Study On The Detection Methods For Aminoglycoside Antibiotics In Milk Based On Broad-Spectrum Aptamer Recognition

The molecular structure of aminoglycoside antibiotics（AAs）is a glycoside compound composed of aminoglycoside and aminocyclic alcohol by oxygen bridging,including kanamycin,neomycin,gentamicin and so on.AAs are widely applied in dairy cattle husbandry because of their low cost,high efficiency and broad-spectrum antibacterial activity.Many kinds of AAs are usually used to treat mastitis and other diseases in cows.Therefore,it often occurs that the multi-residues of AAs are found to exist in food products,which may threaten the health of consumers.In order to protect public health and avoid drug abuse,timely and accurate detection of antibiotic residues in milk is an important link to ensure food safety.If the entire class of antibiotics can be analyzed simultaneously,it will simplify detection process and avoid enormous samples analysis of each antibiotic one by one.Aptamer-based sensing analysis has become an important method for simultaneous detection of AAs,and the breakthrough of multi-residues detection technology of aptasensors depends on the acquisition of broad-spectrum aptamers,whose characteristics determine the sensitivity and accuracy of detection analysis.Therefore,the main research contents of this dissertation are as follows:The broad-spectrum aptamers were screened by the non-immobilized graphene oxide（GO）-SELEX method with kanamycin,neomycin and gentamicin as mixed targets.In the selection process,the target was in a free state,and GO could adsorb the ssDNA that was not bound to the target by π-π conjugation.After 12 rounds of selection,then cloning sequencing,sequence analysis and affinity determination,the best aptamer preliminary sequence AAs 03 was obtained with the dissociation constant（Kd）for neomycin,gentamicin,and kanamycin of 219.69 nM,244.86 nM,and 312.46 nM,respectively.The broad-spectrum and specificity characteristics of aptamer AAs 03 were further analyzed.It was found that aptamer AAs 03 could recognize streptomycin,tobramycin and other AAs with Kd value of 285.57～322.47 nM,showing good broadspectrum characteristics.The binding ability to tetracycline,chloramphenicol and other antibiotics was significantly weaker than AAs,showing good specificity.Because the aptamer AAs 03 contained 79 bases,the long base sequence was easy to form steric resistance.The sequence was truncated and optimized based on the secondary structure of aptamer.The aptamer truncated sequence AAs 03-2 containing 49 bases was obtained with Kd value of 94.44～185.94 nM.Compared with the original sequence AAs 03,the Kd value was reduced by one time and the affinity was improved.In order to explore the binding mechanism of aptamer and target,circular dichroism analysis was performed.It was found that the base packing of aptamer AAs 03-2 decreased,showing that its conformational change occurred after binding to target.Furthermore,the molecular simulation software was used to conduct aptamer modeling and molecular docking,and it was found that the TGCTAT binding region at the base site of AAs 03-2 formed a binding pocket to wrap AAs.The interaction between the aptamer and AAs was mainly through the hydrogen bond,electrostatic salt bridge and van der Waals force interaction between the common structure deoxystreptomyamine of AAs and aptamer,generating the aptamer and AAs to form a stable complex in the way of spatial structure complementarity.In order to verify the application performance of AAs 03-2,a novel electrochemical aptasensor based on ordered mesoporous carbon（OMC）@Ti₃C₂ MXene nanomaterial was constructed by using AAs 03-2 as a recognition element.OMC could be embedded into Ti₃C₂ MXene nanosheets to prevent stacking of Ti₃C₂ MXene,generating good current channels and enhancing electrode conductivity.In addition,OMC@Ti₃C₂ MXene possessed a large surface area and could be functioned as nanocarrier to accommodate numerous aptamers for recognizing and capturing target.In the presence of AAs,the transport of electron charge on the electrode surface was impeded by the bio-chemical reactions of the aptamer and AAs,generating a significant decline in the electrochemical signals.The proposed aptasensor presented a wide linear range of 10～2,000 nM and the limit of detection（LOD）of 3.51 nM.Moreover,the aptasensor with satisfactory stability,reproducibility and specificity,was successfully employed to detect the AAs residuals.In order to further improve the detection sensitivity of the aptasensor,the selected broad-spectrum aptamer AAs 03-2 was used as a recognition element,and the dualmode aptasensor based on enzyme-assisted cyclic amplification strategies was constructed to detect AAs.The electrode surface was modified with OMC@Ti₃C₂ MXene as a substrate,and the Au-Pd@Fc nanocomposite was coupled with signal DNA to prepare nanoprobes.In the presence of AAs,the aptamer was bound to AAs.Then target-induced cyclic amplification reaction run under the action of Exonuclease III,which increased the number of nanoprobes on the electrode surface,promoting the transfer of electrons,and enhancing the electrical signal.AAs was able to be quantitatively detected based on the change of electrical signal,and the LOD was 0.0355 nM.At the same time,the Au-Pd@Fc nanomaterials on the nanoprobe had the characteristics of nano-enzyme,which could catalyze the color reaction of 3,3’,5,5’tetramethylbenzidine.With the change of the concentration of AAs,the color of the solution changed,and the LOD was 0.0458 nM by measuring the absorbance of the solution.The electrochemical and colorimetric detection modes was both applied to detect AAs,and the detection results were verified mutually to improve the accuracy of the detection method.The constructed aptasensor was applied to detect AAs in milk samples.The main components of protein,fat,lactose,Na+ and Ca 2+in milk samples caused the interference to the electrochemical signal and the binding of aptamer to target.Magnetic molecular imprinted polymers（MMIPs）capable of specific adsorption of AAs were prepared.Two milk sample treatment methods including acetonitrile extraction combined with MMIPs magnetic adsorption separation and acetic acid combined with MMIPs magnetic adsorption separation were compared.It was found that the method of acetonitrile combined with magnetic separation was better for the pretreatment of milk samples.Milk samples were further processed by acetonitrile combined with magnetic separation,and AAs were detected by electrochemical aptasensors constructed in Chapter 4 and Chapter 5,with LOD of 3.45～3.97 ng/mL and 0.0439～0.0574 ng/mL,respectively.The recoveries of spiked milk samples were 96.20%～98.21%and 97.19%～98.70%,respectively.It indicated that the pretreatment method of acetonitrile combined with magnetic separation was feasible,and the constructed aptasensor could be used for the simultaneous detection of AAs in milk.This work provides a theoretical basis for the application of aptamer in the simultaneous detection of antibiotics and other food hazards.