Applications Of Aptasensors In The Detection Of Kanamycin

As anti-infective drugs, antibiotics are widely used in the fields of medicine, agriculture, forestry, fisheries and animal husbandry since they has been discovered. Kanamycin is a commonly used aminoglycoside antibiotics in its water-soluble sulfate, with the characteristics of broad antimicrobial spectrum and obvious bactericidal effect. Currently, the overcommitment of antibiotics leads to multidrug resistance, release of endotoxin and residues of food-borne animals, which is a great challenge to the quality and safety of agricultural products. Therefore, it is of great importance to found simple, portable, high-specific, high-sensitive methods without the aid of expensive instruments. In this thesis, two methods were fabricated to detect kanamycin. One is an aptamer-based electrochemical method, the other is a colorimetric method based on aptamer and nano-mimetic enzyme.In aptamer-based electrochemical detection, 5’-SH-modified kanamycin-specific aptamer was self-assembled on the surface of a gold electrode through Au–S chemistry. When the aptamer-functionalized electrode was incubated in the samples containing kanamycin, the binding of kanamycin with its aptamer triggered the conformational change of the aptamer and thus the resistance change of the electrode surface. Using [Fe(CN)6]3-/4- as a probe, differential pulse voltammetry(DPV) was employed to quantify the concentration of kanamycin. The optimal concentration of kanamycin-specific aptamer was 1 μmol·L-1, and the optimal reaction time of the functionalize electrode in kanamycin samples was 30 min. Under the optimized conditions, the linear detection range of the electrochemical detection is from 10 to 2000 nmol·L-1, indicating high sensitivity and broad detection range. The same concentration of gentamicin, ampicillin, streptomycin and ultrapure water, instead of kanamycin, were used to verify the specificity of the method. The method was further applied to detect kanamycin in 5-fold diluted milk samples containing kanamycin directly. The results showed that the matrix of milk do not influence on the detection of kanamycin. The proposed method thus is with good application prospect.In UV-vis colorimetric method, aptamer and nano-mimetic enzyme were cooperated in the detection system. Gold nanoparticles(AuNPs), with its inherent simulation of enzyme nature, can catalyze 3,3’,5,5’-tetramethylbenzidine(TMB) and other substrates to produce blue substance in the presence of H2O2. As ssDNA, aptamer can be adsorbed on the surface of AuNPs, preventing the contact between AuNPs and the substrates. Therefore, the chromogenic reaction is inhibited. In the presence of kanamycin, it binds with the kanamycin-specific aptamer on the surface of AuNPs competitively, and thus exposes the surface of AuNPs and restores its catalytic activity. Then, the concentration of kanamycin is quantified by the absorbance at 650 nm, which is corresponding to the oxidation product of TMB. Under the optimized conditions, the linear range of colorimetric detection is from 5 to 300 nmol·L-1, with the detection limit of 5 nmol·L-1. Then the same concentration of gentamicin, ampicillin, streptomycin and ultrapure water were used to verify enzymatic dynamic and specificity. Nano-mimetic enzyme is proven to be stable, with high catalytic activity, good dispersity and recyclability with the optimal pH 4.0 and optimal temperature of 45℃.