A new matrix was designed and constructed: enabling the physical and chemical co-crosslinking of alginate-biotin (Alg-B) with alginate-pyrrole (Alg-py), namely biotinylated-glucose oxidase (B-GOx) was conjugated to Alg-B through avidin (Av) bridges, thereafter mixed with Alg-Py. The alginate was physically crosslinked via calcium ions followed by pyrrole covalent crosslinking through electro-generation of polypyrrole networks. The pyrrole electropolymerization on the electrode surface created spatial wires which tightened the polymeric coating to the electrode surface, packed the 3D structure and reinforced its structure by the covalent bonds. These matrices exhibited better stability than those without Alg-Py even under vigorous stirring applied during the amperometric measurements. When the concentration of the glucose analyte was determined by hydrogen peroxide oxidation, the current was conducted faster to the electrode Surface, therefore providing a low response time and higher sensitivity value: using a relatively low enzyme load. (C) 2009 Elsevier Ltd. All rights reserved.