适配体传感器在卡那霉素检测中的应用
发布时间：2018-11-02 20:56
【摘要】：抗生素作为一类抗感染药物,自发现以来广泛应用于医药,农林业,渔业,畜牧业等领域。抗生素种类繁多,卡那霉素是其中一种常用的氨基糖苷类抗生素,以水溶性硫酸盐存在,具有抗菌谱广,杀菌效果明显等特点。不合理使用抗生素可导致耐药性增强,内毒素的产生和残留在食源性动物体内,不论是对食品质量还是安全都产生极大的影响和挑战。因此,建立操作简单,便携,特异性好,灵敏度高,不需要借助昂贵仪器的卡那霉素检测方法,对于保障动物源性食品的安全具有重要意义。本研究针对卡那霉素的定性和定量分析建立了两种检测方法:第一种是基于核酸适配体的电化学法;第二种是利用核酸适配体的纳米模拟酶比色法。研究内容主要包括:建立基于核酸适配体的检测卡那霉素的电化学方法,将末端修饰巯基(-SH)的卡那霉素适配体固定在金电极表面得到适配体功能化的金电极,将该电极置于包含卡那霉素的样品溶液中,卡那霉素与电极表面适配体特异性结合产生构象变化及电极表面阻抗变化,溶液中[Fe(CN)6]3-/4-充当信号探针,卡那霉素与适配体结合后产生的电化学信号改变可通过差示脉冲伏安法(DPV)检测,进而实现定量检测。在优化条件下(适配体浓度1μmol·L-1,适配体自组装时间16 h,卡那霉素结合时间30 min),利用电化学法实现卡那霉素的检测范围是0.2到2000 nmol·L-1。以相同浓度(500 nmol·L-1)的庆大霉素,新霉素,链霉素,四环素以及超纯水代替卡那霉素验证该检测方法对卡那霉素的特异性。进一步对稀释5倍的含卡那霉素的牛奶样品进行了直接检测,结果表明牛奶样品中其他组分对卡那霉素残留的检测基本没有影响,该检测方法特异性好,灵敏度,适用于稀释牛奶样品中卡那霉素的直接检测。建立利用核酸适配体和纳米模拟酶的卡那霉素比色检测法。金纳米颗粒的模拟酶的性质可催化H2O2和3,3’,5,5’-四甲基联苯胺(TMB)混合底物产生显色反应。适配体ssDNA能非共价吸附在金纳米颗粒(AuNPs)表面从而阻碍金纳米颗粒与底物的接触,催化作用被抑制,显色反应不能发生;卡那霉素特异性结合金纳米颗粒表面的卡那霉素适配体,使其游离到溶液中,金纳米颗粒重新暴露而催化活性恢复,表现为UV-vis比色法中TMB氧化产物(oxTMB)特征峰的吸收变化,通过650 nm处吸光度的不同来表征卡那霉素浓度。据此建立的卡那霉素检测方法,在优化条件下,对卡那霉素检测的线性范围是5-200nmol·L-1,检测限为5 nmol·L-1。再以相同浓度(500 nmol·L-1)的卡那霉素,庆大霉素,链霉素,新霉素以及超纯水来进行特异性验证,表明该方法具有较强的特异性。所用纳米模拟酶pH值适用范围宽,pH 4.0催化效率最高;温度耐受性范围宽,最适催化温度为45℃,具有催化活性好,稳定性高,分散性强,可重复利用的优点。
[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，
本文编号：2306928
[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，
本文编号：2306928
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