(qiáng)的腎毒性、肝毒性、生殖毒性、免疫毒性、骨骼毒性、心腦血管毒性以及致癌等危害。目前,Cd的檢測方法主要是儀器方法。該方法具有測量精度高,結(jié)果重復(fù)性好,特異性好,但大多需要借助大型儀器,成本較高,且前處理繁瑣,不能滿足現(xiàn)場、在線分析檢測。而ELISA等免疫學(xué)方法,以其簡單、快速、成本低且適應(yīng)環(huán)境及市場的現(xiàn)場檢測等優(yōu)點(diǎn)受到廣泛青睞。 本研究采用雙功能螯合劑p-SCN-Bn-DTPA構(gòu)建了Cd離子的人工抗原,并采用紫外光譜法、三硝基苯磺酸法、原子吸收光譜法、聚丙烯酰胺凝膠電泳以及X射線吸收精細(xì)結(jié)構(gòu)光譜對(duì)其進(jìn)行分析鑒定。然后以構(gòu)建成功的人工抗原免疫BALB/c小鼠,采用ELISA方法對(duì)抗血清的各項(xiàng)特性進(jìn)行了研究,并采用間接競爭ELISA進(jìn)行了加標(biāo)回收和實(shí)際水樣的檢測,具體內(nèi)容如下： 1鎘離子人工抗原的構(gòu)建 Cd是金屬元素,分子量僅為112.4,它既沒有反應(yīng)原性,又沒有免疫原性,只有通過雙功能螫合劑與蛋白質(zhì)等載體偶聯(lián)后,才能刺激機(jī)體體產(chǎn)生抗體。本實(shí)驗(yàn)利用雙功能螯合劑p-SCN-Bn-DTPA (2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid,2-(4-異硫氰酸根芐基)-二乙烯三胺五乙酸)上的DTPA基團(tuán)螯合Cd離子,并通過其SCN與載體蛋白牛血清白蛋白(bovine serum albumin, BSA)和卵清白蛋白(ovalbumin,OVA)的游離氨基偶聯(lián),制備得到免疫抗原Cd-p-SCN-Bn-DTPA-BSA (Cd-BSA)和包被抗原Cd-p-SCN-Bn-DTPA-OVA (Cd-OVA)。同時(shí),為了分析Cd的偶聯(lián)情況,還構(gòu)建了p-SCN-Bn-DTPA-BSA (DTPA-BSA)、p-SCN-Bn-DTPA-OVA (DTPA-OVA)和Cd-DTPA。 2鎘離子人工抗原的表征 通過BSA、OVA、DTPA-BSA和DTPA-OVA的紫外光譜掃描分析,發(fā)現(xiàn)DTPA-BSA和DTPA-OVA的最大吸收波長較BSA和OVA發(fā)生了微弱的藍(lán)移,分別是278 nm到270nm (BSA)和280 nm到268nm (OVA),它們的紫外掃描曲線也發(fā)生了改變。 采用2,4,6-三硝基苯磺酸法(triniro-benzene-sulfonic-acid)法對(duì)Cd-BSA和Cd-OVA中游離氨基含量進(jìn)行了測定,Cd-BSA中游離氨基數(shù)目為BSA的48.8%,而Cd-OVA的為OVA的41.4%。 SDS-PAGE結(jié)果表明,BSA (OVA)、DTPA-BSA (DTPA-OVA和Cd-BSA (Cd-OVA)的分子量是依次遞增的。 擴(kuò)展X射線吸收精細(xì)結(jié)構(gòu)光譜(extended X-ray absorption fine structure)結(jié)果表明,Cd-DTPA、Cd-BSA和Cd-OVA中Cd2+的配位類型及配位數(shù)基本相同。同時(shí),它們的X射線吸收近邊結(jié)構(gòu)光譜(X-ray absorption near edge structure)結(jié)果表明,Cd-BSA和Cd-OVA中Cd2+的吸收邊位置、形狀、吸收峰強(qiáng)度均與Cd-DTPA的類似。吸收邊位置相似表明,Cd-DTPA、Cd-BSA、Cd-OVA三種抗原中Cd離子的局部結(jié)構(gòu)是一致的；吸收邊形狀相似,說明Cd-BSA和Cd-OVA與Cd-DTPA中Cd2+與DTPA形成螫合物的；吸收峰強(qiáng)度一致,則表明兩種人工抗原中的Cd與Cd-DTPA中Cd一樣均呈+2價(jià)。 綜上所述,人工抗原Cd-BSA和Cd-OVA構(gòu)建成功。 根據(jù)Cd-BSA和Cd-OVA的中游離氨基的替換率,結(jié)合它們的蛋白質(zhì)含量,計(jì)算得出Cd-BSA和Cd-OVA中Cd2+與BSA和OVA的摩爾比分為28.7：1和11.7：1。 3抗鎘離子抗血清的制備及其性質(zhì)分析 以Cd-BSA免疫三組BABL/c小鼠,每組三只。免疫周期均為首次免疫21d,后續(xù)加強(qiáng)免疫均為14d,免疫劑量依次為200、100和50μg/只/次。通過抗體產(chǎn)生進(jìn)程曲線可以看出,三組BALB/c小鼠對(duì)Cd-BSA都能產(chǎn)生免疫應(yīng)答,不同小鼠的免疫應(yīng)答水平不一樣,但總體的應(yīng)答趨勢基本一致。