Rapid And Accurate Detection Of β-lactoglobulin In Infant Formula Food Based On Antifouling Peptide-aptamer Interface Modification Technology

Infants are the main population with cow’s milk allergy,andβ-lactoglobulin（β-Lg）is a major sensitizing protein in cow’s milk and its products.Data shows that more than 80%of patients with cow’s milk allergy are allergic toβ-Lg,soβ-Lg can be used as a marker for the detection of cow’s milk allergens.The existingβ-Lg assay is prone to false positive results due to the interference of non-specific proteins,which affects the accuracy of the assay.Given this,in this paper,EK（Glutamic acid E and lysine K are alternately linked to form polypeptides）with antifouling properties and aptamer with high specificity was applied to the construction of theβ-Lg detection method for infant formula from the perspective of suppressing the interference of non-specific protein and enhancing the active recognition ability ofβ-Lg.Firstly,we investigated the anti-protein adsorption interface modification technology of EK peptide and then constructed a capture probe that can specifically recognizeβ-Lg and resist non-specific protein interference,and finally combined with a fluorescent probe to construct a sandwich assay to detectβ-Lg in three commercially available infant formulae,and compared the results with ELISA kits to verify the feasibility of the method.The main study contents and results are as follows.1.Research on anti-protein adsorption interface modification technology based on EK peptide.The hydrophilicity and anti-protein adsorption properties of EK peptides were explored by using surface characterization methods such as Water Contact Angle（WCA）,Atomic Force Microscope（AFM）,Surface Plasmon Resonance（SPR）based on the plane,and characterization methods based on Fe₃O₄@PDA magnetic microspheres.The hydrophilicity and anti-protein adsorption ability of three lengths of EK peptides,CPPPPEKEK（EK₂）,CPPPPEKEKEKEKEK（EK₅）,and CPPPPEKEKEKEKEKEKEKEKEKEK(EK₁₀)were characterized.The results showed that EK₁₀ had good hydrophilicity and anti-protein adsorption ability,which could be used for selecting anti-protein adsorption materials in this study.2.Construction of capture probes for targeted recognition ofβ-Lg with both anti-matrix interference functions.To balance the anti-matrix interference and active recognition ofβ-Lg,two spatial arrangements of EK₁₀ andβ-Lg aptamer（APT）on Fe₃O₄@PDA were investigated to investigate the effects of non-specific protein adsorption and active recognition ofβ-Lg,and the spatial arrangement of EK₁₀ modified on Fe₃O₄@PDA followed by APT grafting was determined for the construction of capture probes.The feed ratios of EK₁₀ and APT were optimized to better enhance the ability of the capture probes to resist non-specific protein adsorption and actively recognizeβ-Lg,and the feed ratios of 1.2 nmol EK₁₀ and 100 pmol APT were determined for the construction of the capture probes.Finally,the optimized spatial arrangement and feeding ratio were used to construct the capture probes with active recognition ofβ-Lg and anti-matrix interference function.3.Research of fluorescence sensing technology based on dual functional domain capture probes.To achieve the quantitative detection ofβ-Lg in infant formula,a fluorescent probe Si-CD-APT was constructed based on silicon-doped carbon quantum dots（Si-CD）and amino APT,and its good fluorescence performance was combined with the capture probe to construct a sandwich-type fluorescence sensing system and optimize its detection conditions.The results of the methodological investigation showed that under the optimal detection conditions,the standard curve equation of the detection system was y=20701x+6442,R²=0.9903,the limit of detection（LOD）was 0.035μg/m L（S/N=3）,the limit of quantitation（LOQ）was 0.116μg/m L（S/N=10）.Finally,β-Lg in commercially available infant formulae was analyzed,and the results were similar to those of the ELISA kit,demonstrating its potential application.Meanwhile,the study of antifouling peptides in food allergen detection in this work provides a new idea for developing an accurate and anti-protein interference food allergen detection method.