Electroactive porous films of myoglobin within calcium alginate

In this work, a novel two-step construction strategy for protein assembly films was proposed. The first step was the preparation of porous calcium alginate (CA) films by spraying calcium chloride (CaCl₂) solution over the mixture surface of sodium alginate and polyethylene glycol on various solid substrates. The second step involved the cast of myoglobin (Mb) onto the porous CA films and then formed the electroactive porous Mb-CA films. The nitrogen adsorption desorption isotherm, scanning electron microscope, alternating current impendence and cyclic voltammetry were used to characterize the porous films. Fully hydrated porous CA films had nearly 90 wt% water contents and UV–vis showed that Mb in the porous films retained its near native conformation at medium pH. The stable films modified on glassy carbon electrode demonstrated good electroactivity in protein-free buffer, which was originated from protein heme Fe(III)/Fe(II) redox couples. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k _s) and formal potential (E ^o′) were estimated by fitting the data of square-wave voltammetry with nonlinear regression analysis. It was observed that the formal potential of the Mb Fe(III)/Fe(II) couple in porous CA films shifted linearly between pH 4.0 and 11.0 with a slope of −52.7 mV/pH, suggesting that one proton transfer was coupled to each electron transfer in the electrochemical reaction. The porous Mb-CA films showed the electrocatalytic activity toward dioxygen, hydrogen peroxide, and nitrite with significant decreases in the electrode potential required, and exhibited good operational and storage stability, reproducibility and fast response time for H₂O₂ detection. It is showing the possible future application of the films for biosensors and biocatalysis.