This paper describes a novel, simple, and versatile protocol for covalent immobilization of enzyme on electrode. The immobilization method is based on the combination of diazonium salt electrografting and click chemistry. The ethynyl-terminated monolayers are obtained by diazonium salt electrografting, then, in the presence of copper (I) catalyst, the ethynyl modified surfaces reacts efficiently and rapidly with enzyme bearing an azide function (azido-enzyme), thus forming a covalent 1,2,3-triazole linkage by means of click chemistry. The ethynyl-terminated film preserves the activity of the immobilized enzyme. The click chemistry along with binary film of diazonium salts offers a variety of good characteristics including high sensitivity, good repeatability and reusability, rapid response and long term stability of the system. Thus, because of the chemoselective reactivity and quantitative yield of the click reaction, an ethynyl-terminated monolayer can be treated as a general platform for obtaining reliable coverage of a wide range of azido-terminated species of interest for various sensing applications.
