Screening Of Diethylstilbestrol Aptamers And Establishment Of Au Nanoparticles Colorimetric Detection

As a synthetic non-steroidal estrogen,diethylstilbestrol(DES)has been widely used in animal husbandry to promote the growth of animal and decrease fat.Currently,DES has been identified as a human carcinogen.DES is difficult to degrade in water or soil and easy to reside in fat or animal body.It even can be enriched in human body through food chain,with significant reproductive toxicity.Therefore,it was ruled strictly not be used in breeding industry by any way in many countries.However,it is still used illegally to reduce the production cost of animals.It is not only a serious threat to human health,but also a huge loss to the national economy.It is necessary to establish simple,sensitive and effective detection methods of DES for effectively monitoring safety of food and guaranteeing safety of the general public.At present,the methods for detecting DES residues mainly include instrumental analysis methods and rapid detection methods.The instrumental analysis methods cannot meet the requirements of on-site rapid detection,because of their shortcomings of high cost,expensive instruments,high requirements for operators and so on.The activity of immune enzymes and high cost of antibodies are unstable in rapid detection methods.Thus,it is particularly important to establish a new rapid detection method with excellent performance,simple operation and convenient sensitivity.There are many advantages of aptamers,for instance,high stability,high specificity,high affinity and large-scale synthesis in vitro.The thesis researches the rapid detection of DES and the screening of aptamers.One part is to establish a method for detecting DES based on the colorimetric determination using Au nanoparticles(Au NPs)and an aptamer known to bind to phenolic hydroxyl groups.Another part is to establish a method to screen aptamers for DES by SELEX based on the magnetic bead immobilized target.The former work effectively achieves the detection of targets,which have phenolic hydroxyl groups.The latter work is about the screening of aptamers for DES and lays a good foundation for the research of aptamer-based DES detection method.The paper mainly consisted of four parts,as follows:In the first chapter,the physicochemical properties and hazards of DES,advantages and disadvantages of various detection methods were summarized.The aptamer and its characteristics,the basic principle,screening methods and application progress of SELEX were briefly introduced.The screening and application of aptamers for detecting DES in food were mainly introduced.In the second chapter,a method was established for the determination of targets which have phenolic hydroxyl groups,based on aptamers-Au nanoparticles colorimetric method.Au nanoparticles would aggregate under the action of sodium chloride solution.The solution changes from red to purple.Au nanoparticles are used as a colorimetric probe,and the aptamers are used as a recognition component.Single stranded DNA could adsorb on the surface of Au nanoparticles,which could prevent the aggregation of Au nanoparticles.Then,the solution turned red.While in the presence of DES,aptamers could specifically bind with DES accompanied with configuration change.The Au nanoparticles lose the aptamers'protection,the aggregation occurred.The solution appears purple.The DES is detected by visually recognizing the color change of the solution.At the same time,the corresponding solution was tested by UV absorption spectroscopy.When the concentration of DES increases,Au nanoparticles will aggregate.A standard curve between?A₆₅₀/A₅₂₂ and DES concentration was established.The binding time of Au nanoparticles to aptamers,sodium chloride concentration,aptamers concentration and the binding time of DES to aptamers were optimized.The optimum experimental conditions were as follows:the binding time of Au nanoparticles to the aptamers was 40 min,the concentration of sodium chloride was 140 m M,the concentration of aptamers was 800 n M,and the binding time of DES to the aptamers was 30 min.The established method could be applied to detect DES in the linear range between 100 n M to 600 n M.The standard curve equation:y=6.81×10^-4x-0.02,R²=0.9743.The limit of this method was 400 n M by the naked eyes.Spiked recoveries were 84%to 105%.In the third chapter,a method was established to screen aptamers for DES based on the magnetic bead immobilized target SELEX method.The method in the second chapter will respond to many phenolic hydroxyl compounds,and the specificity of the method is not good enough.So,this chapter screens specific aptamers for DES.The DES was carboxylated and coupled with the aminated magnetic beads to achieve target fixation.Separation of the complex was achieved by magnetic separation.The aminated magnetic beads were used as the negative selection targets,the 17?-estradiol and bisphenol A were used as the counter selection target.Combined with SELEX and PCR,eleven rounds of in vitro screening were performed to obtain aptamers from random libraries(10¹³ to 10¹⁵)with different sequences.The optimal aptamers for DES was obtained by sequence analysis,secondary structure analysis,affinity and specificity study.The number of cycles and annealing temperature of PCR were optimized.The optimum experimental conditions were as follows:the circle round number of PCR was 25,and the annealing temperature of PCR was 57?.Under the optimal experimental conditions,21aptamers were screened.The overall homology was more than 60%.Two aptamers(DES-01 and DES-03)bind well to DES.The values of dissociation constants(Kd)of the most stable aptamers(DES-01 and DES-03)were determined by fluorescent method,which were176.33 n M and141.25 n M,respectively.In the fourth chapter,the established aptamers-Au nanoparticles colorimetric method and he selection of DNA aptamer specifically recognizing DES based on magnetic separation were summarized.The established detection method in the thesis has the advantages of simple operation and low cost.The selected aptamers laid a good foundation for the rapid detection of DES.