Research On The Detection Of Neonicotinoid Pesticide Acetamiprid By Aptamer-modified Nanoprobe

Pesticides make great contributions to the development of agriculture,forestry and animal husbandry because of their high efficiency in insecticide and weeding.However,the excessive use of pesticides causes a certain amount of pesticide residues in agricultural products,which seriously threatens people’s food safety.How to quickly and accurately determine whether pesticide residues and residual levels exist in agricultural products is an important means to ensure food safety.Conventional pesticide residue detection technologies include high performance liquid chromatography,liquid chromatography-mass spectrometry,etc.These techniques are highly sensitive and accurate,but rely on complex sample preparation,long detection cycles,and require professional operation,can not meet the needs of rapid detection of pesticide residues in life.Therefore,it is necessary to develop a simple and rapid method for detecting pesticide residues.In view of this,this thesis focuses on the nano-probes modified by nucleic acid aptamers.Three kinds of detection methods,such as colorimetric method,up-conversion fluorescence method and peroxidase mimic enzyme method,were constructed to detect the new neonicotinoid pesticide acetamiprid,which is widely used in agriculture.The specific research contents are as follows:（1）A colorimetric method was constructed based on gold nanoparticles（Au NPs）modified by nucleic acid aptamer for the detection of acetamiprid.First,the complementary strand of aptamer was modified on the surface of Au NPs by Au-S bond,and then the aptamer was immobilized on the surface of Au NPs by the principle of complementary base pairing to obtain Au NPs probes modified by double-stranded nucleic acid（ds DNA-Au NPs）.In the presence of Na Cl at a concentration of 0.15 M,the state of ds DNA-Au NPs changes from dispersion to aggregation,accompanied by a visible absorption peak shifting from 524 nm to 650 nm.When acetamiprid is present in the environment,the aptamer preferentially recognizes and binds to acetamiprid,causing dissociation of double-stranded nucleic acids on the surface of Au NPs to form partially complementary single-stranded nucleic acid-modified Au NPs（c DNA-Au NPs）.The salt-induced aggregation ability of c DNA-Au NPs was enhanced compared to ds DNA-Au NPs.In 0.15 M Na Cl,c DNA-Au NPs were still in a dispersed state,and the maximum absorption peak was still at 524 nm.Therefore,based on the change of the single-double strand of the Au NP surface-modified nucleic acid,the detection of acetamiprid can be achieved.The ratio of absorbance at 524 nm and 650 nm(A₅₂₄/A₆₅₀)was used as a signal,which showed a good linear relationship with the concentration of acetamiprid in the range of 0.001-10μM（correlation coefficient R²=0.99）,and the detection limit was 9 n M.（2）An up-conversion fluorimetric method for the detection of acetamiprid was constructed in combination with upconverting nanoparticles（UCNPs）.First,the random nucleic acid strand was modified to the surface of Na YF4:Yb,Er UCNPs by an amide bond to obtain nucleic acid-modified UCNPs probes（r DNA-UCNPs）.There is an internal filtration effect between r DNA-UCNPs（fluorescence emission）and ds DNA-Au NPs（absorption）,which results in changes in the emission fluorescence of UCNPs at 980 nm excitation light.The presence of acetamiprid increase the anti-aggregation ability of ds DNA-Au NPs in 0.15 M Na Cl,and the aggregation effect affect the absorption intensity of ds DNA-Au NPs at 524 nm and 650 nm,and quenching the emission fluorescence of r DNA-UCNPs at 540 nm and 645 nm,respectively.Therefore,the detection of acetamiprid was achieved based on the comparison of the quenching intensity of the fluorescent peaks at 540 nm and 645 nm of r DNA-UCNPs.The change in the ratio of the emission fluorescence at 540 nm and 645 nm(F₆₅₄/F₅₄₀)is a good linear relationship with the concentration of acetamiprid in 0.5-1000 n M（correlation coefficient R²=0.99）,and the limit of detection is 0.36 n M.The method also has good selectivity and anti-interference,and has good effects in the detection of acetamiprid in leaves and tea samples,the recovery rate reaches95.93-102.83%.