Covalent electropolymerization of glucose oxidase in polypyrrole. Evaluation of methods of pyrrole attachment to glucose oxidase on the performance of electropolymerized glucose sensors

Abstract

Methods for covalently immobilizing glucose oxidase in polypyrrole are investigated. The enzyme was chemically modified with pyrrole using one of three different reactive side chains found in the protein. The reactions involve carbodiimide coupling to either lysyl or carboxyl residues on the enzyme and Schiff base reaction of the carbohydrate moiety. O ptimal coupling was achieved with the carbodiimide reaction, 15-20 mol of pyrrole/mol of enzyme compared with 6 mol of pyrrole/mol of enzyme for the Schiff base method. The pyrrole-substituted enzymes were electrochemically active, showing that the pyrrole moieties were oxidizable. Electropolymerized enzyme films deposited from solutions of free pyrrole and amounts of native or pyrrole-modified enzyme of equivalent activity resulted in covalently immobilized enzymes showing both higher enzyme activities and amperometric glucose responses than polypyrrole-entrapped native enzyme. The apparent Michaelis constant (Km* 1) and pH optimum of the modified enzymes electrodes correlated with that of the native enzyme electrode. Enzyme films generated from carbodiimide-modified enzymes were 6-fold more stable to thermal denaturation than native enzyme electrode.

Synthesis of polymeric microcapsule arrays and their use for enzyme immobilization

“…14 Immobilized enzymes are used in bioreactors and biosensors (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Current methods for immobilizing enzymes include adsorption or covalent attachment to a support (2)(3)(4), microencapsulation (5,6), and entrapment within a membrane/film (7,8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) or gel (9 The synthetic methods developed are related to our work on polymeric microtubules (21)(22)(23)(24). While such tubules (21)(22)(23)(24)(25)(26)(27...…”

Biofabrication Using Pyrrole Electropolymerization for the Immobilization of Glucose Oxidase and Lactate Oxidase on Implanted Microfabricated Biotransducers

“…The monomerization of enzymes [48][49][50][51] and biomolecules [52,53] is a well-practiced art and the sulfonization of GOx using PEG spacer arms to a poly(2-acrylamido-2-methylpropane sulfonic acid) has likewise recently been described [54,55]. In this work, the amine groups of lysine residues of GOx were covalently coupled to either 4-(3-pyrrolyl)butyric acid or 4-sulfobenzoic acid using EDC-NHS coupling chemistry, Figure 2.…”

“…Covalently monomerized GOx did not perform statistically different from PPy-GOx. Glucose oxidase that is lysyl modified with an N-substituted 1H-Pyrrole-1-propionic acid has been shown to have higher electrode activity compared to native GOx in polypyrrole membranes [59]. Chemical modification of GOx lysine groups with 3-carboxymethyl pyrrole has also been shown to exhibit higher enzyme activity and storage stability than systems using N-substituted pyrrole [60].…”

Application of Conducting Polymers in Electroanalysis

“…A stable modified enzyme-pyrrole with an average of 30 pyrrole units was obtained thanks to the formation of amide bonds between caboiimide-derivated N-(2-caboxyethyl)pyrrole derivatives and lysyl residues at the surface of protein (Wolwacz et al, 1992). Similarly, N-(3-aminopropyl)pyrrole has been bound to carbodiimide-activated carboxylic residue of the enzyme (Yon-Hin et., al 1993). A similarly obtained glucose-functionalized pyrrole monomer was also polymerized with bithiophene to achieve bio-polymers with higher redox potential compared to that of pyrrole; this assures film with more stability toward degradation effect of peroxide product of GOX (Hiller et al, 1996).…”