Herein, a novel label-free electrochemical immunosensor was fabricated via immobilizing specific anti-β-lactoglobulin (β-LG) antibodies (Abs) onto an integrated electrode of gold nanoparticles (AuNPs) / Prussian Blue (PB) / CMK-8. This immunosensor allowed for the quantitative detection of the major milk allergen β-LG. CMK-8 with excellent electrical conductivity and uniformly adjustable pore structure was modified on the glassy carbon electrode (GCE) and served as the sensitive substrate for the electro-polymerization of PB, forming the redox-active layer. AuNPs were subsequently electrochemically deposited on PB/CMK-8/GCE to improve the electrical conductivity and utilized as the connector for Abs immobilization. During β-LG detection, the Abs-modified AuNPs/PB/CMK-8/GCE exhibited a significant reduction in DPV current signal when exposed to β-LG, displaying an inverse dose-dependent relationship. The developed electrochemical immunosensor demonstrated good detection performance for β-LG, with a wider linear range of 0.01 - 100 ng mL^-1 and a lower detection limit of 4.72 pg mL^-1. Meanwhile, the sensor exhibited remarkable repeatability, reproducibility, stability and anti-interference capabilities, which was further applied to detect β-LG in dairy food, achieving satisfactory recoveries (89.2% - 98.8%) and lower relative standard deviation ( ≤ 3.1%). Therefore, this innovative electrochemical method for food allergens detection holds great potential application in food safety determination and evaluation.

In this study, a label-free, portable and reproducible immunochip based on quartz crystal microbalance (QCM) was developed for the qualitative detection of β-lactoglobulin (β-LG), an allergen, in milk products. Experimental parameters in the fabrication and regeneration procedure such as pH of the coupling microenvironment, amount of anti-β-LG antibody and regeneration reagent were optimized in detail. Under optimal conditions, the proposed QCM immunochip exhibited good recognition of β-LG, with a calibration curve of ΔF = 12.877C_β-LG^0.4809 (R² = 0.9982) and limit of detection of 0.04 μg/mL. Additionally, this portable QCM immunochip had good stability, high specificity, and no obvious cross-reaction to three other milk proteins (α-casein, α-lactalbumin, and lactoferrin). It could compete a qualitative measurement within 5 min, and could be reused at least ten times. In the β-LG analysis of actual milk samples, the developed QCM immunochip yielded reliable and accurate results, which correlated strongly with those from the standard HPLC method (R² = 0.9969). Thus, the portable, stable, and reproducible QCM immunochip developed in this study allowed the rapid, cost-effectively and sensitively measure the β-LG in milk products.