Establishment And Evaluation Of Visual Detection Method For Immunoglobulin Of Yolk And Lysozyme

Objective:In recent years,protein food additives such as Immunoglobulin of yolk and Lysozyme have been widely used in foods and medicines,the common methods of protein include Enzyme-linked immunosorbent assay?ELISA?and Western Bolt?WB?,etc.However,these methods have some problems,such as expensive and long detection time.Due to the complicated composition of most food samples,these methods are characterized by poor stability and sensitivity in practical application.Therefore,the establishment of rapid,accurate and sensitive on-site protein detection technology is an urgent need for production and operation enterprises,quality control personnel and government management departments.The purpose of this paper is to construct a rapid,specific and visual detection method for protein substances,and to evaluate the method.The research results can provide some technical support for the detection of protein substances in the fields of foods and medicines.Methods:In this paper,a visual detection method of Immunoglobulin of yolk?IgY?and Lysozyme?Lyz?was established.1.The IgY was detected visually based on molecular imprinting technology?MIT?.In this method,using Carboxyl-functioned Fe₃O₄ nanoparticles?Fe₃O₄-COOH?as the core,dopamine?DA?containing catechol and amine functional groups as the functional monomers and crosslinkers,self-polymerizes occurs in a slightly alkaline condition and polydopamine?PDA?coating with catechol and quinone active groups were formed on the surface of Fe₃O₄ nanoparticles.The surface was coupled with IgY to form molecularly imprinted polymers.Sodium dodecyl sulfate?SDS?was used to wash the polymers to removed template molecules,and used magnetic field to separate them.The prepared Magnetic molecularly imprinted polymers?MMIPs?had specific“holes”that complement to IgY structure on its surface.Combined with the action of peroxidase of Fe₃O₄ nanoparticles at the same time,that is,in the presence of H₂O₂,it can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine?TMB?to produce blue oxidized TMB?oxtmb?with the maximum absorption peak at 652 nm.When the target protein was contained in the sample to be tested,the MMIPs specifically combines with it,reducing the specific“holes”and catalytic activity.According to the content of the target and the ratio between the blue depth,the goal of rapid,specific and visual detection of IgY can be achieved.2.The Lysozyme?Lyz?was detected visually based on MIT.In this method,using Lyz as the template protein,Carboxyl-functioned Fe₃O₄ nanoparticles?Fe₃O₄-COOH?as the core,and DA as the functional monomers and crosslinkers.Under weakly alkaline conditions,the negatively charged Fe₃O₄ nanoparticles adsorbed the positively charged Lyz.The PDA layers with a large number of non-covalent groups were formed on the surface of Fe₃O₄ nanoparticles by the self-polymerization of DA.Lyz was embedded into the PDA layer with Fe₃O₄@PDA as carrier.After the elution of the embedded Lyz using 5%glacial acetic acid-SDS,the imprinted polymer microspheres with the specific recognition sites that complement to Lyz can be obtained.The separation was carried out in an external magnetic field,and the samples containing Lyz were concentrated and separated specifically,rapidly and efficiently.At the same time,combined with magnetic nano enzyme catalytic colorimetry technology,according to the inverse proportion relationship between the content of the target protein and the blue product,the results of visual observation and UV-Vis detection were analyzed to achieve the rapid and visual detection of Lyz in the samples to be tested.Results:1.Visual detection of IgY based on MIT.The characterization results of Fe₃O₄nanoparticles and imprinted polymers showed that the Fe₃O₄ nanoparticles synthesized in this study were spherical-like with good dispersion and uniform size,The average particle size of 190±20 nm.The surface of MMIPs and MNIPs had an extra layer of PDA film on the surface of Fe₃O₄ nanoparticles due to the self-polymerization of DA.Compared with MMIPs,the surface of MNIPs had a smoother PDA layer.Under the optimized detection conditions,the linear correlation existed between the concentration of IgY from 0.002 to 0.2 mg·mL^-1.The equation of linear regression was y=-3.0504x+0.9311?R²=0.9852?.The detection of the instrument was 0.013 mg·mL^-1,and the visual detection limit was 0.02 mg·mL^-1.The recoveries of standard addition in the milk powder matrix was 86.7%¹02.6%,and the relative standard deviation was 2.4%⁵.1%?n=3?.The results showed that the method had good specificity,simple operation,the whole detection process was completed within 2.5 hours,and the results can be judged with naked eyes.2.Visual detection of Lyz based on MIT.The characterization results showed that the MMIPs were successfully prepared.When the content ratio of DA to Fe₃O₄nanoparticles were 3:5 and the enrichment time was 30 minutes,the imprinted polymer film had the best enrichment effect on Lyz and the adsorption capacities were138.77 mg·g^-1.Under the optimized conditions,the method had a good linear relationship in the concentrations of Lyz range from 0.008 to 0.08 mg·mL^-1,and linear regression equation was y=-5.0537x+0.9034?R²=0.9356?.The detection limit of the instrument was 0.025 mg·mL^-1,and the visual detection limit was 0.02mg·mL^-1.The recoveries rate of lysozyme enteric coated tables was between 96.82%¹03.03%,and the relative standard deviation was between 2.24%⁷.57%?n=3?.The whole detection process can be completed within 1 hour.Conclusions:1.The sample of Immunoglobulin of yolk and Lysozyme separation based on magnetic separation technology without centrifugation or filtration,which simplified the experimental steps and achieves better separation effect.2.Based on the MIT to detection IgY and Lyz,that is,the PDA layer was modified on the surface of magnetic nanoparticles and the specific“holes”were produced by imprinting template molecules,which improves the selectivity of the imprinted polymers to the target protein.3.A new colorimetric method for IgY and Lyz detection was established in this project,which was based on the molecularly imprinted technology,nano-enzyme colorimetry and magnetic separation technology.According to the inverse proportional relationship between the content of target protein and the blue oxidized TMB product in the actual sample,the target protein of rapid and visual detection was achieved.The detection method has the advantages of good selectivity,high accuracy,simple operation,low cost and so on.