Penicillin-Binding Proteins and Graphene/Chitosan Nanocomposite-Based Electrochemical Assay for Multiple Beta-Lactam Antibiotics Detection in Milk

Abstract

Beta-lactam antibiotics (BLAs) are not only important in animal husbandry but are also unavoidable contaminants in milk. The gradual accumulation of BLAs in the human body due to increased milk intake can cause direct harm to human health. Therefore, developing selective and sensitive methods for rapid BLA detection is essential. In this study, a specific electrochemical receptor sensor based on penicillin-binding proteins (PBPs) was established. PBP1a and PBP2x, which have high affinity and specificity for the six BLAs, were used as the receptors and their performance was compared. The sensor was fabricated by sequentially immobilizing a graphene/chitosan nanocomposite, penicillin G (PENG), and Nafion on the surface of a glassy carbon electrode using a convenient casting approach. The mechanism of the electrochemical receptor sensor involved direct competitive inhibition of the binding of the graphene/chitosan/PENG composite to the PBPs by the free BLAs in the solution. The PENG-PBPs complex resisted the interaction between [Fe(CN)6]3−/4− ions and the glassy carbon electrode, thereby decreasing the current signal of the BLAs. The BLA concentration was quantified using differential pulse voltammetry mode. The graphene/PENG/chitosan/Nafion-based electrochemical assay demonstrated good selectivity against other antibiotic residues frequently detected in milk. Under optimized conditions, the detection limits of this method ranged from 0.24 to 1.39 ng·mL−1, which are much lower than the maximum residue limits set by China and the EU. Moreover, the sensor showed a satisfactory recovery rate of 98.8 ± 5.8%–103.2 ± 7.1%. The total analysis time involved only one incubation step, and the entire analysis could be completed within 35 min.