The Immunoassay For Sudan Ⅰ And The Study Of Fluorescent Probe For Mg²⁺Detection Based On Aminated Sudan Ⅰ

Sudan Ⅰ is a lipophilic azo compound that belongs to the Sudan dyes family.As a synthetic colorant,it is extensively applied in cellulose ester varnishes and floor polishes to improve the brightness of tints.Meanwhile,Sudan Ⅰ is also used in some foods as health-threatening additives.Nevertheless,numerous studies have confirmed that Sudan Ⅰ has a strong carcinogenicity and teratogenicity.The International Agency for Research on Cancer(IARC)has classified Sudan Ⅰ as a category 3 carcinogen.Abusing Sudan dyes in the food industry has been prohibited by law in many countries.Therefore,developing a simple and convenient method to detect Sudan Ⅰ is strongly required.The main purpose of this study is to explore the important factors that may affect the enzyme-linked immunosorbent assay(ELISA)and establish the immunoassay method for quick and efficient detection of Sudan Ⅰ.In this paper,the research work is divided into two parts based on Sudan Ⅰ:I Modification of Sudan Ⅰ and establishment of the immunoassay for Sudan ⅠThis section includes design and synthesis of Sudan I,polyclonal antibody production,surface charge calculation of hapten derivatives,monoclonal antibody preparation,homogeneous and heterogeneous immunoassay,as well as real samples detection.(1)First,according to the structural characteristics of Sudan I and the principle of designing an ideal hapten derivative,we designed and synthesized three hapten derivatives:hapten-M,hapten-T and hapten-X,and connected them to BSA and OVA to prepare the immunogens and coating antigens,respectively.(2)Second,polyclonal antibodies were produced and the combinations of different antibodies and coating antigens were designed to explore the relationship between hapten structure and antibody.Meanwhile,theoretical calculation was also performed.The results show that antibodies derived from different hapten derivatives exhibited different characteristic recognition.Antibodies with high titers were obtained against three hapten derivatives.We found that antibodies against hapten-M and hapten-T had good affinities of Sudan Ⅰ,while antibodies against hapten-X,almost does not recognize Sudan Ⅰ.Results mentioned above lead to the preliminary conclusion that the hydroxyl of naphthol ring played an indispensable role in inducing specific antibody production for Sudan Ⅰ and the spacer length of hapten did not affect the characteristics of the antibody significantly.(3)Next,we further verified the experimental results theoretically by calculating the charge distribution of atoms in Sudan Ⅰ and three hapten derivatives within the framework of density fuctional theory.Finally,we selected hapten-M as the optimal derivative to produce the monoclonal antibody and the homogeneous and heterogeneous immunoassays were carried out to explore the sensitivity and specificity of immunoassay.From the experiments,we can draw that heterogeneous immunoassay was more sensitive than homogeneous immunoassay.And the optimal combination of antibody and coating antigen was selected to carry out the following research.In addition,we found that goat IgG has the best blocking effect,but the type and the pH of coating buffer had no significant effect for immunoassay.(4)Thirdly,standard curve of Sudan Ⅰ was drawn in a series of different concentrations by indirect competitive ELISA and the equation was acquired according to logistic curve fitting(four-parameter).Furthermore,the recognition abilities of monoclonal antibodies towards Sudan Ⅱ,Sudan Ⅲ,Sudan Ⅳ and acid orange 7 were also analyzed.The ICso value of antibodies against Sudan Ⅰ was 0.65 ng·mL~-1,and it was almost no cross-reactivity between antibodies and Sudan Ⅱ and IV,respectively.The results that 11.2%of cross-reactivity of antibodies against acid orange 7(structure is very similar to Sudan Ⅰ)showed that the monoclonal antibodies displayed high specificity.(5)Finally,we chose the chili sauce,chili powder and tomato sauce to detecting Sudan Ⅰ by ELISA after extraction by methanol.The spiked recoveries were ranged from 82.2%to 116.0%and the CVs were ranged from 4.3%to 10.9%.The LOD and LOQ were 0.06ng·inL~-1 and 0.62ng·inL~-1,respectively.These results revealed that this method had high accuracy and precision and may be used for detecting Sudan Ⅰ in food samples.Ⅱ Selective fluorescent probe based on aminated Sudan Ⅰ for magnesium ion detectionIn this section,a fluorescent probe SU-CM was designed and synthesized based on hydroxymethyl coumarin and aminated Sudan Ⅰ.SU-CM belongs to "Turn-On" sensor and has a typical "Salen" skeleton,it may coordinate magnesium ion(Mg~2+)specifically by Schiff base ligand.At First,in the absence of Ma~2+.there was almost no fluorescence when SU-CM was excited by light(wavelength:372 nm)in isopropyl alcohol solution.After adding Mg~2+ to the solution,the blue light with wavelength of 474nm was emitted.But it did not respond to other metal ions which shown that SU-CM displayed a good selectivity to Mg~2+.Secondly,with the gradual increase of Mg~2+,a good linear relationship between the fluorescent intensity and concentration of Mg~2+ was observed.By drawing the Job’s plot,we could find that a complex of 1:1 stoichiometry was formed between SU-CM and Mg~2+.Next,we also studied the influence of different solvents on the fluorescence of SU-CM-Mg~2+.In addition,the results that there was no apparent change of fluorescent intensity in the presence of twelve other ions revealed that the SU-CM had a strong anti-jamming capability.In summary,SU-CM could be as a potential fluorescence sensor for rapid Mg~2+ detection with high selectivity and sensitivity.In this study,we have synthesized and selected the best hapten derivative,prepared the monoclonal antibody and finally established the heterogeneous ELISA for Sudan I.This method can be used for detecting Sudan I in food samples with simplicity,high specificity and sensitivity.Meanwhile,we first designed and synthesized a fluorescent probe SU-CM based on anminated Sudan I.It may also provide a good theoretical basis for further application through exploring the fluorescence behavior in mixed solution.