Screening And Application Of Aptamers Against Three Veterinary Drug Residues Based On Capture-SELEX

Veterinary drugs are absorbed into the human body through the food chain and accumulate in the body,causing a variety of toxic hazards,including damage to the liver,kidneys,digestive system,nervous system and blood circulatory system,affecting the physiological functions of the human body.Aptamers are a new type of recognition molecules that can specifically bind to targets.They have similar affinity and specificity to antibodies,and have the advantages of a wide range of target molecules,and they are easy to be synthesized and modified.As veterinary drug aptamers are successively screened,their application in the field of veterinary drug residue detection has become more and more extensive.In this study,the specific aptamers of levamisole,trimethoprim and halofuginone were screened by Capture-SELEX for the first time.Based on the obtained aptamers combined with colorimetric/surface-enhanced raman scattering(SERS)/fluorescence method,the detection methods of levamisole,trimethoprim and halofuginone in food were constructed respectively.Firstly,the aptamers of levamisole,trimethoprim and halofuginone were screened by Capture-SELEX technology.The screening process included library immobilization,target incubation,polymerase chain reaction(PCR)amplification,and single-strand preparation,and the progress of the reaction was monitored by fluorescence.High-throughput sequencing was performed on the circulating products when the enrichment rate reached saturation,and the top 5 sequences with the highest abundance were selected as candidate aptamers.The affinity and specificity of candidate aptamers were investigated by SYBR Green I(SGI)dye detection and isothermal titration calorimetry(ITC).Among them,LEV-5,TMP-1 and HAL-4 showed good affinity and specificity for levamisole,trimethoprim and halofuginone,respectively,and the dissociation constants(K_d)were 66.15±11.86 n M and 48.86±5.807 n M,96.74±11.22 n M.Circular dichroism(CD)was further used to explore the conformational changes before and after the aptamer binds to the target,mainly manifested as the enhancement of deoxyribonucleic acid(DNA)helicity at 245 nm and the enhancement of base stacking at 275nm.Molecular simulation docking was further used to analyze the binding mechanism of the aptamer and the target.The results showed that the aptamer mainly achieved non-covalent binding to the target through hydrophobic interaction,van der Waals force and hydrogen bonding.Secondly,a method for detecting levamisole in milk based on colorimetric and SERS dual-mode aptamer sensing was constructed.The aptamer-modified magnetic nanomaterials were used as capture probes,and the AuNPs/Cu-TCPP(Fe)nanosheets modified with complementary DNA with peroxidase-like activity and SERS effect were used as signal probes.The detection of levamisole was realized by colorimetric and SERS dual modes.The detection limits of the two modes were 5 n M and 1.12 n M,respectively.The colorimetric-SERS dual-mode aptamer sensing method has higher sensitivity and more intuitive detection performance than single colorimetric method or single SRES detection method,and can adapt to more practical application scenarios.The method was successfully used for the detection of levamisole in milk,with recoveries ranging from 94.95%to 111.2%.Thirdly,a method for detecting trimethoprim in milk by aptamer sensing based on complementary strand competition chromogenic method was constructed.The aptamer immobilized on the surface of the microplate was used as the capture probe,and the complementary strand modified by horseradish peroxidase(HRP)was used as the signal probe.The detection of trimethoprim was realized by utilizing the discoloration characteristic of the TMB substrate oxidized by HRP.The color of solution is negatively correlated with the target concentration.The method exhibited good linearity with absorbance values in the target concentration range of 0.1μM～12μM.The detection limit was 0.1μM.It was successfully applied to the determination of trimethoprim in milk samples,with recoveries ranging from85.5%to 112.0%.Finally,a fluorescence-based aptamer-sensing method for the detection of halofuginone in eggs was constructed.The biotin-modified complementary strand immobilized on the surface of the magnetic beads was used as the capture probe,and the FAM-modified aptamer was used as the signal probe.Due to the change of the fluorescence intensity of the signal probe in the supernatant after adding the target,the detection of halofuginone can be realized.The method showed a linear relationship with the fluorescence intensity in the target concentration range of 0.1μM to 50μM,and the detection limit was 0.13μM.The method can be successfully applied to the determination of halofuginone in egg samples,and the recovery rate was 87.40%～109.9%.