本文選題：卡那霉素 + 適配體��； 參考：《江南大學(xué)》2017年碩士論文

【摘要】：抗生素主要用于防治致病微生物感染類疾病,自發(fā)現(xiàn)以來廣泛應(yīng)用于醫(yī)藥、養(yǎng)殖業(yè)、農(nóng)林業(yè)等多個(gè)領(lǐng)域。然而,過度使用抗生素已經(jīng)成為目前社會(huì)的重大問題,可導(dǎo)致微生物的耐藥性增強(qiáng),并在食源性動(dòng)物體內(nèi)殘留,這對(duì)于生態(tài)安全以及食品安全造成了極大的威脅。而卡那霉素(kanamycin)作為一種較為常用的氨基糖苷類抗生素,被廣泛應(yīng)用,其殘留問題比較突出。故,構(gòu)建可視化、特異性好、成本低的卡那霉素檢測方法,對(duì)保障環(huán)境和食品安全具備深刻意義。同時(shí),金納米顆粒(Au NPs)作為一種新型的材料,在物質(zhì)檢測中應(yīng)用頗廣,這主要?dú)w功于其所具備的固有的納米模擬酶活性(過氧化物酶)。本研究以卡那霉素特異性核酸適配體(aptamer)為識(shí)別元件,基于金納米材料所固有的模擬酶特性和粒徑在光吸收等方面的影響,從而,建立了三種卡那霉素檢測方法,具體如下:首先,建立了一種基于Au NPs納米模擬酶活性和適配體檢測卡那霉素的電化學(xué)方法。當(dāng)無卡那霉素存在時(shí),Au NPs被適配體所包裹,其過氧化物酶活性被抑制,整個(gè)體系處于信號(hào)“關(guān)閉”狀態(tài);而當(dāng)目標(biāo)物卡那霉素存在時(shí),Au NPs重新暴露催化過氧化氫(H_2O_2)和還原態(tài)硫堇(Thi)發(fā)生反應(yīng),利用產(chǎn)生的氧化態(tài)硫堇,而氧化態(tài)硫堇在電極等作用下,被還原成還原態(tài),在電化學(xué)工作站中得到還原峰電流信號(hào)實(shí)現(xiàn)檢測目的。在最適條件下,該方法在0.1-60 nmol·L~(-1)存在著良好的線性關(guān)系,線性方程為y=0.0214x+1.2757,R~2=0.9942,最低檢測限達(dá)到0.06 nmol·L~(-1)。其次,為了實(shí)現(xiàn)可視化檢測,建立了一種卡那霉素比色分析方法。在目標(biāo)物卡那霉素存在時(shí),其與復(fù)合結(jié)構(gòu)中的適配體競爭結(jié)合,使適配體和c DNA去雜交,Au NPs釋放。Au NPs被釋放,加入3,3',5,5'-四甲基聯(lián)苯胺(TMB)和過氧化氫,在Au NPs的過氧化物酶活性催化下溶液變藍(lán),用濃硫酸終止反應(yīng)后,溶液變黃,在450 nm處有明顯吸收。無目標(biāo)物時(shí),無釋放的Au NPs,在450 nm處無吸收。通過比較有無目標(biāo)物時(shí)450 nm處的吸收值的差值,實(shí)現(xiàn)對(duì)卡那霉素的定量檢測。該方法優(yōu)化后在卡那霉素濃度為5-100nmol·L~(-1)中有良好的線性關(guān)系。線性回歸方程為y=0.0014x+0.0656,R~2=0.9954,檢測限為5.37 nmol·L~(-1)。最后,建立了一種以Au NPs為信號(hào)探針,卡那霉素特異性適配體為識(shí)別元件,基于目標(biāo)物卡那霉素誘導(dǎo)Au NPs生長的UV-vis光譜分析方法。將卡那霉素適配體與Au NPs孵育,無卡那霉素時(shí),Au NPs因表面覆蓋適配體而被封閉,不發(fā)生生長反應(yīng);添加卡那霉素,Au NPs重新暴露,發(fā)生生長反應(yīng),表現(xiàn)為UV-vis光譜吸收峰的位移和溶液顏色的變化。通過建立卡那霉素濃度和吸收峰偏移程度的關(guān)系,實(shí)現(xiàn)了對(duì)目標(biāo)物的檢測。在最適反應(yīng)條件下,本方法的線性范圍為20-100 nmol·L~(-1),檢測限為9.21 nmol·L~(-1)。該方法也成功應(yīng)用于對(duì)洋槐花蜂蜜中卡那霉素的檢測,說明其能夠較好地應(yīng)用到食品、醫(yī)學(xué)及水環(huán)境等諸多實(shí)際樣品的檢測中。
[Abstract]:Antibiotics are mainly used to prevent and cure diseases of pathogenic microorganism infection, which have been widely used in many fields such as medicine, aquaculture, agriculture and forestry since its discovery. However, excessive use of antibiotics has become a major problem in the society, which can lead to enhanced microbial resistance and residues in food borne animals, which is ecological safety and food. As a more commonly used aminoglycoside antibiotic, kanamycin is widely used, and its residual problems are relatively prominent. Therefore, the construction of kanamycin detection method with visual, specific and low cost is of profound significance for the protection of the environment and food safety. At the same time, gold nanoparticles (A U NPs, as a new kind of material, is widely used in material detection, which is mainly due to its inherent activity of nanoscale enzyme (peroxidase). This study takes kanamycin specific aptamer (aptamer) as an identification element, based on the intrinsic analog enzyme properties and particle size of gold nanoparticles in light absorption and so on. Three kinds of kanamycin detection methods were established. First, an electrochemical method based on Au NPs nanoscale enzyme activity and aptamer was established. When no kanamycin was present, the Au NPs was encapsulated by the aptamer, its peroxisase