A novel and universal dual-functionalized Hazo-POPs@COOH-apt/PGE-based electrochemical biosensor for detection of aflatoxin M1 (AFM1) in raw milk sample: A versatile peroxidase-mimicking aptananozyme approach

Abstract

A dual-functionalized electrochemical biosensor was developed for ultrasensitive detection of aflatoxin M₁ (AFM₁) in milk. AFM₁, a hydroxylated derivative of aflatoxin B₁ (AFB₁), poses significant health risks, including reduced liver function, stunted growth in children, and immunosuppression. With no FDA-approved decontamination methods, a point-of-care (POC) device for rapid and precise AFM₁ detection is crucial. The electrochemical biosensor combines specificity from COOH-functionalized AFM₁-Aptamer (COOH-Apt) and o-hydroxyazobenzene polymers (Hazo-POPs) as a nanozyme probe at pencil graphite electrode (PGE). Hazo-POPs exhibiting integrated electroactive potential and peroxidase-like activity, independently utilized for AFM₁ detection. Hazo-POPs were characterized using XRD, FTIR, UV–Vis, DLS, Raman spectroscopy, and FESEM and BET. At the same time, PGE surface modification was analyzed through optical microscopy, contact angle measurement, FESEM, micro-CT, CV, and EIS. In method, I, PGE was modified with Hazo-POPs@COOH-Apt for an optimized electrochemical response. COOH-Apt immobilization (Hazo-POPs@COOH-Apt/PGE) exhibited increased current, whereas AFM1 incubation (Hazo-POPs@COOH-Apt/PGE/AFM1) blocked the electron transfer, leading to decreased current. Method II employed Hazo-POPs' peroxidase-like activity, measuring the DPV response of 3,3′,5,5′-tetramethylbenzidine (TMB), confirming aptananozyme (Hazo-POPs@COOH-Apt/PGE) specificity for AFM₁. H₂O₂ generated hydroxyl radicals (°OH) in the presence of Hazo-POPs and TMB was oxidized to diimine by these radicals, leading to reduced TMB peak values, indicating oxidation. The electrochemical biosensor (Hazo-POPs@COOH-Apt/PGE) exhibited a broad linear range (0.005–500 nM) and low LOD (0.004 nM in method I; 0.003 nM in method II). Validated in spiked milk samples, it showed high recovery (101.21–104 %), exceptional sensitivity, reproducibility, and stability for three weeks.