Chapter 4
Electrochemical Characterization of Ferrocene Derivatives in a Perfluoropolymer Glucose Oxidase Electrode
Downloaded by EAST CAROLINA UNIV on September 20, 2015 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch004
Jian Wei Chen, Daniel Bélanger, and Guy Fortier Groupe de Recherche en Enzymologie Fondamentale et Appliquée, Département de Chimie et Biochimie, Université du Québec à Montréal, C.P. 8888, Succursale A, Montreal, Quebec H3C 3P8, Canada Glucose oxidase, G O D , was immobilized into Nafion film, into which artificial electron transfer mediators were incorporated. The mediators were ferrocene derivatives with various hydrophobicity obtained by varying the alkyl chain length. It was demonstrated that they were incorporated in the film with different maximum loadings and at different rates depending on the hydrophobicity. The diffusion coefficients for ferrocene derivatives in Nafion-GOD films decreased when the hydrophobicity of the redox couples increased. The rapid decrease of the catalytic current of glucose oxidation is related to the leaching of the ferrocene derivatives from the Nafion-GOD layer into the solution. However, hexadecanyldimethylaminomethyl ferrocene bromide, the most hydrophobic mediator used, gave a low but stable catalytic current in presence of glucose. It is well demonstrated that glucose oxidase can not directly exchange electrons with a metallic electrode because its redox center is too far from the glucose oxidase's surface (7). Therefore, during the last decade, many considerations were directed to the redox mediators that can be used for the transport of electrons from the active center of the enzyme to the electrode surface. The first generation of amperometric biosensors uses the oxygen as the electron acceptor. In this particular case, the enzymatic reaction rate corresponds to the consumption of oxygen or to the electrochemical oxidation of hydrogen peroxide. The fluctuation of oxygen pressure and the high anodic potential required to oxidize hydrogen peroxide (0.7 V vs SCE) severely limited the application, especially in presence of blood interferents. Consequently, it has been proposed to substitute oxygen by artificial electron acceptors such as ferrocenes (2-72), quinones (13), ruthenium hexaamine (14), osmium complexes (15-17) and components of organic salts (18) which are characterized by lower redox potentials. The approaches that have been proposed to immobilize artificial mediators include the adsorption of the redox mediator (9), the immobilization in carbon paste (73), the covalent linkage on electroinactive (75) or conducting polymer backbone (10), the covalent attachement to the enzyme structures (3) and 0097-6156/92/0487-0031$06.00/0 © 1992 American Chemical Society
In Biosensors and Chemical Sensors; Edelman, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
32
BIOSENSORS AND CHEMICAL SENSORS
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entrapment in ion-exchange polymer (19-22). DuPont's Nafion perfluoronated sulfonated ion-exchange polymer has been extensively used as the immobilization matrix of cationic species (23) and as the covering membrane for enzyme electrodes (24-26). We have recently reported our work on the entrapment of glucose oxidase in Nafion film on a platinum electrode in an aerobic environment (79) and with dimethylaminomethyl ferrocene as an electron acceptor for reduced glucose oxidase (27). In the latter, incorporation of mediators into Nafion film without leakage to the aqueous solution is essential for long term operation of the Nafionglucose oxidase-mediator systems. Considering the structure of Nafion, which possesses hydrophobic fluorocarbon chains and negatively charged sulfonated groups, we synthesized a série of positively charged ferrocene derivatives with variable hydrocarbon length chain with the aim of improving their retention in the Nafion film (28) containing the enzyme. Their electrochemical behavior inside Nafion film has been studied and characterized in terms of incorporation and desorption rates, diffusion coefficients and, amperometric current generated in presence of glucose.
