纳米模拟酶活性研究及其在抗生素检测中的应用

发布时间：2018-05-20 17:16

本文选题：卡那霉素 + 适配体　；参考：《江南大学》2017年硕士论文

【摘要】：抗生素主要用于防治致病微生物感染类疾病,自发现以来广泛应用于医药、养殖业、农林业等多个领域。然而,过度使用抗生素已经成为目前社会的重大问题,可导致微生物的耐药性增强,并在食源性动物体内残留,这对于生态安全以及食品安全造成了极大的威胁。而卡那霉素(kanamycin)作为一种较为常用的氨基糖苷类抗生素,被广泛应用,其残留问题比较突出。故,构建可视化、特异性好、成本低的卡那霉素检测方法,对保障环境和食品安全具备深刻意义。同时,金纳米颗粒(Au NPs)作为一种新型的材料,在物质检测中应用颇广,这主要归功于其所具备的固有的纳米模拟酶活性(过氧化物酶)。本研究以卡那霉素特异性核酸适配体(aptamer)为识别元件,基于金纳米材料所固有的模拟酶特性和粒径在光吸收等方面的影响,从而,建立了三种卡那霉素检测方法,具体如下:首先,建立了一种基于Au NPs纳米模拟酶活性和适配体检测卡那霉素的电化学方法。当无卡那霉素存在时,Au NPs被适配体所包裹,其过氧化物酶活性被抑制,整个体系处于信号“关闭”状态;而当目标物卡那霉素存在时,Au NPs重新暴露催化过氧化氢(H_2O_2)和还原态硫堇(Thi)发生反应,利用产生的氧化态硫堇,而氧化态硫堇在电极等作用下,被还原成还原态,在电化学工作站中得到还原峰电流信号实现检测目的。在最适条件下,该方法在0.1-60 nmol·L~(-1)存在着良好的线性关系,线性方程为y=0.0214x+1.2757,R~2=0.9942,最低检测限达到0.06 nmol·L~(-1)。其次,为了实现可视化检测,建立了一种卡那霉素比色分析方法。在目标物卡那霉素存在时,其与复合结构中的适配体竞争结合,使适配体和c DNA去杂交,Au NPs释放。Au NPs被释放,加入3,3',5,5'-四甲基联苯胺(TMB)和过氧化氢,在Au NPs的过氧化物酶活性催化下溶液变蓝,用浓硫酸终止反应后,溶液变黄,在450 nm处有明显吸收。无目标物时,无释放的Au NPs,在450 nm处无吸收。通过比较有无目标物时450 nm处的吸收值的差值,实现对卡那霉素的定量检测。该方法优化后在卡那霉素浓度为5-100nmol·L~(-1)中有良好的线性关系。线性回归方程为y=0.0014x+0.0656,R~2=0.9954,检测限为5.37 nmol·L~(-1)。最后,建立了一种以Au NPs为信号探针,卡那霉素特异性适配体为识别元件,基于目标物卡那霉素诱导Au NPs生长的UV-vis光谱分析方法。将卡那霉素适配体与Au NPs孵育,无卡那霉素时,Au NPs因表面覆盖适配体而被封闭,不发生生长反应;添加卡那霉素,Au NPs重新暴露,发生生长反应,表现为UV-vis光谱吸收峰的位移和溶液颜色的变化。通过建立卡那霉素浓度和吸收峰偏移程度的关系,实现了对目标物的检测。在最适反应条件下,本方法的线性范围为20-100 nmol·L~(-1),检测限为9.21 nmol·L~(-1)。该方法也成功应用于对洋槐花蜂蜜中卡那霉素的检测,说明其能够较好地应用到食品、医学及水环境等诸多实际样品的检测中。
[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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.84

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