| Pesticide residues pose a potential danger to food safety.Traditional detection methods of pesticide residue mainly are related to sample collection,sample comminution,extraction and purification with the long detection cycle,leading whole test process being time-consuming.This brings a lot of inconvenience to supervise the work based on the entire processing process of agricultural products.Therefore,it is necessary to develop a highly efficient,convenient,new method for detecting pesticide residues.Due to the superiority of great sensitivity,high selectivity,minimal sample preparation and nondestructive nature,surface-enhanced Raman scattering(SERS)technology has been widely pursued in various fields mainly on the detection of complex biological structures,monitoring mycotoxins,and detecting virus and so on.This paper combined SERS and nanomaterials with aptamer modification to receive a higher sensitivity and specificity of detection.Specific work content is as follows:First of all,IP6-Fe3O4@SiO2@Au gold magnetic nanoparticle was synthesized,acetamiprid aptamer coupling IP6-Fe3O4@SiO2@Au gold magnetic nanoparticles was regarded as capture probe I.Then,Cys-SiO2@Au nanoparticle was also synthesized,chlorpyrifos aptamer coupling Cys-SiO2@Au nanoparticle was considered as capture probe Ⅱ.Uv-vis spectrophotometer,infrared spectrum,grain size analysis and transmission electron microscopy(TEM)were used to characterize the quality of these product above.Results showed that spherical amination Fe3O4 nanoparticles with 45 nm was synthesized,showing better homogeneity and relatively uniform dispersion.IP6-Fe3O4@SiO2@Au in aqueous solution had a good monodispersity with 65 nm nanoparticle size.Gold seed grew in the dot shaped onto the SiO2 layer to form a shell.The enhancement factor is 1.25×107 and lasts 21 days.According to the results of Uv-visible absorption spectrum,capture probe Ⅰ included characteristics of ultraviolet absorption peak of both IP6-Fe3O4@SiO2@Au gold magnetic nanoparticles and aptamer concentrate,suggesting that successful preparation of capture probe Ⅰ.Cys-SiO2@Au gold shell nanoparticle showed certain dispersion in aqueous solution with full gold shell growth and nanometer particle size with about 210 nm.The enhancement factor is 1.02×107 and lasts 28 days.Uv-visible absorption map showed that capture probe Ⅱ contained characteristics of ultraviolet absorption peak of both Cys-SiO2@Au gold shell nanoparticles and aptamer concentrate,suggesting that successful preparation of capture probe Ⅱ.Then,a SERS acetamiprid detection model was built based on the capture probe I.Based on the single factor analysis and Box-Behnken optimization test,the best optimized condition was obtained.The quadratic multinomial regression equation of acetamiprid SERS effect on volume(A)of capture probe Ⅰ,concentration of coagulant(B),the incubation time(C): Y =-9424.86458 + 88.83750×A + 7603.75000×B + 56.81979×C-8.25000×AB-0.17937×AC + 99.00000×BC-0.21792×A2-45366.66667×B2-0.26604×C2.Model F value is 9.96,the response to the regression model showed significant difference(P = 0.0105 < 0.05),difference of loss of quasi item was not significant(P = 0.1065 > 0.05),the model decision coefficient R2 = 0.9472,which could explain 94.72% change of response values.This model could achieve a better fitting.Order of influence on acetamiprid SERS relative peak based on various factors is: coagulant concentration(B)> volume of capture probe Ⅰ(A)> incubation time(C).The best SERS detection condition of acetamiprid was determined as follows: the volume of capture probe Ⅰ of 170 μL,the coagulant concentration of 0.15 M,the incubation time of 80 min.SERS relative peak intensity was 883 with the theoretical value of 883.213,which was consistent with the model prediction.There is a good linear relationship(y=1499.0616x-1546.0905,R2=0.9978)between SERS relative peak area(1480-1520 cm-1)and logarithms of acetamiprid concentration within 4.5 to 1500 nM.The detection limit was 1.3 nM.Next,the specificity experiment with acetamiprid was conducted.Capture probe I was used to capture acetamiprid and carboxyl fluorescein(FAM)coupling acetamiprid aptamer was considered as a signal probe Ⅱ,this built a SERS acetamiprid detection specificity model.In addition,malathion,phosphorus,carbofuran,phoxim,abamectin,trichlorfon,thiophanate-methyl,carbendazim,chlorpyrifos were chosed to conduct the specificity experiments with acetamiprid at the same time.The standard addition recovery experiment was subsequently implemented,(recovery was 93.3%-102.7%).The results proved that this method is specific,which can be used in the acetamiprid detection of samples.Next,chlorpyrifos aptamer coupling Cys-SiO2@Au nanoparticle was considered as capture probe Ⅱ.Based on the single factor analysis and Box-Behnken optimization test,the best optimized condition was obtained.The quadratic multinomial regression equation of chlorpyrifos SERS effect on oscillation frequency(D),incubation temperature(E),aging time(F)and pH(G): Y’ =-4492.60417 + 11.39000×D + 4.82500×E +28.98611×F + 917.8333×G-0.014000×DE-0.020833×DF-0.15500×DG +1.37500×EF-2.00000×EG-4.50000×FG-0.019567×D2-0.94417×E2-0.59838×F2-64.29167×G2.Model F value is 2.97,the response to the regression model showed significant difference(P=0.0252<0.05),difference of loss of quasi item was not significant(P=0.9901>0.05),the model decision coefficient R2=0.7483,which could explain 74.83% change of response values.This model could achieve a better fitting.The order of influence on chlorpyrifos SERS relative peak of various factors is: incubation temperature(E)> aging time(F)> ioscillation frequency(D)> pH(G).The best SERS detection conditions of chlorpyrifos was determined as follows: oscillation frequency of 260 rpm,incubation temperature of 33 ℃,aging time of 30 min,pH of 6.3,After three parallel experiment,SERS relative peak intensity was 470 with the theoretical value of 469.139,which was consistent with the model prediction.There is a good linear relationship(Y=276.2172X-94.9788,R2=0.9863)between SERS relative peak area(765-805 cm-1)and logarithm of chlorpyrifos concentration within 10 to 3000 nM.The detection limit was 2.0 nM.Then,carboxyl tetramethyl rhodamine(TAMRA)modified chlorpyrifos aptamer was regarded as a signal probe Ⅱ.A double layer SERS model for detecting the chlorpyrifos was built.The standard addition recovery experiment in red bell pepper was subsequently implemented(chlorpyrifos recovery was 93.8%-98.9%).The results prove that this method is specific,which can be used in the detection of real samples. |
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