基于石墨烯的抗生素特异性适配体筛选和传感器构建
发布时间：2018-12-07 18:00
【摘要】：抗生素作为兽药过量使用会造成食源性动物食品中抗生素的残留,损害人类身体健康,从而成为食品安全领域的重要议题。为进一步提高动物源食品的安全性,建立高效灵敏、快速经济的抗生素检测方法是非常重要的。基于适配体(aptamer)的传感检测方法具有特异性、灵敏度高的特点,在抗生素检测中具有良好的应用前景。氧氟沙星和卡那霉素都是常用的临床抗生素和兽药,本文在抗生素特异性适配体的筛选和适配体传感器的构建方面开展了工作,具体如下:建立了基于功能化的石墨烯,氧化石墨烯与指数富集的配体系统进化(SELEX)技术结合的氧化石墨烯筛选技术(GO-SELEX)。利用氧化石墨烯作为结合ss DNA与分离的介质,从全长为79 nt,中间含有35个随机序列的初始ss DNA文库中筛选与氧氟沙星高亲和、高特异的ss DNA适配体。初始文库预处理后与氧氟沙星孵育,加入氧化石墨烯,不能与氧氟沙星结合的ss DNA结合在氧化石墨烯上,离心,上清液中能与氧氟沙星结合的ss DNA纯化后进行PCR扩增,链霉亲和素磁珠法分离制备单链次级文库。每轮筛选结束计算ss DNA的回收率,经过6轮筛选回收率趋于平稳后进行一轮负筛选,除去能与同类其他抗生素结合的ss DNA,终轮筛选结束整个筛选过程。将终轮筛选后的PCR扩增产物克隆、测序获得12条适配体序列。将同源性较高的四条序列ap1、ap3、ap4、ap5进行二级结构模拟,测得它们的解离常数(Kd)值分别为251.3、130.1、159.1、304.4 nmol·L-1。考察Kd值较小的ap3、ap4对氧氟沙星的特异性,发现特异性良好。构建了一种基于石墨烯(Gr)及适配体的卡那霉素传感检测方法。卡那霉素适配体(kana-aptamer)可以吸附在石墨烯修饰的电极表面,从而阻碍电化学探针[Fe(CN)6]3-/4-与电极表面的电子传递,然而与含有卡那霉素的样品反应后,卡那霉素能与适配体结合并使其从电极上解吸下来,对界面电子传递的阻碍作用降低,探针的电化学信号得到回复。通过循环伏安法和原子力显微镜法对该过程进行了表征。该原理被用来对卡那霉素进行电化学检测,结果表明在优化条件下用差分脉冲伏安法(DPV)检测卡那霉素,在1×10-6~1×10-5 mol·L-1浓度范围内呈现良好的线性关系,检出限为5×10-7 mol·L-1。该方法被进一步应用于牛奶样品中卡那霉素的检测,显示出与标准样品中相似的线性范围。稳定性实验所得相对标准偏差表明方法稳定性较好。
[Abstract]:Excessive use of antibiotics as veterinary drugs will result in residues of antibiotics in foodborne animal food, which will harm human health and become an important issue in the field of food safety. In order to further improve the safety of animal food, it is very important to establish an efficient, sensitive, rapid and economical method for the detection of antibiotics. The sensing method based on aptamer (aptamer) has the characteristics of specificity and high sensitivity, and has a good application prospect in antibiotic detection. Ofloxacin and kanamycin are common clinical antibiotics and veterinary drugs. In this paper, the screening of antibiotic specific aptamers and the construction of aptamer biosensor were carried out. Graphene oxide combined with exponentially enriched ligand phylogenetic (SELEX) screening technique (GO-SELEX). Using graphene oxide as a medium for binding ss DNA and isolation, a highly compatible and specific ss DNA aptamer with ofloxacin was screened from an initial ss DNA library with 35 random sequences in the middle of 79 nt,. After the initial library was pretreated with ofloxacin, the ss DNA which could not bind to ofloxacin was incubated with ofloxacin and added graphene oxide. After centrifugation, the ss DNA which could bind to ofloxacin in supernatant was purified and amplified by PCR. Single chain secondary library was isolated by streptavidin magnetic bead method. The recovery rate of ss DNA was calculated at the end of each round of screening. After 6 rounds of screening the recovery rate was stabilized and one round of negative screening was carried out. The whole screening process was completed with the removal of ss DNA, which could combine with other antibiotics of the same kind. The PCR amplified product was cloned and sequenced, and 12 aptamer sequences were obtained. The secondary structure of four ap1,ap3,ap4,ap5 sequences with high homology was simulated and their dissociation constant (Kd) values were determined to be 251.3130.1159.1304.4 nmol L-1, respectively. The specificity of ap3,ap4 with low Kd value to ofloxacin was investigated. A kanamycin sensing method based on graphene (Gr) and aptamer was proposed. Kanamycin aptamer (kana-aptamer) can be adsorbed on the surface of graphene modified electrode, which hinders the electron transfer between [Fe (CN) 6] 3 / 4-and the electrode surface. However, it reacts with the sample containing kanamycin. Kanamycin binds to the aptamer and desorbs it from the electrode. The blocking effect of kanamycin on the electron transfer at the interface is reduced and the electrochemical signal of the probe is recovered. The process was characterized by cyclic voltammetry and atomic force microscopy. The principle has been applied to the electrochemical detection of kanamycin. The results show that the linear relationship of kanamycin with differential pulse voltammetry (DPV) is linear in the concentration range of 1 脳 10 ~ (-6) to 1 脳 10 ~ (-5) mol 路L ~ (-1). The detection limit is 5 脳 10 ~ (-7) mol L ~ (-1). The method has been further applied to the determination of kanamycin in milk samples, showing a linear range similar to that in standard samples. The relative standard deviation obtained from the stability experiment shows that the stability of the method is good.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.796