對(duì)三組小鼠第五次免疫后的抗血清進(jìn)行性質(zhì)分析,其效價(jià)從2.6×104到4.2x104不等,差異不大。 經(jīng)方陣滴定實(shí)驗(yàn)確定的三組小鼠抗原-抗體反應(yīng)最佳工作條件后,采用間接競爭ELISA方法對(duì)三組小鼠抗血清進(jìn)行分析,測得1號(hào)小鼠靈敏度為120ng/mL,3、4、5、6、7、8和9號(hào)小鼠靈敏度分別為200、70、59、58、155、150和167 ng/mL。選取三組小鼠中的1號(hào)、6號(hào)和8號(hào)小鼠抗血清進(jìn)行特異性分析,結(jié)果顯示,抗血清均對(duì)汞離子有一定的交叉反應(yīng),但是與鉛、鐵、銅、鋅等其他參試金屬離子沒有交叉反應(yīng)。 4鎘離子ELISA方法的建立與湖水中鎘離子的檢測 選取6號(hào)小鼠的抗血清為材料,通過間接競爭ELISA分析,建立了Cd2+的間接競爭ELISA方法。采用本方法,在自來水中添加Cd2+的濃度為10-200 ng/mL時(shí),其線性方程為y=-20.62 x+87.069(R2=0.9919),加標(biāo)回收率為85.3%-90.3%,平均為86.59%。從武漢市的三個(gè)湖泊中共采集3個(gè)樣品進(jìn)行Cd2+的ELISA,檢測結(jié)果顯示A湖中Cd2+濃度達(dá)到6.08ng/mL,與AAS分析結(jié)果的相符性為105.7%。
[Abstract]:Cadmium (Cd) is one of the most common heavy metals in contaminated food and beverages..Cd can cause food and water pollution through environmental pollution, biological concentration and the use of Cd chemical fertilizer. The detection rate of Cd in rice and vegetables in China is high and the phenomenon is serious. It has strong renal toxicity, hepatotoxicity, reproductive toxicity and immunity. At present, the method of detection of Cd is mainly instrument method. This method has high precision, good repeatability and good specificity, but most of them need large instrument, and the cost is high, and the preprocessing is tedious. It can not meet the site, on-line analysis and detection. And ELISA and other immunological parties The method is widely favored because of its simple, fast, low cost and adaptability to environmental and market site detection.
In this study, a double functional chelating agent p-SCN-Bn-DTPA was used to construct an artificial antigen of Cd ion, and it was identified by UV spectrometry, three nitrobenzene sulfonic acid method, atomic absorption spectrometry, polyacrylamide gel electrophoresis and X ray absorption fine structure spectrum. Then, a successful artificial antigen was used to immunize BALB/c mice and to use E. LISA method was used to study the characteristics of anti serum, and indirect competitive ELISA was used for the recovery of standard addition and the detection of actual water samples.
