Fe₃O₄ Nanoparticle-Enhanced Laccase-Copper Hybrid Nanoflower-Based Enzyme Sensor for Rapid Determination of Polyphenols in Foods

A novel biosensor, Lac-Cu/Fe₃O₄/MWCNTs-COOH/GCE, was prepared for the rapid and sensitive determination of polyphenols in foods by immobilizing magnetic nanosized ferric oxide (Fe₃O₄), carboxylated multi-wall carbon nanotubes (MWCNTs-COOH) and laccase-Cu (Lac-Cu) onto the surface of a glassy carbon electrode (GCE). The morphological structure of the modified material was characterized by scanning electron microscopy (SEM). This nanoenzyme sensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The CV results showed that the oxidation peak current (I_p) of gallic acid at GCE, Lac-Cu/GCE, Lac-Cu/MWCNTs-COOH/GCE and Lac-Cu/Fe₃O₄/MWCNTs-COOH/GCE was 19.38, 38.87, 46.61 and 59.95 μA, respectively, indicating that the biosensors of Fe₃O₄/MWCNTs-COOH and Lac-Cu had a significant catalytic effect on the electrochemical oxidation of gallic acid, the oxidation peak current increased by 2.10 times compared to the glassy carbon electrode. The experimental conditions optimized by linear sweep voltammetry (LSV) were as follows: 0.1 mol/L citric acid at pH 3 as the electrolyte solution, 4:1 of MWCNTs-COOH to Fe₃O₄ ratio, 5 μL of the composite solution, 4 U of Lac-Cu hybrid nanoflowers, and 12 min of enrichment time. Under these conditions, the I_p exhibited a linear relationship with polyphenol concentration (C_pp) over the range of 7 to 118 μmol/L, described by the equation I_p = 0.6797 C_pp + 38.978 (R² = 0.9993). The limit of detection (LOD) was 1.5 × 10^-9 mol/L (signal-to-noise ratio, R_SN = 3). For the detection of oat polyphenols, this method outperformed the Folin phenol method in accuracy, with spiked recovery rates of 94.45% to 103.5%. With its advantages of rapidity, high accuracy, low LOD and good interference resistance, this method is practical for the rapid and sensitive determination of polyphenols in foods.