AFM1 Dual-signal Sensing Technology In Dairy Products Based On Split Aptamer And Au-rGo

With the improvement of living standards,the demand for dairy products is increasing.Aflatoxin M1（AFM1）is one of the main factors affecting the safety of dairy products.AFM1 is obtained by hydroxylation and metabolism in vivo after animal intake of aflatoxin B1（AFB1）contaminated feed,and its toxicity and carcinogenicity are second only to AFB1.In addition,it is difficult to eliminate the toxicity of AFM1 in dairy processing.Therefore,accurate identification and detection of AFM1 are the primary and key steps to control AFM1 pollution in dairy products.Electrochemical aptamer sensors are widely used in environmental monitoring,food safety and other fields due to their high sensitivity,fast response,low instrument cost and simple operation.However,the flexible structure of aptamers can easily lead to false positive results,and also affect the detection sensitivity.In this paper,AFM1 was taken as the research object,and the complete AFM1 aptamer chain was split into two segments to identify AFM1.The recognition ability and conformation of split aptamer were studied by electrochemical method and circular dichroism.At the same time,gold-graphene composite was used to modify the electrode to establish a highly sensitive and selective dual-signal electrochemical sensing detection method for the efficient detection of AFM1 in dairy products.This study provides a new idea for the application of aptamers in food safety pollutant detection.The main research contents and results are as follows:（1）Study the recognition ability of split aptamer to target.The complete AFM1aptamer chain ACTGCTAGATTCCACAT was split in different proportions,namely mode1（3:1）:ACTGCTAGATTC,CACAT;mode2（2:1）:ACTGCTAGAGATTT,TCCACAT;mode3（1:1）:ACTGCTAGAGA,TTTTCCACAT;mode 4（1:2）:ACTGCTA,GAGATTTTCCACAT;mode5（1:3）:ACTGC,TAGAGATTTTCCACAT.The recognition ability of split aptamer to AFM1 was studied.The results showed that the linear ranges of the sensors established under the five modes were 0.06～0.4μg/L,0.06～0.4μg/L,0.06～0.5μg/L,0.1～0.4μg/L and 0.1～0.4μg/L,respectively.The detection limits were 0.02μg/L,0.02μg/L,0.03μg/L,0.03μg/L and 0.03μg/L,respectively.The calculated Kd values of the five mode split aptamers were 128.77±41n M,262.80±53 n M,87.12±29 n M,339.78±101 n M and 454.19±112 n M,respectively.Mode 3（1:1）has the best recognition effect of split aptamer.The specificity of split aptamer was studied by choosing the structural analogues of AFM1,such as AFM2,AFB1,AFG1 and AFG2,as the competition.The results showed that split aptamer had high specificity for AFM1.In addition,the conformation of split aptamer was studied by circular dichroism.The results showed that the AFM1 aptamer was a B-type structure.After splitting the aptamer,the circular dichroism diagram did not change after the interaction of two separate fragments with AFM1.After the interaction of the two fragments with AFM1,the positive peak intensity of the circular dichroism diagram increased significantly,the negative peak moved and the intensity increased,and the conformation of the aptamer changed.（2）In order to improve the performance of the electrode,the surface of the electrode was modified by Au-reduced graphene oxide（Au-r GO）composite materials,and the controllable preparation of the composite materials was studied.Firstly,r GO was coated on the surface of glassy carbon electrode by dripping,and then Au was deposited by electrochemical deposition to obtain Au-r GO composite modified electrode.The effects of r GO drop concentration and electrodeposition conditions on composite materials were studied by electrochemical method and Scanning Electron Microscope（SEM）.The results showed that the electrode conductivity increases with the increase of r GO concentration.When the r GO concentration was 3 mg/m L,the conductivity of the electrode was the best.With the increase of r GO concentration,the conductivity of the electrode remains unchanged.On this basis,Au-r GO composites were prepared by Au deposition.It was found that Au nanoparticles（Au NPs）grew as agglomerated spherical particles on the surface of r GO at positive potential.With the increase of deposition potential,the particle size of agglomerated spherical particles increased.When the deposition potential was+0.3 V,a uniform agglomeration sphere with a particle size of about 1μm was formed,and the active area of the electrode was the largest.When the voltage continues to increase to+0.5 V,the spherical structure was destroyed,and the agglomeration particle size increased to 4μm;when the deposition potential was negative,the Au NPs are attached to the r GO surface as small particles,and the Au NPs deposited on the r GO surface decrease with the increase of the negative deposition potential.When the deposition voltage reached-0.5 V,bubbles appear on the electrode surface,which hinders the deposition of Au NPs.With the increase of deposition time,the Au NPs deposited on the r GO surface gradually agglomerate into spheres and the particle size gradually increased.When the deposition time was 60 min,the uniform spherical structure can be formed,and the particle size was about 1μm.At this time,the electrode had the largest active area and the best conductivity.When the deposition time increased to 90 min,the spherical structure was gradually destroyed,showing a trend of cauliflower-like development.When the concentration of chloroauric acid increased from 2 m M to10 m M,the number of agglomerated Au NPs deposited on the electrode surface gradually increased,but the particle size gradually decreased from 3μm to 1μm.When the concentration of chlorauric acid was 10 m M,the electrode has a large active area.（3）In this study,a dual-signal electrochemical sensor based on split aptamer and Au-r GO nanocomposites was studied for the detection of AFM1 in dairy products.Based on the above research,Au-r GO composite nanomaterials were used to modify the working electrode.Mode 3（1:1）split aptamers（S1 and S2）were used as recognition elements.S1modified thiol（SH-S1）,which was modified on the surface of the working electrode by Au-S bond.S2 modified methylene blue（MB-S2）was used as the sensing signal,and the complementary chain CS1 of S1 modified ferrocene（Fc-CS1）was used as another sensing signal.In the absence of AFM1,double stranded structure is formed by S1 and CS1,and Fc signal had a response;after adding AFM1,the double chain was opened,and CS1 was released.S1 and S2 formed a certain conformation to recognize AFM1.At this time,the Fc signal decreased,and the MB signal increased.The effects of S1concentration,AFM1 incubation time and recognition environment on the sensor were studied.The results showed that the response signal(I_MB/I_Fc)was the best when the sensor was prepared with 1.0μM S1modified electrode and incubated with AFM1 for 1.0 h at p H 7.4 and Na⁺concentration of 200 n M.Under the condition,the linear range of the sensor was 0.03μg/L～2.00μg/L,the regression equation was I_MB/I_Fc=3.17*C+0.06,the determination coefficient was R²=0.99,and the detection limit was 0.015μg/L.The specificity,stability and practical application of the sensor were also studied.The results showed that the dual-signal electrochemical sensor had a certain anti-interference ability for AFM1 structurally similar mycotoxins（AFM2,AFB1,AFG1,AFG2）.In four weeks,the signal value of the sensor was reduced by 23%,illustrating that the sensor has good stability.The linear range of actual milk samples were 0.20μg/L～1.00μg/L,which conformed to the linear equation I_MB/I_Fc=3.76*C+0.61,the determination coefficient R²=0.98,and the detection limit was 0.05μg/L.Pure milk and skim milk were used as actual samples to study the recovery rate.The results showed that the average recoveries were 86.23%～120.30%and the standard deviations were 5.43%～9.51%.