EXPERIMENTAL Enzyme and substrate: Glucose oxidase, type VII-S(E.C. #1.1.3.4) was supplied from Sigma Chemicals Co. A stock solution of glucose oxidase (24,000 U/ml) was prepared by dissolving the appropriate amount of the enzyme in water. A stock solution of glucose (2M) (BDH Chemicals) solutions was prepared 24 hours prior to use in order to obtain the α anomer. Mediators: Dimethylaminomethyl ferrocene (DMAFc) from Strem Chemicals was used as received. Trimethylaminomethyl ferrocene ( C i F c ) chloride was obtained by metathesis of the corresponding iodide salt (Strem Chemicals) with AgCl. A g i precipitate was filtered from the solution and cyclic voltammetry was employed to verify if I- was completely removed. Hexanyldimethylaminomethyl ferrocene bromide (QFcBr) and hexadecanyl dimethylaminomethyl ferrocene bromide (CioFcBr) were synthesized from D M A F c with bromohexane and bromohexadecane, respectively (29). The crude products were recrvstallized from acetone-ether. Their structure and purity were verified by * H - N M R spectroscopy. D M A F c , CeFcBr and Ci6FcBr are lightly soluble in aqueous solution whereas CiFcCl has relatively higher solubility. +
Buffer: A l l experiments were performed in 0.1M K2HPO4-KH2PO4 (pH 7.0) buffer solution. Distilled water from a Barnstead water system was used throughout. Electrode and Nafion film: Platinum disk electrode (area = 0.28 cm^) was fabricated as previously described (27). A solution of Nafion (EW = 1100) 5% w/v in a mixture of lower aliphatic alcohols and 10% water was obtained from Aldrich and diluted with methanol to give a stock solution of 0.35% w/v. Films of Nafion-glucose oxidase were formed by syringing aliquot (20 μΐ) of the mixed solution (86.7 μΐ of Nafion stock solution and 13.3 μΐ of glucose oxidase solution) at the surface of the platinum electrode. The solvents were left to evaporate at room temperature for at least 30 min. The extra amount of GOD was
In Biosensors and Chemical Sensors; Edelman, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
4. CHEN ET AL.
33
Characterization of Ferrocene Derivatives
removed from the film by immersing the electrode in 0.1 M phosphate buffer for about 10 min. The film thickness is calculated based on the density 1.58 g/cm^ (19).
Downloaded by EAST CAROLINA UNIV on September 20, 2015 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch004
Glucose measurement : The amperometric response to glucose was evaluated in a deaerated 0.1 M phophate buffer solution under mild stirring conditions. Prior to glucose measurement, the enzyme electrode was potentiostated at 0.7 V for 1 min and an appropriate aliquot of the stock solution of glucose was added to the buffered solution. The reading was taken at steady state. Apparatus: Cyclic voltammetry and amperometric current-time curves were obtained with a Pine Instrument Inc., Model RDE4 bipotentiostat and Kipp & Zonen B D 91 X Y Y ' recorder equipped with a time base module. A l l measurements were performed in a conventional single-compartment cell with a saturated calomel electrode as the reference electrode and a Pt mesh as the auxiliary electrode at room temperature. Chronoamperometry was made with E G & G Princeton Applied Research potentiostat/galvanostat Model 273 equipped with Model 270 Electrochemical Analysis Software. RESULTS AND DISCUSSION 1. Electrochemistry of mediators in the Nafion-GOD film. 1.1 Incorporation of mediators in the Nafion-GOD film. The incorporation of ferrocene derivatives into Nafion-GOD films was carried out by immersion of a Nafion-GOD coated electrode in a solution containing the ferrocene derivative. The ferrocenes such as DMAFc, C i F c and C 6 F c in the aqueous solution, respectively, partitionned or preconcentrated into mostly hydrophobic part of the Nafion-GOD films. A continuous cyclic voltammogram was performed during the incorporation and showed an increase in the anodic and cathodic currents. The increased peak current between each scan monitored the incorporation rate. The loading of ferrocene derivatives in the film was evaluated from the peak area on the slow scanned voltammogram. The steadystate cyclic voltammograms, corresponding to an equilibrium partition concentration, were obtained after approximately 30 min depending on the mediator concentration in the soaking solution. When the same concentration of D M A F c , C i F c + and C6Fc+ is used in the soaking solution, the initial incorporation rate is in the order D M A F c > C i F c > C 6 F c , respectively. The final mediator concentration in the film of Nafion-GOD vs. the concentration for the different mediators in the solution are compared in Table T. It is interesting to note that when the mediator concentration in the soaking solution is small (
