【摘要】：抗生素作為獸藥過(guò)量使用會(huì)造成食源性動(dòng)物食品中抗生素的殘留,損害人類(lèi)身體健康,從而成為食品安全領(lǐng)域的重要議題。為進(jìn)一步提高動(dòng)物源食品的安全性,建立高效靈敏、快速經(jīng)濟(jì)的抗生素檢測(cè)方法是非常重要的�；谶m配體(aptamer)的傳感檢測(cè)方法具有特異性、靈敏度高的特點(diǎn),在抗生素檢測(cè)中具有良好的應(yīng)用前景。氧氟沙星和卡那霉素都是常用的臨床抗生素和獸藥,本文在抗生素特異性適配體的篩選和適配體傳感器的構(gòu)建方面開(kāi)展了工作,具體如下:建立了基于功能化的石墨烯,氧化石墨烯與指數(shù)富集的配體系統(tǒng)進(jìn)化(SELEX)技術(shù)結(jié)合的氧化石墨烯篩選技術(shù)(GO-SELEX)。利用氧化石墨烯作為結(jié)合ss DNA與分離的介質(zhì),從全長(zhǎng)為79 nt,中間含有35個(gè)隨機(jī)序列的初始ss DNA文庫(kù)中篩選與氧氟沙星高親和、高特異的ss DNA適配體。初始文庫(kù)預(yù)處理后與氧氟沙星孵育,加入氧化石墨烯,不能與氧氟沙星結(jié)合的ss DNA結(jié)合在氧化石墨烯上,離心,上清液中能與氧氟沙星結(jié)合的ss DNA純化后進(jìn)行PCR擴(kuò)增,鏈霉親和素磁珠法分離制備單鏈次級(jí)文庫(kù)。每輪篩選結(jié)束計(jì)算ss DNA的回收率,經(jīng)過(guò)6輪篩選回收率趨于平穩(wěn)后進(jìn)行一輪負(fù)篩選,除去能與同類(lèi)其他抗生素結(jié)合的ss DNA,終輪篩選結(jié)束整個(gè)篩選過(guò)程。將終輪篩選后的PCR擴(kuò)增產(chǎn)物克隆、測(cè)序獲得12條適配體序列。將同源性較高的四條序列ap1、ap3、ap4、ap5進(jìn)行二級(jí)結(jié)構(gòu)模擬,測(cè)得它們的解離常數(shù)(Kd)值分別為251.3、130.1、159.1、304.4 nmol·L-1�？疾霮d值較小的ap3、ap4對(duì)氧氟沙星的特異性,發(fā)現(xiàn)特異性良好。構(gòu)建了一種基于石墨烯(Gr)及適配體的卡那霉素傳感檢測(cè)方法�？敲顾剡m配體(kana-aptamer)可以吸附在石墨烯修飾的電極表面,從而阻礙電化學(xué)探針[Fe(CN)6]3-/4-與電極表面的電子傳遞,然而與含有卡那霉素的樣品反應(yīng)后,卡那霉素能與適配體結(jié)合并使其從電極上解吸下來(lái),對(duì)界面電子傳遞的阻礙作用降低,探針的電化學(xué)信號(hào)得到回復(fù)。通過(guò)循環(huán)伏安法和原子力顯微鏡法對(duì)該過(guò)程進(jìn)行了表征。該原理被用來(lái)對(duì)卡那霉素進(jìn)行電化學(xué)檢測(cè),結(jié)果表明在優(yōu)化條件下用差分脈沖伏安法(DPV)檢測(cè)卡那霉素,在1×10-6~1×10-5 mol·L-1濃度范圍內(nèi)呈現(xiàn)良好的線(xiàn)性關(guān)系,檢出限為5×10-7 mol·L-1。該方法被進(jìn)一步應(yīng)用于牛奶樣品中卡那霉素的檢測(cè),顯示出與標(biāo)準(zhǔn)樣品中相似的線(xiàn)性范圍。穩(wěn)定性實(shí)驗(yàn)所得相對(duì)標(biāo)準(zhǔn)偏差表明方法穩(wěn)定性較好。
[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.
【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】：2015
【分類(lèi)號(hào)】：S859.796

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