【摘要】：抗生素作為一類抗感染藥物,自發(fā)現(xiàn)以來廣泛應(yīng)用于醫(yī)藥,農(nóng)林業(yè),漁業(yè),畜牧業(yè)等領(lǐng)域。抗生素種類繁多,卡那霉素是其中一種常用的氨基糖苷類抗生素,以水溶性硫酸鹽存在,具有抗菌譜廣,殺菌效果明顯等特點(diǎn)。不合理使用抗生素可導(dǎo)致耐藥性增強(qiáng),內(nèi)毒素的產(chǎn)生和殘留在食源性動(dòng)物體內(nèi),不論是對(duì)食品質(zhì)量還是安全都產(chǎn)生極大的影響和挑戰(zhàn)。因此,建立操作簡單,便攜,特異性好,靈敏度高,不需要借助昂貴儀器的卡那霉素檢測方法,對(duì)于保障動(dòng)物源性食品的安全具有重要意義。本研究針對(duì)卡那霉素的定性和定量分析建立了兩種檢測方法:第一種是基于核酸適配體的電化學(xué)法;第二種是利用核酸適配體的納米模擬酶比色法。研究內(nèi)容主要包括:建立基于核酸適配體的檢測卡那霉素的電化學(xué)方法,將末端修飾巰基(-SH)的卡那霉素適配體固定在金電極表面得到適配體功能化的金電極,將該電極置于包含卡那霉素的樣品溶液中,卡那霉素與電極表面適配體特異性結(jié)合產(chǎn)生構(gòu)象變化及電極表面阻抗變化,溶液中[Fe(CN)6]3-/4-充當(dāng)信號(hào)探針,卡那霉素與適配體結(jié)合后產(chǎn)生的電化學(xué)信號(hào)改變可通過差示脈沖伏安法(DPV)檢測,進(jìn)而實(shí)現(xiàn)定量檢測。在優(yōu)化條件下(適配體濃度1μmol·L-1,適配體自組裝時(shí)間16 h,卡那霉素結(jié)合時(shí)間30 min),利用電化學(xué)法實(shí)現(xiàn)卡那霉素的檢測范圍是0.2到2000 nmol·L-1。以相同濃度(500 nmol·L-1)的慶大霉素,新霉素,鏈霉素,四環(huán)素以及超純水代替卡那霉素驗(yàn)證該檢測方法對(duì)卡那霉素的特異性。進(jìn)一步對(duì)稀釋5倍的含卡那霉素的牛奶樣品進(jìn)行了直接檢測,結(jié)果表明牛奶樣品中其他組分對(duì)卡那霉素殘留的檢測基本沒有影響,該檢測方法特異性好,靈敏度,適用于稀釋牛奶樣品中卡那霉素的直接檢測。建立利用核酸適配體和納米模擬酶的卡那霉素比色檢測法。金納米顆粒的模擬酶的性質(zhì)可催化H2O2和3,3’,5,5’-四甲基聯(lián)苯胺(TMB)混合底物產(chǎn)生顯色反應(yīng)。適配體ssDNA能非共價(jià)吸附在金納米顆粒(AuNPs)表面從而阻礙金納米顆粒與底物的接觸,催化作用被抑制,顯色反應(yīng)不能發(fā)生;卡那霉素特異性結(jié)合金納米顆粒表面的卡那霉素適配體,使其游離到溶液中,金納米顆粒重新暴露而催化活性恢復(fù),表現(xiàn)為UV-vis比色法中TMB氧化產(chǎn)物(oxTMB)特征峰的吸收變化,通過650 nm處吸光度的不同來表征卡那霉素濃度。據(jù)此建立的卡那霉素檢測方法,在優(yōu)化條件下,對(duì)卡那霉素檢測的線性范圍是5-200nmol·L-1,檢測限為5 nmol·L-1。再以相同濃度(500 nmol·L-1)的卡那霉素,慶大霉素,鏈霉素,新霉素以及超純水來進(jìn)行特異性驗(yàn)證,表明該方法具有較強(qiáng)的特異性。所用納米模擬酶pH值適用范圍寬,pH 4.0催化效率最高;溫度耐受性范圍寬,最適催化溫度為45℃,具有催化活性好,穩(wěn)定性高,分散性強(qiáng),可重復(fù)利用的優(yōu)點(diǎn)。
[Abstract]:Antibiotics, as a class of antiinfective drugs, have been widely used in medicine, agriculture and forestry, fishery, animal husbandry and so on. There are many kinds of antibiotics, kanamycin is one of the commonly used aminoglycoside antibiotics. Irrational use of antibiotics can lead to increased drug resistance, endotoxin production and residues in foodborne animals, both food quality and safety have a great impact and challenges. Therefore, the establishment of a simple, portable, specific and sensitive kanamycin detection method does not need to use expensive instruments, which is of great significance to ensure the safety of animal food. In this study, two methods were established for the qualitative and quantitative analysis of kanamycin: the first was electrochemical method based on aptamer of nucleic acid; the second was nano-mimic enzyme colorimetry using aptamer of nucleic acid. The main contents are as follows: an electrochemical method for the detection of kanamycin based on aptamer of nucleic acid was established, and the ligand functionalized gold electrode was obtained by immobilization of kanamycin aptamer with terminal modified mercapto (- SH) on the surface of gold electrode. The electrode was placed in a sample solution containing kanamycin, where kanamycin specifically binds to the aptamer on the electrode surface to produce conformation changes and changes in the surface impedance of the electrode. [Fe (CN) 6] 3 / 4- acts as a signal probe in the solution. The change of electrochemical signal produced by the binding of kanamycin with aptamer can be detected by differential pulse voltammetry (DPV), and then quantitative detection can be realized. Under the optimized conditions (aptamer concentration 1 渭 mol L -1, aptamer self-assembly time 16 h, kanamycin binding time 30 min), the detection range of kanamycin by electrochemical method was 0. 2 to 2000 nmol L ~ (-1). The specificity of kanamycin was verified by the same concentration (500 nmol L-1) of gentamicin, neomycin, streptomycin, tetracycline and ultrapure water instead of kanamycin. The results showed that the other components in the milk samples had no effect on the detection of kanamycin residues, and the method was specific and sensitive. It is suitable for direct detection of kanamycin in diluted milk samples. A kanamycin colorimetric assay using aptamer and nanomimetic enzyme was established. The mimic enzyme properties of gold nanoparticles can catalyze the chromogenic reaction of H2O2 and (TMB) mixed substrates. The aptamer ssDNA can adsorb on the surface of gold nanoparticles (AuNPs), which hinders the contact between gold nanoparticles and substrate, the catalytic action is inhibited, and the color reaction can not take place. Kanamycin specifically binds to the kanamycin aptamer on the surface of gold nanoparticles to free them into the solution, and the gold nanoparticles are exposed again and the catalytic activity recovers. The results showed that the characteristic peak of TMB oxidation product (oxTMB) was absorbed by UV-vis colorimetry, and the concentration of kanamycin was characterized by absorbance at 650 nm. Under the optimized conditions, the linear range of kanamycin detection is 5-200nmol L -1 and the detection limit is 5 nmol L -1. The specificity of kanamycin, gentamicin, streptomycin, neomycin and ultrapure water was verified with the same concentration of kanamycin (500 nmol L-1). The pH value of nanometer mimic enzyme is wide, the catalytic efficiency of pH 4.0 is the highest, the temperature tolerance range is wide, and the optimum catalytic temperature is 45 鈩，

本文編號(hào)：2306929