Development Of Immunosensor For Detecting Sulfamethoxydiazine Residues

Sulfonamide are synthetic bacteriostatic drug,widely used in animal husbandry and as feed additives.As a result,food derived from animals treated with sulfonamides may be contaminated with those drugs,and will influence human health, cause allergic reactions, and could result in resistance in pathogeic organisms because of the extensive use of sulfonamides.To safeguard the public heath,many countrys and organisations have established the Maximum Residues Limits(MRLS) of 0.1ug/g or even none.At present,the primary methods for detecting Sulfonamides Residues, high performance liquid chromatography(HPLC),gas chromatography(GC) and mass chromatography, are not suitable for screening a large number of samples,for they are time-consuming,costly and needing high skill.The study is developing the immunosensor,which is a kind of biosensor, for examining Sulfamethoxydiazine. This topic mainly contains several works ,such as glucose oxidase coupling with sulfamethoxydiazine ,measuring activation of glucose oxidize coupling with sulfamethoxydiazine, checking antigen- monoclonal antibody interaction, immobilising monoclone antibody on layers of nylon. Immobilising protein on nylon film is a key process of assembling of immunoelectrode . By the way, sulfamethoxydiazine is one sort of sulfonamides.During the experiment process,Some archivements were obtained, which are described in detail below:First,glucose oxidase coupling with sulfamethoxydiazine is a key step in this experiment.The traditional method is utilized for the coupling reaction is using glutaraldehyde or in order to form diazo between protein and sulfamethoxydiazine, but it is short of efficience because diazo compound is very difficult to produce.So this reserch didn't use the usual method . The author is directed by Professor Rubinggen who is a famous expert on protein chemisty belonging to Peking University and use EDC as catalyst to produce an new amide bond between glucose oxidase (abbreviation:GOD) and sulfamethoxydiazine . This experiment has proved that EDC is an efficient catalyst of coupling reaction because the operation sequence of the reaction is very simple and the rate of the reaction is rapid. The author insists that the coulping reaction is worthy being expanded.Second,the resercher efficiently detect whether covalent linkage between sulfamethoxydiazine and glucose oxidize is produced or not .The abbreviation of new substantane is GOD-SMD.After coulping reaction, the worker sends GOD-SMD and GOD to the analytical centre of medicine in PKU for the purpose of measuring their molecular weight through mass spectrum. Results of measuremnent manifest that average each glucose oxidize binds 6 sulfamethoxydiazines. Later, the ultraviolet spectrometrum scanining , Fourier transform infrared spectrum scanining, circular dichroism spectrum scanining of GOD-SMD and GOD show the three-deminison structure of GOD-SMD is different from the three-deminison structure of GOD.These facts have proved that new amide bond between Glucose oxidase and sulfameth -oxydiazine existed.Third, the author measured the the activity of GOD-SMD. The operation sequence base on the methods named improvement of Amano Methods. The luminosity scanning of visible Spectroscopy whose wavelength range of spectrum is from 400 nm to 800nm and o-Dianisidine are essential.The result displayed that GOD-SMD still has the enzyme activty of glucose oxidase and the enzyme activty is rather high. Therefore, the experiment reveal that point that someone said glucose oxidase coupling with sulfamethoxydiazine was sure to lost enzyme activity is completely wrong.Fourthly, the author complete the immuno reaction between GOD-SMD and monoclonal antibody . It is an easy task if the antigen of monoclonal antibody is GOD-SMD .However, the antigen of antibody is provided by Yangzhou University is not the GOD-SMD which is produced but bovine serum albumin which couples sulfamethoxydiazines(abbreviation :BSA-SMD) .In a word, the protein carrier of BSA-SMD is different from GOD-SMD which is produced by me but two antigens have the same hapten. The experiments show that the immune reaction between GOD-SMD and monoclonal antibody has existed. The author didn't use the Chromatographic analysis ,which is common method for checking antigen-antibody interaction. The worker used FTIR, CD, the UV spectrum for verifying the antigen-antibody recognition between interaction GOD-SMD and monoclonal antibody.Fifthly, Using Amnano method to measure the enzymatic activty of GOD-SMD binded with monoclonal antibody (anti-BSA-SMD), the result shows that enzymatic activty is enough.Assembling of signal converter:Ion analysator was used to transform chemical sigals to electrical signals。The activities of GOD-SMD and GOD was analysed and the graph of voltage changes in different time was drawed successively.