Highly sensitive determination of putrescine in pork by an electrochemical biosensor constructed with unfolded hemoglobin and diamine oxidase

Abstract

Based on the principle that diamine oxidase (DAO) catalyzes putrescine to produce H₂O₂ and unfolded hemoglobin (uHb) has the good catalytic ability to H₂O₂, a highly sensitive electrochemical biosensor for the determination of putrescine was constructed. The biosensor was prepared through immobilizing uHb on the surface of clay modified glassy carbon electrode (GCE) by physical adsorption method, and then using 2.5% glutaraldehyde as cross-linking agent to fix DAO. In order to achieve the best results, the concentration of DAO, the type of peroxidase, the stability of modified electrode and the test conditions were optimized. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the preparation process of the sensor. Under the optimized conditions, the difference of reductive peak currents had good linear relationships with putrescine concentration in the ranges of 1.0 × 10⁻¹¹-1.0 × 10⁻¹⁰ mol L⁻¹ and 1.0 × 10⁻¹⁰-1.0 × 10⁻⁹ mol L⁻¹ (r ≥ 0.997), respectively. The limit of detection (LOD) was 3.3 × 10⁻¹² mol L⁻¹ (S/N = 3). The developed electrochemical biosensor had good reproducibility, stability and sensitivity, and was successfully applied for the determination of putrescine in real pork samples.