Construction of 1 cadmium ion artificial antigen
Cd is a metal element with a molecular weight of only 112.4. It does not respond to the original and immunogenicity. Only through the coupling of the double functional chelating agent to the protein and other carriers can the body produce antibodies. This experiment uses the bifunctional chelating agent p-SCN-Bn-DTPA (2- (4-isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid, 2- (4- different). DTPA group of thiocyanate benzyl) - two ethylene three amine five acetic acid chelate Cd ion, and through its SCN and carrier protein bovine serum albumin (bovine serum albumin, BSA) and ovalbumin (ovalbumin, OVA) free amino coupling, preparation of immune antigen Cd-p-SCN-Bn-DTPA-BSA (Cd-BSA) and inclusion antigen Cd-p-SCN-Bn-DTPA-OVA. OVA. At the same time, in order to analyze the coupling of Cd, p-SCN-Bn-DTPA-BSA (DTPA-BSA), p-SCN-Bn-DTPA-OVA (DTPA-OVA) and Cd-DTPA. were also constructed.
Characterization of 2 cadmium ion artificial antigen
Through the UV spectrum scanning analysis of BSA, OVA, DTPA-BSA and DTPA-OVA, it was found that the maximum absorption wavelength of DTPA-BSA and DTPA-OVA had a weak blue shift than that of BSA and OVA, which were 278 nm to 270nm (BSA) and 280 nm, and their UV scanning curves were also changed.
The content of free amino groups in Cd-BSA and Cd-OVA was determined by 2,4,6- three nitrobenzene sulfonic acid method (triniro-benzene-sulfonic-acid). The number of free amino groups in Cd-BSA was 48.8% of BSA, and Cd-OVA was 41.4%. of OVA.
SDS-PAGE results show that the molecular weights of BSA (OVA) and DTPA-BSA (DTPA-OVA and Cd-BSA (Cd-OVA) are increasing sequentially.
The extended X ray absorption fine structure spectra (extended X-ray absorption fine structure) results show that the coordination type and coordination number of Cd2+ in Cd-DTPA, Cd-BSA and Cd-OVA are basically the same. The placement, shape, and absorption peak intensity are similar to that of Cd-DTPA. The absorption edge position similarity indicates that the local structure of the Cd ions in the three antigens of Cd-DTPA, Cd-BSA and Cd-OVA is the same; the shape of the absorption edge is similar, indicating that Cd-BSA and Cd-OVA are formed by Cd2+ and DTPA in Cd-DTPA, and the peak intensity of the absorption peak is consistent with that of the two kinds of artificial antigens. Cd in Cd-DTPA is the same as +2 price.
In conclusion, artificial antigens Cd-BSA and Cd-OVA have been successfully constructed.
According to the substitution rate of the middle reaches of Cd-BSA and Cd-OVA and the content of their protein, it is calculated that the molar ratio of Cd2+ to BSA and OVA in Cd-BSA and Cd-OVA is 28.7:1 and 11.7:1..
Preparation and characterization of 3 anti cadmium antiserum
Three groups of BABL/c mice were immunized with Cd-BSA, with three mice in each group. The immune cycle was 21d for the first time, and the subsequent enhanced immunization was 14d. The immunization dose was 200100 and 50 mu g/ only. The immune response of three groups of BALB/c mice could be produced by the antibody production process curve. The immune response level of different mice was different, but the total immune response of different mice was different, but the total number of immune responses in different mice was different. The response rate of the body was basically the same. The antisera of the three groups of mice were analyzed after fifth times of immunization, and their titers ranged from 2.6 * 104 to 4.2x104, with little difference.
After the best working conditions of three mice antigen antibody reaction determined by the square titration experiment, three groups of mice were analyzed by indirect competitive ELISA method. The sensitivity of No. 1 mice was 120ng/mL, and the sensitivity of 3,4,5,6,7,8 and 9 mice was 200,70,59,58155150 and 167 ng/mL. respectively, 1, 6 and 8 of the mice were selected. The specific analysis of antiserum in mice showed that the antiserum had a certain cross reaction to the mercury ion, but there was no cross reaction with other metal ions, such as lead, iron, copper and zinc.
4 establishment of cadmium ion ELISA method and detection of cadmium ion in lake water
Using the antiserum of No. 6 mice as material, the indirect competitive ELISA method of Cd2+ was established by indirect competitive ELISA analysis. The linear equation of Cd2+ was y=-20.62 x+87.069 (R2=0.9919) and the recovery rate was 85.3%-90.3%, and the average of 86.59%. from three lakes in Wuhan. 3 samples were collected for ELISA of Cd2+. The results showed that the Cd2+ concentration in A Lake reached 6.08ng/mL, and the consistency with AAS analysis was 105.7%..

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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