An Electrochemical Immunosensor Based On Nanobody And AuNPs-OMC For The Detection Of CP4-EPSPS Protein

Objective:In recent years,the environmental security problems and potential risks on human health brought by transgenic crops have attracted more and more attention.However,few works have been reported about the highly sensitive and selective detection of transgenic proteins.Herein,a new type of electrochemical immunoassay was developed for the qualitative and quantitative detection of transgenic protein CP4-EPSPS by using Au nanoparticles?AuNPs?-ordered mesoporous carbons?OMC?nanocomposite as carriers for the redox probe,thionine?Th?,and nanobody?Nb?instead of conventional monoclonal antibody.Methods:First,AuNPs-OMC nanocomposite was prepared with optimized mass ratios of AuNPs to OMC,and the as-obtained nanocomposite was characterized and analyzed by a series of techniques such as Transmission electron microscopy?TEM?and scanning electron microscopy?SEM?and so on.The properties of Nb such as thermal stability was investigated and compared with those of conventional monoclonal antibody.The electrochemical immunosensor was constructed as follows:first,the AuNPs-OMC composites were drop casted on the surface of the glassy carbon electrode?GCE?.Then,the mixture of Nb and Th was modified onto the electrode surface.Finally,the solution containing CP4-EPSPS protein was incubated on the surface of the modified electrode.Each step for the immunosensor fabrication was monitored by cyclic voltammetry?CV?.After the successful construction of the sensor,the differential pulse voltammetry?DPV?was applied for the quantitative detection of CP4-EPSPS protein,and the standard curve was derived from the experimental data corresponding to different concentrations of CP4-EPSPS protein.Specificity,reproducibility,stability,and reliability of this sensor were tested to verify the feasibility of the method.Results:TEM analysis showed that AuNPs had a diameter of about 13 nm.For better electrochemical performance,a series of AuNPs-OMC composites with different AuNPs/OMC ratio of 1:5,2:5,3:5 and 4:5 were successfully prepared,and a AuNPs/OMC ratio of 2:5 was selected as the optimized ratio for the nanocomposite preparation.The results of TEM and SEM showed that AuNPs were uniformly attached on OMC surface,and the specific surface area of this nanocomposite from Brunauer-Emmett-Teller?BET?analysis was about 1125 m²/g.Thermal stability study showed that Nb exhibited enhanced thermal stability compared with conventional monoclonal antibody.Finally,The regression equation was obtained as?I₀-I_i?/I₀=0.0754LgC?ng/mL?+0.3628 with a correlation coefficient of 0.9932 and the CP4-EPSPS protein could be determined with high sensitivity and efficiency by DPV in a wide linear range from 0.001 to 100 ng/mL with a limit of detection?LOD?of 0.72 pg/mL.In addition,we obtained that the response signal of CP4-EPSPS protein for this sensor was five-fold as much as that of other transgenic proteins.The relative standard deviation?RSD?was 4.2%?n=5?at10 ng/mL CP4-EPSPS solution.The response signal remains more than 80%after 14 days of placement of the immunosensor,and the recoveries of the spiked samples between 96.18%and102.7%with a RSD less than 3.6%.Conclusions:In summary,a simple electrochemical immunoassay with high sensitivity and efficiency was developed for the detection of CP4-EPSPS by coupling Nb,OMC,and Th.OMC not only had excellent conductivity and large surface area,but also provided abundant binding sites by cooperating with AuNPs for the conjugation of Nb and Th that acted as capture agent and electrochemical probe,respectively.The as-constructed immunosensor exhibited a wide linear range,high stability,and satisfactory reproducibility for CP4-EPSPS detection,which is a new approach of genetically modified crop screening and detection with practical significance and application prospect.