Research On The Activity Of Nano-mimic Enzyme And Its Application In Detection Of Antibiotics

Antibiotics have been used in the prevention and treatment of diseases,which caused by pathogenic microbial infections,so they are widely used in the fields of medicine,aquaculture,agriculture and forestry since they had been discovered.However,overuse of antibiotics has become a major social problem,which can lead to increased resistance to microbial,and there are amounts of antibiotics residue in food-borne animals’bodies,which caused a great threat for ecological security and food safety.Kanamycin as a kind of aminoglycoside antibiotics,which has been used widely.However,the residual problem of kanamycin becomes more prominent.Meanwhile,gold nanoparticles（Au NPs）,as a new type of material,are widely used in the detection of substances,due to their inherent nano-mimetic enzyme activity.Therefore,it is great importance to find visualization,specificity and low cost methods to detect kanamycin.In this study,based on the kanamycin-specific nucleic acid aptamers used as recognition element,and the inherent mimetic enzyme of Au NPs,several methods of the detection of kanamycin had established.Firstly,an enzyme-free,ultrasensitive electrochemical detection of kanamycin residue was achieved based on mimetic peroxidase activity of gold nanoparticles（Au NPs）and target-induced replacement of the aptamer.Au NPs exhibited mimetic peroxidase activity.In the absence of kanamycin,however,Au NPs were blocked by the kanamycin aptamers（ss DNA）,which caused the peroxidase activity of Au NPs was inhibited.So,the whole system was in the closed state.While in the presence of target kanamycin,exposed the surface of Au NPs and recovered the peroxidase activity.Then Au NPs catalyzed the reaction between H₂O₂ and reduced thionine to produce oxidized thionine and the formation of an electron transport system to form a corresponding current value,which achieved the detection of kanamycin through the reduction peak current of oxydic thionine.Under the optimized conditions,the proposed electrochemical assay showed an extremely high sensitivity towards kanamycin,with a linear relationship between the peak current and the concentration of kanamycin in the range of 0.1-60 nmol·L^-1,and a detection limit of 0.06 nmol·L^-1.Moreover,the linear regression equation of y=0.0214x+1.2757（R²=0.9942）can be derived.Secondly,in order to facilitate visualization operation,this study had established a colorimetric detection of kanamycin.In the presence of kanamycin,the combination between aptamer with c DNA breaks.Through magnetic separation,taking out the supernatant included gold nanoparticles,and then the substrates and buffer were joined into the above solution,with the color of the above solution became blue.Finally,the concentrated sulfuric acid was added into the above solution worked as a terminated solution,the color became yellow,with the obvious absorption peak at 450 nm.In the absence of target,there is no absorption peak at 450nm.Kanamycin was qualitatively and quantitatively determined by observing the yellow depth of the solution after the reaction.Comparing with the values at 450 nm before and after adding the kanamycin,to realize the detection of kanamycin.A linear relationship between the absorption peak at 450 nm and the concentration of kanamycin in the ranges of 5-100 nmol·L¹ was obtained and the linear regression equation of y=0.0014x+0.0656,R²=0.9954,with a detection limit of 5.37 nmol·L^-1.Finally,a UV-visible spectroscopic method for the detection of kanamycin was successfully developed based on target-induced growth of gold nanoparticles（Au NPs）,using Au NPs as probe and kanamycin-specific aptamer as recognition element.Firstly,the aptamer was incubated with Au NPs,in the absence of kanamycin,the surface of Au NPs was blocked,which inhibited the further growth of Au NPs.In the presence of target kanamycin,Au NPs were re-exposed and the growth reaction occurred,the color and the shift of the maximum absorption wavelength of Au NPs solution were changed,which was utilized to determine the concentration of kanamycin.Under the optimized conditions,the proposed UV-visible spectroscopic assay showed high sensitivity and high specificity for kanamycin,with a linear detection range from20 to 100 nmol·L^-1,and the detection limit of 9.21 nmol·L^-1.Moreover,the assay has been successfully applied to detect kanamycin in honey samples without pretreatment.Therefore,it has great application prospect to detect residual kanamycin in food,medicine and water environment.