Stabilization of Wired Glucose Oxidase Anodes Rotating at 1000 rpm at 37°C

Abstract

Anodes on which glucose is directly electro-oxidized can be based on the electrical wiring of reaction centers of glucose oxidase to carbon or gold electrodes. The "wires" are hydrated, cross-linked redox polymer networks, which conduct electrons and are permeable to water soluble reactants and products such as glucose and its oxidation product, gluconolactone. 1-10 Upon cross-linking and hydra-tion, the wiring redox polymer and the enzyme form a hydrogel, the swelling of which depends on the extent of cross-linking. If insufficiently cross-linked and excessively swollen, part or all of the hydro-gel is removed from the electrode upon the application of a sufficient shearing stress. Such a stress is produced by a fast flowing fluid, or by movement of the electrode if it touches soft biological tissue. The stability of a wired horseradish peroxidase coating in a fast flowing liquid was earlier improved by increasing the density of cross-links. To form the denser cross-links, alcohol functions of oligosaccharides of the peroxidase were oxidized by periodate to aldehydes, then the aldehydes were condensed with amines of the redox polymer and of the enzyme itself forming multiple Schiff bases. 11 Here we examine and address the rotation and volume dependent causes of instability of glucose electro-oxidizing anodes. We find that such anodes based on the wiring of glucose oxidase loose their electrocatalytic activity rapidly when rotated at 1000 rpm at 37C. There are two potential causes for the observed decline: loss of polymer under the shear stress produced by the rotation, 12 an effect similar to that observed in fast flowing liquids in the case of the wired horseradish peroxidase cathodes, or accelerated leaching of low molecular weight components of the coating, present if the cross-linking is inadequate. In the latter case leaching is accelerated because of shrinkage of the interfacial boundary layer when the angular velocity is increased. 13 We show that the stability of the glucose electro-oxidizing anodes under shear stress is improved when oligosaccha-rides of glucose oxidase are periodate oxidized to aldehydes and linked, by forming multiple Schiff bases, to amine functions of the wiring polymer and the enzyme. The resulting network is further cross-linked with a water soluble diepoxide. Following such linking, the major residual causes of instability is the attack of the wired enzyme layer by H 2 O 2 , 14-16 which is alleviated by overcoating the electrode with a film of cross-linked catalase. Experimental Chemical.-Poly(4-vinylpyridine) (PVP) of 5 10 4 Mw, pyridines of which were complexed in part with [Os(bpy)2Cl] /2 , and were in part quaternized with 2-bromoethylamine was used as the enzyme wiring redox polymer. The composition, synthesis, and characterization of the redox polymer were reported earlier. 4 The dry polymer was stored refrigerated in the dark. Glucose oxidase (GOx), Fluka catalog no. 49182 from Aspergillus niger, bovine liver cata-lase catalog no. C10 and bovine albumin catalog no. A-2153, and glutaraldehyde (25% aqueous solution) from Sigma were used. In addition to cross-linking by Schiff-base formation, the redox polymer and the enzyme were cross-linked with poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) (Polysciences) or sulfosuccin-imidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB) (Pierce). Other reagents were from Aldrich, Sigma, or Alfa-Aesar and were used as received. Coating solutions.-The coatings were made using volumes of a 10 mg/mL aqueous redox polymer solution and of a 13.33 mg/mL aqueous GOx solution. The polymer was dissolved within the 24 h preceding the coating of the electrodes. A 2.5 mg/mL solution of the cross-linker PEGDGE was prepared within 30 min of its use. For the sulfo-SiAB-containing coated anodes 2.5 mg/mL aqueous solution of the cross-linked material was premixed with a 13.33 mg/mL enzyme solution in a 0.7:1 volume ratio and allowed to react in the dark for 30 min prior to combination with the PVP. To oxidize primary alcohol functions of GOx to aldehyde functions the enzyme was dissolved (20 mg/mL) in 0.1 M NaHCO 3. Sodium periodate (12 mg/mL) was added, to bring the enzyme concentration to 13.3 mg/mL. The oxidation was allowed to proceed in the dark, at room temperature, for 1 h. The periodate oxidized GOx is denoted as GOxox. The catalase solution used contained 13.3 mg/mL of enzyme. The periodate oxidation procedure for catalase was similar to the procedure for forming GOxox. The albumin solutions contained 10 mg/mL of bovine serum albumin. Volumes of these solutions were mixed to provide the stated compositions of films. Electrodes.-Glassy carbon electrodes of 3 mm diam were used. 11 These were sanded with ultrafine sand paper, polished with a 1 m particle-size alumina slurry, and sonicated for 5 min. The polishing process was repeated until no voltammetric peaks were observed at 50 mV/s scan rate throughout the measurement region. The electrodes were then washed with deionized (DI) water, coated with 5 L of the premixed solution containing the redox polymer, enzyme, and cross-linker, and allowed to cure for at least 24 h. Instrumentation and cell.-The measurements were performed using a CH-Instruments electrochemical detector model no. CH1832, and the data were collected on an attached computer. A Pine Instruments rotator was used to rotate the electrodes. The water-jacketed electrochemical cell had a Pt wire counter electrode and a Ag/AgCl or saturated calomel (SCE) reference electrode. It The stability of glucose electro-oxidizing anodes rotating at 1000 rpm at 37C was investigated. The anodes were made by coating 3 mm diam vitreous carbon disks with a cross-linked, electron conducting redox hydrogel, which electrically connected reaction centers of glucose oxidase to the electrode. The endurance of the hydrogel films under the shear stress resulting from the rotation depended on their cross-linking. Periodate oxidation of enzyme oligosaccharides, thereby forming multiple Schiff bases which cross-linked the enzyme and the redox polymer, combined with additional cross-linking by a diepoxide, resulted in toughened elec-trocatalytic films that survived the shearing caused by the rotation. The main residual cause of damage to the toughened films was chemical attack by H 2 O 2 , an undesired by-product. Overcoating the "wired" enzyme film with a layer of immobilized catalase reduced such damage. The current density of glucose electro-oxidation of the stabilized rotating electrodes was at 50 mM concentration , 420 A cm 2 , decaying in the 11 h test period by 28 A/cm 2 .