[Abstract]:Excessive use of antibiotics as veterinary drugs will result in residues of antibiotics in foodborne animal food, which will harm human health and become an important issue in the field of food safety. In order to further improve the safety of animal food, it is very important to establish an efficient, sensitive, rapid and economical method for the detection of antibiotics. The sensing method based on aptamer (aptamer) has the characteristics of specificity and high sensitivity, and has a good application prospect in antibiotic detection. Ofloxacin and kanamycin are common clinical antibiotics and veterinary drugs. In this paper, the screening of antibiotic specific aptamers and the construction of aptamer biosensor were carried out. Graphene oxide combined with exponentially enriched ligand phylogenetic (SELEX) screening technique (GO-SELEX). Using graphene oxide as a medium for binding ss DNA and isolation, a highly compatible and specific ss DNA aptamer with ofloxacin was screened from an initial ss DNA library with 35 random sequences in the middle of 79 nt,. After the initial library was pretreated with ofloxacin, the ss DNA which could not bind to ofloxacin was incubated with ofloxacin and added graphene oxide. After centrifugation, the ss DNA which could bind to ofloxacin in supernatant was purified and amplified by PCR. Single chain secondary library was isolated by streptavidin magnetic bead method. The recovery rate of ss DNA was calculated at the end of each round of screening. After 6 rounds of screening the recovery rate was stabilized and one round of negative screening was carried out. The whole screening process was completed with the removal of ss DNA, which could combine with other antibiotics of the same kind. The PCR amplified product was cloned and sequenced, and 12 aptamer sequences were obtained. The secondary structure of four ap1,ap3,ap4,ap5 sequences with high homology was simulated and their dissociation constant (Kd) values were determined to be 251.3130.1159.1304.4 nmol L-1, respectively. The specificity of ap3,ap4 with low Kd value to ofloxacin was investigated. A kanamycin sensing method based on graphene (Gr) and aptamer was proposed. Kanamycin aptamer (kana-aptamer) can be adsorbed on the surface of graphene modified electrode, which hinders the electron transfer between [Fe (CN) 6] 3 / 4-and the electrode surface. However, it reacts with the sample containing kanamycin. Kanamycin binds to the aptamer and desorbs it from the electrode. The blocking effect of kanamycin on the electron transfer at the interface is reduced and the electrochemical signal of the probe is recovered. The process was characterized by cyclic voltammetry and atomic force microscopy. The principle has been applied to the electrochemical detection of kanamycin. The results show that the linear relationship of kanamycin with differential pulse voltammetry (DPV) is linear in the concentration range of 1 脳 10 ~ (-6) to 1 脳 10 ~ (-5) mol 路L ~ (-1). The detection limit is 5 脳 10 ~ (-7) mol L ~ (-1). The method has been further applied to the determination of kanamycin in milk samples, showing a linear range similar to that in standard samples. The relative standard deviation obtained from the stability experiment shows that the stability of the method is good.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.796
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