The Research Of Upconversion Immunoassay For Imidaclothiz Residue

Imidaclothiz,as a efficient and low-toxic,broad-spectrum neonicotinoid insecticides,was autonomously exploited by China.The environment polloution that caused by its widespread use has attracted more and more attention.At present,the traditional instrumental analysis for imidaclothiz is high performance liquid chromatography(HPLC)and high performance liquid chromatography-mass spectrometry(HPLC-MS/MS).However,there are many defects such as high cost,used large amount of organic solvents,complicated operation,time consuming and so on.Compared with instrumental analysis,because of its convenient and swift,large analysis capacity,and low detection cost,immunoassay has become one of the most competitive analysis technology in 21st century.Fluorescence immunoassay has the advantages of high sensitivity and strong specificity,and is widely used in pharmaceutical analysis.As a novel fluorescence proble,Ln3+-doped upconversion nanoparticles(UCNPs)can eIite high-energy light when excited by low-energy near-infrared light through the mechanism dual-photon or multi-photon,which also called anti-Stokes mechanism.Fluoroimmunoassay is a high sensitivity and specificity method,and has been widespread used in pharmaceutical analysis.Compared with traditional fluorescence labeling materials such as organic fluorescent materials and quantum dot,the UCNPs have many unique radiance properties,such as higher fluorescence intensity and longer fluorescent life,narrower emission peak,larger stokes shift,better chemical stability,lower toxicity,and the same time,becaused of used 980 nm near-infrared light as excited light,which eliminated the interference of background fluorescence and improved the detection sensitivity.Recently,UCNPs have attracted extensive attention in biological detection,drug screening and clinical medicine.But so far,the synthetic UCNPs also exists many problem,such as heterogeneity of particle size,weak of fluorescence intensity and poor water soluble.So how to solve the above problem and make the UCNPs successful used in biological analysis are also the primary problems of researcher faced at present.In this study we synthesized a high fluorescent efficiency of UCNPs.Using imidaclothiz monoclonal antibody,with UCNPs as fluorescent probe,based on inner filter effect,magnetic separation principle and immunochromatograghy method,we established three imidaclothiz upconversion immunoassay.The established immunoassays were also used to detect imidaclothiz residues in agricultural and environmental samples.The main results were as follows:1.The UCNPs was synthesized successfully by hydrothermal method.We also optimized the reaction conditions,including the choice of complexing agent,the ratio of rare earth elements,the pH,reaction time and reaction temperature.The results showed that when we used sodium citrate as complexing agent,the molar ratio of rare earth elements was 79%:19%:2%,the pH at 5.0,reaction time at 6h and reaction temperature at 180?,could obtain the high luminous efficiency of hexagonal phase UCNPs(?-UCNPs).At the same time,the synthesized UCNPs was characterized by transmission electron microscopy(TEM),X-ray diffraction(XRD)and fluorescence spectrophotometer.2.We synthesized a gold nanoparticles(AuNPs)with particle size of 20 nm,and used the above synthesized UCNPs and the available antibody of our laboratory,an upconversion fluorescence immunoassay was established based on IFE.The NaYF4,Yb,Er UCNPs and AuNPs were coupled with anti-imidaclothiz antibody and antigen,respectively.At the same time,the dosage of AuNPs and buffered solution were optimized.Under the optimal conditions,the standard curve was established.The sensitivity,specificity,precision and accuracy were assessed by testing the cross-reactivity(CR),spiked recovery and correlation with HPLC.Under the optimal conditions,the concentration of analyte producing 50%saturation of the signal(SC50)and limit of detection(LOD,SC10)were 18.92 ng/mL and 2.09 ng/mL,respectively,and the relative standard deviation(RSD)were 1.9%-10.3%.The CRs were negligible with the analogues except for imidacloprid with CR of 92.19%.The average recoveries were 67.4%-104.6%in paddy water,soil,pear,rice,apple,tomato,pakchoi and cabbage.Compared the detection results of the analysis with these of HPLC,they showed highly correlation,the correlation equation was y = 0.8904x +4.2483,and the coefficient of determination(R2)was 0.9811.The research showed that the IFE immunoassay had many advantages,such as simplicity of operator,rapid,high specificity,great accuracy and sensitivity,which could rapidly and accurately detect the residue of imidaclothiz in environmental and agricultural samples.3.We first synthesized the amino functionalized Fe3O4 magnetic nanomaterials(MNPs),and then established a immunoassay based on UCNPs labeling and magnetic separation.The imidaclothiz antibody and antigen were coupled with UCNPs and MNPs,respectively,then the dosage of nanomaterials and working buffer solution conditions were optimized.Under the optimal conditions,the linear equation of this method was y=122.73x+56.119,R2=0.9981,the linear range(IC10-IC90)was in the range of 0.74-289.30 ng/mL,the median inhibitory concentration(IC50)was 14.59 ng/mL,the minimum detection limit(LOD,IC10)was 0.74 ng/mL.The method has no obvious CR with other imidaclothiz analogues except imidacloprid(89.18%).The recovery and RSD for paddy water,soil,pear,peach,tomato,wheat,rice and pakchoi were in the range of 75.7%-105.2%and 0.9%-11.2%.The results for real sample detection indicated that the established method has a high degree of correlation with HPLC,the correlation equation is y = 1.0322x-0.8721,and the R2 was 0.9958.The established competitive immunoassay that based on magnetic separation was more sensitive than enzyme-linked immunoassay(ELISA),fluorescence polarization immunoassay(FPIA)and quantum dots-based fluoroimmunoassay(QDFIA).It is expected to be a high sensitive analytical tool for detecting the residues of imidaclothiz in environmental and agricultural samples.4.Based on immunochromatography,a upconversion fluorescence immunoassay for imidaclothiz was developed.Under the optimal conditions(pH=8,the Na+ concentration was 0.3 mol/L,the content of methanol and PEG20000 were 2.5%and 1%,respectively),the IC50 was 97.37 ng/mL,the LOD was 26.30 ng/mL,the linear range was 26.30-363.08 ng/mL and the detection time was 25 min.There were no obvious CRs for imidaclothiz analogues except for imidacloprid.The average recovery for paddy water,soil,pear,peach,wheat,cucumber,tomato and rice were 71.8-97.2%and RSD were 0.7-10.7%.The detection results for real samples of this method showed a high degree of correlation with HPLC.The established imidaclothiz upconversion fluorescence immunochromatographic method has the advantages of simple operation,little time consumed,which could realize the rapid detection of imidaclothiz residue samples,and provides a new choice for the rapid screening method of imidaclothiz residue.