A novel, highly selective and sensitive voltammetric bi-enzyme biosensor for sensing dopamine (DA), in presence of H2O2 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 MnO2 nanoparticles (MnO2 NPs) enriched poly thiophene (PTh). Cyclic voltammetry was applied for electropolymerization of thiophene on GCE surface and MnO2 NPs dispersed on PTh network by soaking PTh/GCE in potassium permanganate (KMnO4) solution. Excellent catalytic properties and large surface area of MnO2 NPs/PTh composite caused it was used as an enzymes immobilization host in this developed bi-enzyme biosensor. The biosensor was characterized by SEM, EDX and CV techniques. 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/MnO2 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 limit 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).
