New Strategies for the Fabrication of Enzyme Electrodes
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CH3
H2N
\
NH2
H3CO Fig. 1: Chemical structure of o-dianisidine. Authors for correspondence
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Mat. Res. Soc. Symp. Proc. Vol. 414 01996 Materials Research Society
MATERIALS AND METHODS The phycobiliprotein, R-phycoerythrin (PE) was obtained from Molecular Probes, Inc., Eugene, OR. Horseradish peroxidase and glucose oxidase were obtained from Sigma Chemicals, St. Louis, MO. Dianisidine hydrochloride and glutaraldehyde (25%) were obtained from Aldrich Chemicals, Milwaukee, WI and were used without further purification. The spectroscopic studies were carried out using a Perkin-Elmer Lambda-9 UV-Vis-Near IR spectrophotometer (Norwalk, CT) and SLM 8000C spectrofluorometer (SLM Instruments, Inc., Urbana IL). Electrochemical experiments were done using a Potentiostat (EG&G Potentiostat/Galvanostat Model 263, Princeton Applied Research, Princeton, NJ). Approximately 0.25% of o-dianisidine in 0.2 M KCI solution was used for both analytical and preparative experiments. Ag/AgC1 and platinum mesh electrodes were used as reference and counter electrode, respectively. Various electrodes such as platinum and ITO (indium doped tin oxide) coated glass were used as the working electrodes. The platinum electrodes were annealed and treated with dilute nitric acid and ITO glass electrodes were cleaned with de-ionized water prior to use. The diffusion
coefficient of o-dianisidine was calculated using chronoamperometric technique at microelectrodes. The cyclic voltammetry experiments were done using both microelectrodes and conventional electrodes, in 0.2 M KCI solution. The potential was scanned at a speed of 50 mV/s between 0.5 V and 1.5 V, with respect to Ag/AgCl reference electrode. The polymerization was achieved at constant potential and varying potential conditions from both aqueous and organic solutions. Immobilization of protein on to the polymer surface was using the bifunctional coupling agent, glutaraldehyde (OHCCH2CH2CH2CHO). This has been carried out using two different methods: coupling to a pre-formed polymer and coupling to the monomer prior to polymerization. The film was treated with glutaraldehyde and then exposed to the protein phycoerythrin (PE), which resulted in the covalent coupling of the protein to the polymer. The protein film was then characterized by fluorescence spectroscopy. Although this technique allows chemical coupling of the protein to the conjugated polymer, it does not allow sufficient loading of the protein. Therefore, another approach was investigated where the enzyme was a priory coupled to the monomer using the bifunctional coupling agent and then polymerized along with the unmodified monomer. Enzyme electrodes were fabricated with proteins horseradish peroxidase and glucose oxidase and their analytical applications were demonstrated. Protein function is retained in the second approach. RESULTS AND DISCUSSION The polymerization of o-dianisidine-hydrochloride was achieved by repeated cycling of the potential between 0.5V and 1.5V. The polymerization was found to start almost instantaneous
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