Construction Of Aptamer-functionalized Magnetic Probes And Application In Acetamiprid Detection

Acetamiprid,as a broad-spectrum neonicotinoid insecticide,is mainly used to protect fruits and vegetables from pests.Because acetamiprid can be absorbed across the epithelium through intestinal cells,if acetamiprid accumulates too much in the body,it may be toxic and harmful to health.Conventional acetamiprid detection methods are usually based on instrumental techniques,such as high performance liquid chromatography（HPLC）,gas chromatography（GC）or combination of chromatography and mass spectrometry（MS）detector,which have the advantages of high accuracy and high sensitivity.On the other hand,colorimetric,fluorescence and electrochemical techniques provide a relatively convenient and highly sensitive strategy for the determination of acetamiprid.Although the accuracy and precision of rapid methods are not as good as instrument technology,these methods can be used as a supplement to instrument methods,especially as a pre screening method for large-scale sample detection.Therefore,we need a new analytical method to detect acetamiprid quickly and sensitively.In view of this,two aptamer functionalized magnetic probes,aptamer modified Au NPs-magnetic beads probes and aptamer modified peptide-magnetic beads probes,were studied in this paper.（1）Based on aptamer modified Au NPs-magnetic beads probes,a syringe assisted aptamer colorimetric sensing method for acetamiprid detection was developed.Firstly,the aptamer modified Au NPs-magnetic beads probes（MBs@Au NPs-ds DNA）with peroxidase activity was prepared by using gold nanoparticles with peroxidase activity.Then it was loaded into a syringe with magnetic rings,and the detection of acetamiprid was easily completed with the assistance of air and magnetic separation.When acetamiprid was captured,the double stranded aptamers on the MBs@Au NPs-ds DNA in the syringe were dissociated to form single stranded aptamers,and the modified state of aptamers on the surface of Au NPs would directly affect the peroxidase activity of the probes,and further affect the catalytic activity of 3,3’,5,5’-tetramethylbenzidine（TMB）.Therefore,the presence of acetamiprid can inhibit the peroxidase activity of the probe,affect the catalytic effect on TMB,and realize the detection of acetamiprid.The linear relationship between A/A₀ and acetamiprid concentration was established by using the ratio of the absorption value of ox TMB at 652 nm（A/A₀）.The linear relationship was good in the range of 2-300 nm（correlation coefficient R²=0.99）,and the LOD was 1.53 n M.The method has good selectivity,anti-interference,stability and reusability.It also has good effect in the actual celery leaves and tea samples,and the recovery rate reaches 98.90%-99.90%.（2）Based on aptamer modified peptide-magnetic beads probes,a biotin triggered aptamer electrochemical sensing method for acetamiprid detection was developed.Firstly,aptamer modified peptide-magnetic beads probes（STMBs@Strep-tag II-ds DNA）was prepared by Strep-tag system.The aptamer modified electrode（MCH/ABA/Au N/GCE）was prepared by electrodeposition of gold nanoparticles on the surface of glassy carbon electrode,using Au-S bond to modify the aptamer on the surface of Au NP,and using 6-mercapto-1-ethanol（MCH）to block the excess sites on the surface.Because biotin can compete for the binding sites between Strep-Tactin and Strep-tag II,when STMBs@Strep-tag II-ds DNA is exposed to acetamiprid,under biotin triggering,the probes will release peptide-aptamers（Strep-tag II-c ABA）.At this time,Strep-tag II-c ABA will be fixed to the surface of MCH/ABA/Au N/GCE under the force of base complementary pairing,which will increase the surface steric resistance,hinder the electron transfer,and increase the Rct value on the surface of the modified electrode.In order to further amplify the signal of increased Rct value,Strepavdin-HRP is continuously modified on the surface of the electrode to realize the detection of acetamiprid.The linear relationship betweenΔRct and acetamiprid concentration was established by using the change value of Rct（ΔRct）as the signal.The linear relationship was good in the range of 0.0005-100 n M（R²=0.99）.The LOD was 0.15 p M.Good results were also obtained in the detection of apple,tomato and tea samples,and the recoveries were 99%-100.75%.