| Milk allergy is an allergic disease caused by protein allergens in milk and dairy products.β-lactoglobulin(β-LG)is abundant in milk,accounting for 50% of whey protein in milk.β-LG is not present in breast milk and cannot be hydrolyzed by pepsin,so it can enter the blood circulation through the gastrointestinal tract.As the main allergen in milk,β-LG has strong sensitization and stimulates the infant’s immune system to produce hypersensitivity reactions.It belongs to the Lipocalin strong allergen family.In this study,the β-LG allergen in dairy products was taken as the research object,and two methods based on quartz crystal microbalance(QCM)immunoassay and molecularly imprinted polymer(MIP)fluorescence sensing assay were established.The study first prepared a QCM-based immunosensor for label-free quantitative detection of β-LG allergens in dairy products.During the experiment,the experimental parameters such as the p H value of the coupled microenvironment,the amount of immobilized β-LG antibody and the type of regeneration solution were optimized in detail.Under the optimal experimental conditions,the prepared QCM immunosensor can detect the β-LG allergen as low as 0.04 μg/m L,and the quantitative detection can be completed within 5.0 min,and the detection can be repeated at least 10 times.The response to frequency changes produced by β-LG was stronger than that produced by the other three milk proteins(α-casein,α-lactalbumin and lactoferrin),which illustrates that it has good specificity.In the analysis and detection of β-LG allergens in actual milk samples,the prepared QCM immunosensor has high accuracy and has a good correlation with the detection results of the standard HPLC method(correlation coefficient: 0.9969).In this study,a temperature-sensitive MIP fluorescence sensor(UCMPs@MIL-100@MIP)was further prepared for the specific detection of β-LG allergens in dairy products.The UCMPs@MIL-100@MIP can achieve the purpose of adsorbing and releasing β-LG by controlling the temperature of the sample solution to be detected.Its fluorescence intensity has a good linear correlation with the concentration of β-LG allergen,and the detection limit is0.043 mg/m L,the imprinting factor reached 3.415,indicating that the prepared fluorescence sensor has good specificity and selectivity for β-LG allergens.In the analytical detection of real samples such as milk,the prepared fluorescence sensor obtained reliable and accurate results(recovery: 86.0-98.4%,RSD: 2.8-6.8%),which was comparable to standard HPLC The results obtained by the method have a good correlation(correlation coefficient: 0.9949). |
