Potentiometric enzyme electrode for urea based on electrochemically prepared polypyrrole membranes

Potentiometric enzyme electrodes for urea were prepared by the covalent attachment of urease to a functionalized polypyrrole film. In particular, a polypyrrole film was first deposited on the surface of a glassy-carbon electrode and then functionalized to incorporate nitro groups that were later electrochemically reduced to amine groups. The enzyme urease was attached to the film's amino groups through its carboxyl functionalities (i.e., C-terminus, aspartic and glutamic acid residues) by a carbodiimide reaction. The pH response of the polypyrrole film after incorporation of the enzyme was evaluated potentiometrically, resulting in a slope of -54+/-1 mV/pH, Urease catalyzes the hydrolysis of urea, which causes a change in pH that can be detected by the polypyrrole film. The film containing the immobilized enzyme was tested for response to urea, typically showing detection limits of 1.4 x 10(-4) +/- 1 x 10(-5) M (n = 3). Interference studies with N-methylurea and hydroxyurea were also conducted. The electrodes exhibited no response to the non-competitive inhibitor N-methylurea. The response characteristics of the electrodes towards urea remained unaffected after N-methylurea additions. In the case of hydroxyurea, the electrodes responded slightly, but urea response was not restored afterwards.