Study On The Construction Of Dual-signal Aptasensor For The Detection Of Kanamycin Residue In Milk

Kanamycin(KAN)is an important aminoglycoside antibiotic that is widely used in animal husbandry,not only to treat animal diseases caused by bacterial infections,but also to promote animal growth.However,the abuse or incorrect use of KAN leads to excessive residues in animal-derived foods such as milk,and accumulate in the human body through the food chain,which seriously affects human health.Therefore,the development of sensitive and accurate rapid detection methods for KAN residues is essential to ensure food safety.Aptamer(Apt)is widely used as an identification element in the field of sensor detection because of its unique advantages such as editability,high stability,in vitro synthesis,and specific identification of different targets.The electrochemical aptasensor has the advantages of low sample consumption,high sensitivity and fast response,and has broad application prospects.In order to further improve the sensitivity of low-content target detection,traditional electrochemical sensors often use different signal amplification techniques.However,in the process of signal amplification,interference is prone to occur due to changes such as the environment and electrode surface modification materials.Therefore,improving sensitivity and enhancing immunity to interference are challenges for electrochemical sensors.Based on this,two dual-signal aptasensors are designed.Firstly,a two-signal ratiometric electrochemical sensor is constructed to improved the anti-interference based on the dual-electric signal.Then,combine the electrical signal and optical signal to build a dual-mode sensor.The sensor not only verifies each other based on two different signals to improve accuracy,but also achieve visualization and high-sensitivity dual mode detection.In addition,the sensitivity of detection is increased through nanomaterials and nuclease amplification signal.And verify the application effect of two dual-signal sensors in the actual sample of milk.The main contents are as follows:(1)Based on ferrocene(Fc)labeled primers and graphdiyne-methylene blue(GDY-MB)nanocomposites,and simultaneously generating Fc and MB dual-electric signal,a ratiometric electrochemical aptasensor that can detect KAN residues in milk was prepared.Primers labeled Fc at the 5’end of the primer can hybridize with the aptamer fractional sequence to form a complementary double-stranded rigid structure such that the Fc labeled at the end of the primer was away from the electrode surface.When KAN was present and specifically bound to Apt,primers designed with their own complementary sequences were released,resulting in a hairpin structure(HP)that brought Fc closer to the electrode surface and enhanced Fc electrical signals.Single-stranded Apt was cleaved using exonuclease I(Exo I)and the target was released to achieve cyclic amplification of the target signal.And the 3’end of primers was phosphorylated to protect HP from digestion.The amplification signal was further realized by modifying the chitosan-antimony tin oxide(CS-ATO)on the surface of the bare gold electrode,then modifying the GDY-MB nanocomposite to generate MB electrical signal,and finally modifying gold nanoparticles(Au NPs)to fix the cyclic product Fc-HP.Under optimal experimental conditions,the current intensity values of Fc(IFc)and MB(IMB)generated by different concentrations of KAN were obtained.The correlation coefficient(R1~2)between the logarithm of KAN concentration and the IFc/IMB standard curve was 0.9967,and the R2~2 with the IFc standard curve was 0.9599.R1~2>R2~2indicated that the ratio electrochemical sensor has good robustness.The limit of detection(LOD)was 6.044 n M(S/N=3).The recovery of actual samples in milk was 92.6%～109.25%.(2)Based on a large number of G-quadruplex generated by rolling ring amplification,a dual-mode aptasensor with colorimetric and electrochemical dual signal combination was designed.Specific binding between the target and Apt was used to release nucleic acid chains(Pr)that complement Apt.Under the action of DNA ligases and polymerase,free Pr combined with the padlock probe to drive roll ring amplification to produce a large number of G-quadruplets.Mimic peroxidase was generated based on the combination of G-quadruplex and hemin,which changed the color effect of catalytic 3,3’,5,5’-tetramethylbenzidine(TMB)to achieve visualization and semi-quantitative detection of KAN.Based on the strong adsorption of methylene blue(MB)by G-quadruplex,the electrochemical quantitative detection of KAN with high sensitivity was realized.Multi-walled carbon nanotubes and gold nanoparticles(MWCNTs CS/Au NP)nanocomposites were modified on the electrode surface to increase the electroactive area and conductivity,and further amplified electrochemical signals.Under optimal conditions,the LOD of the colorimetric aptasensor for KAN was 1.949 n M(S/N=3),and the actual sample recovery range was 89.43%～111.18%.The LOD of the electrochemical aptasensor was 0.333 p M(S/N=3),and the actual sample recovery range was 90.52%～111.15%.