Study On The Rapid Detection Of Upconversion Fluorescence Method Based On Aptamer And Antibody Of Acetamiprid

Acetamiprid,a new type chloronicotinic insecticide of broad-spectrum,has strong touch and stomach toxicity.It is developed and commercialized by the Japanese Cao Da Corporation.It has been used widely in controlling sucking-type insects.The excessive residual acetamiprid can destroy ecosystem and harm human's health.Hence,it is vital to detect its residue in environment and agricultural products.At present,analyses of acetamiprid mainly depend on instrumental detections which usually are time-consuming,high expense and difficult to realize high-throughput detection.Therefore,it is necessary to develop a rapid detecting method for the detemination of residual acetamiprid.Upconversion nanoparticles(UCNPs)has been used as a good fluorescence signal in rapid detection by its advantages in biological safety and optical property.Aptamers and antibody have high affinity to their targets which can be used as recognition tools in rapid detection.The detections of small molecule compounds are always relied on molecular recognition elements.Antibody as molecular recognition elements for detection of small molecule compounds has already been well known.In recent years,aptamers have been widely used in the detection of small molecular compounds for their characteristic of easy synthesis,stability and high specific affinity to target.In this study,aptamer and monoclonal antibody(MAb)of acetamiprid were choosed to build three rapid detecting methods of upconversion fluorescenceby combining with UCNPs respectively.1.We synthesized the amino functionalized Fe3O4 magnetic nanomaterials(MNPs)and UCNPs,and then established an upconversion fluorescence DNA probe which consists of aptamer-conjugated magnet nanoparticles(apt-MNPs)and complementary DNA-conjugated upconversion nanoparticles(cDNA-UCNPs)to detect acetamiprid based on magnetic separation principle.Acetamiprid can specifically conjugate with the apt-MNPs to dissociate the cDNA-UCNPs from the apt-MNPs and resulted in reduced fluorescence intensity through an external magnet.The change of fluorescence intensity(?I)is positively related to the concentration of acetamiprid,which can be applied for the quantification of acetamiprid.Under optimal conditions,a linear detection range and detection limit are 0.89-114.18 ?g/L and 0.65 ?g/L,respectively.The probe was successfullyused to detect acetamiprid in spiked paddy water,soil,pear,apple,wheat and cucumber.Average recoverieswere 78.2%-103.5%with intra-day relativestandard deviations(RSDs)of 2.6%-10.9%and inter-day RSDs of 4.3%-10.2%.The amounts of acetamiprid in the authentic paddy water and pear samples detected by the DNA probe are significantly correlated with that detected by high-performance liquid chromatography(HPLC).The correlation equation is y=0.9768x-0.189(R2=0.9974)which proved the upconversion DNA probe is reliable.2.We synthesized gold nanoparticles(AuNPs)with particle size of 20 nm and utilize it and UCNPs to build an upconversion fluorescence probe based on IFE to the quantitative detection of acetamiprid.Under the optimal conditions,the SC50 is 92.7 ?g/L.The linar range of the method is 2.23-285.4?g/L.This method had no obvious cross reaction with other analogues.The average recoveries in paddy water,soil,pear,apple,heat and cucumber were 75%-105.6%,and the relative standard deviation were 1.3%-10.1%.The method is highly correlated with HPLC,and the correlation equation is y=0.9347x+21.412(R2=0.9798).This method is homogeneous reaction without elution steps,simple operation and has high sensitivity which can be used for sensitive,rapid detection and analysis of acetamiprid residues in environmental and agricultural samples.3.The acetamiprid immune antigen and coating antigen were obtaind through active ester method.The immune antigen was used to immune BALB/c female.Three monoclonal antibody(MAb)cell lines named as 2F11B12,2F2B10 and 4D10B8 were obtained through cell fusion.The sensitivity of antibody were investigated by ELISA and the sensivity of 4D10B8 was best.Therefore,the sensivity and specificity of 4D10B8 were studied thoroughly by ic-ELISA.Under the optimal condition,the IC50 and the linar range was 0.15?g/L and 0.003-7.5 ?g/L,respectively.The study of specificity shows it has low cross reacitivity(CRs)with analogues of acetamiprid except thiacloprid.These results demonstrate Mab of 4D10B8 has high sensitivity and specificity.4.We utilized antigen and antibody of acetamiprid to bind with AuNPs and UCNPs respectively and build an upconversion fluorescence method to detect acetamiprid based on IFE.The dosage of AuNPs,the icon intensity,pH and content of methanol of buffer solution were optimized.Under the best condition,the SC50 and SC10 of the method are 0.03 ug/L and 0.001 ug/L,respectively.IFE shows cross reactivity to thiacloprid while it has nearly no cross reactvity to other analogues.The spiked recoveries of soil,pear,wheat and cucumber were 75.1%-104.7%with relative standard deviations(RSDs)of 1.4%-10.1%.HPLC was used to velidate the method,the result shows high correlation between two methods.The correlation equation was y=1.1305x-1.0695(R2=0.9796),which demonstrate the IFE method can be a potential tool to detect acetamiprid.