Polythiophene supported MnO_2 nanoparticles as nano-stabilizer for simultaneously electrostatically immobilization of D-amino acid oxidase and hemoglobin as efficient bio-nanocomposite in fabrication of dopamine bi-enzyme biosensor

摘要

A novel, highly selective and sensitive voltammetric bi-enzyme biosensor for sensing dopamine (DA), in presence of H_2O_2 which resulted as D-alanine enzymatic oxidation, was fabricated on the basis of simultaneously electrostatically immobilization of D-amino acid oxidase (DAAO) and hemoglobin (Hb) on MnO_2 nanoparticles (MnO_2 NPs) enriched poly thiophene (PTh). Cyclic voltammetry (CV), technique was applied for electropolymerization of thiophene on glassy carbon electrode (GCE) surface and MnO_2 NPs dispersed on PTh network by soaking PTh/GCE in potassium permanganate (KMnO_4) solution. Excellent catalytic properties and large surface area of MnO_2 NPs/PTh composite caused it was used as an enzymes immobilization host in this developed bi-enzyme biosensor. The developed biosensor was characterized by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and CV. The performance of the modified bi-enzyme biosensor was investigated in terms of its response time, detection limit, sensitivity, stability and selectivity in a lab environment. The composite of DAAO-Hb/MnO_2 NPs/PTh to construct a bi-enzyme biosensor in this study showed a linear response with DA in the concentration range of 0.04-9.0 μM with R-squared value of 0.994 (for S/N = 3) and its sensitivity and detection limite were about 12.801 μA/μM and 41 nM respectively. Also this bi-enzyme biosensors exhibited high selectivity, rapid response (5 s) and long-term stability (42 days). At the end, the proposed biosensor was applied successfully in human serum as real sample.