activity was suppressed, and the whole system was "closed". When the target kanamycin is present, Au NPs reacts to catalyze the reaction of hydrogen peroxide (H_2O_2) and the reduced state thio (Thi), and is reduced to a reductive state by the oxidation state thio ion, while the oxidized thio is under the action of the electrode. The peak current signal is detected in the electrochemical working station. Under conditions, the method has a good linear relationship in 0.1-60 nmol L~ (-1). The linear equation is y=0.0214x+1.2757, R~2=0.9942, and the minimum detection limit reaches 0.06 nmol. L~ (-1). Secondly, in order to realize visual detection, a colorimetric analysis method of kanamycin is established. When the target kanamycin is present, it is suitable for the composition of the complex structure. In combination with the body competition, the aptamers and C DNA are hybridized, and Au NPs releases.Au NPs to be released, adding 3,3', 5,5'- four methyl diphenyl amine (TMB) and hydrogen peroxide. The solution becomes blue under the activity of Au NPs peroxidase, and after the termination of the reaction with concentrated sulfuric acid, the solution becomes yellow and has obvious absorption at the 450 nm. No release There is no absorption. The quantitative detection of kanamycin is achieved by comparing the difference of the absorption value at 450 nm without target. The method has a good linear relationship with the concentration of kanamycin in 5-100nmol L~ (-1) after optimization. The linear regression equation is y=0.0014x+0.0656, R~2=0.9954, and the detection limit is 5.37 nmol. L~ (-1). Finally, a kind of method is established. With Au NPs as a signal probe, kanamycin specific aptamer is a identification element, UV-vis spectral analysis method based on kanamycin induced Au NPs growth based on target kanamycin. The kanamycin aptamer is incubated with Au NPs, without kanamycin, Au NPs is closed because the surface is covered with aptamers, and no growth reaction occurs; the addition of kanamycin, Au NPs is reproduced. Exposure, the occurrence of growth reaction, the displacement of the absorption peak of the UV-vis spectrum and the change of the color of the solution. By establishing the relationship between the concentration of kanamycin and the shift degree of the absorption peak, the target detection is realized. Under the optimum reaction conditions, the linear range of this method is 20-100 nmol. L~ (-1) and the detection limit is 9.21 nmol. L~ (-1). It has been successfully applied to the detection of kanamycin in acacia honey, indicating that it can be applied to the detection of many practical samples in food, medicine and water environment.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S859.84

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 ;Direct electrochemistry and electrocatalysis of horseradish peroxidase in MnO_2 nanosheet film[J];Chinese Science Bulletin;2008年08期

2 龍朝陽;許秀敏;;動(dòng)物源性食品中氨基糖苷類抗生素獸藥殘留分析[J];中國食品衛(wèi)生雜志;2006年02期

3 董連青,呂呈剛;幾種不同抗生素對(duì)飼喂肉雞的促生長效果[J];動(dòng)物科學(xué)與動(dòng)物醫(yī)學(xué);2001年02期

4 丁大連;羅德峰;皇甫慕三;;卡那霉素腎毒性與耳毒性的關(guān)系[J];臨床耳鼻咽喉頭頸外科雜志;1990年03期

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本文編號(hào)：1915537