Polypyrrolebiotin Film Characterization

The structure of the film was assessed by scanning electron microscopy. The polymerization of the pyrrole-biotin monomer was carried out on a carbon felt. Figure 25 shows photographs of polymer deposition on the carbon surface. The latter illustrates that the electrochemical addressing of polymer films allows the spatially controlled functionalization of surfaces whatever their shape and size are. We recently demonstrated by gravimetric measurements that the immobilization of avidin on biotinylated polypyr-role films via bioaffinity interactions provides a compact avidin monolayer (Cosnier et al. 2001). Figure 25C shows this specific binding event of avidin onto polymerized biotin leading to a smoothed polypyrrole-air interface.

Peroxidase Immobilization Amount of Immobilized Enzyme

The amount of POD immobilized on the carbon felt after electropoly-merization were determined by enzymatic activity measurements. The

PODox + pyrogallol ^ purpurogallin + PODred

Fig. 25. Micrographs of carbon felt before (A) and after modification by electropolymeriza-tion of pyrrole-biotin monomer (B) and deposited on platinum surface (C) before (left) and after (right) complexation with avidin

enzyme solutions were analyzed before and after contact with the carbon tissue and the amount of enzyme actually grafted in the polymer matrix was calculated in the same way as described previously in the experimental section. The results demonstrate that 1.6 mg of enzyme was anchored in the polymeric matrix, corresponding to 83% of the initial amount of enzyme (2 mg) put in contact with the carbon felt modified by the polymer. The amount of fixed active enzyme in the membrane is around 4.1 mg/cm3, which corresponds to approximately 0.1 mg/cm2 of the effective surface. Taking into account the fact that the theoretical maximum surface coverage for a close-packed avidin monolayer corresponds to 3.3-5X 1012 molecules/cm2 (Cosnier 1999), the maximum amount of an chored POD should be 0.22-0.33 mg/cm2. The specific anchoring of POD by avidin-biotin bridges thus constitutes an efficient approach for the func-tionalization of the whole structure of a carbon felt. The specific recognition between avidin and biotinylated enzyme allows a strong increase in enzyme grafting as compared to other matrices (Cosnier and Innocent 1993). Thus, the complexation between biotin and avidin appears to be limited by a steric interaction, whereby the smaller the enzyme size, the more efficient the immobilization. The same result has been reported for enzyme immobilization in the case of polypyrrole derivatives for biosensor elaboration (Cosnier and Innocent 1993).

Membrane Working

An equimolecular aqueous solution of H2O2 and pyrogallol (5 X 10-3 M) passed through the POD membrane and was analyzed by absorption spec-troscopy. In the course of the transfer across the membrane, H2O2 is reduced by the immobilized POD, which in turn oxidizes pyrogallol into purpurogallin (see Eqs. 1 and 2). The amount of purpurogallin detected corresponds to the quantity of H2O2 consumed by the enzymatic reaction. The chemical yield of the membrane process corresponds to the ratio between the number of purpurogallin molecules produced and the number of H2O2 molecules introduced into the column. A value of 0.8 was obtained for this ratio. This value, although satisfying by itself, can be improved by a recirculation of the feed solution through the enzymatic membrane.

After a recirculation time of 30 min (which corresponds to six passages of the feed solution volume through the membrane), all of the initial H2O2 was oxidized by the enzymatic catalytic membrane. Figure 26 shows the rate of consumption of H2O2 (initial concentration 5 X 10-3 M) versus time, whereby the exit solution is continuously injected into the column, thus allowing a constant flow through the enzymatic membrane. The remarkable efficiency of this H2O2 degradation process is explained by the peculiar molecular interaction process used for the enzyme attachment in the immobilization procedure. In this way the enzyme was neither altered nor denatured as a result of avidin-biotin complexation and the catalytic activity was maintained in the membrane. The measurement of the specific activity of the free POD in solution related to pyrogallol transformation into purpurogallin indicates 200 U/mg of enzyme (a value not very far from that notified by the supplier, 240 U/mg). The membrane enzyme activity determined in the same experimental conditions was 117 U/mg. This value corresponds to 58% of free enzyme activity. Thus, the remaining activity of the immobilized enzyme is markedly higher than those reported previously with other immobilization methods based on electrogenerated polymer films (only 4% of free peroxidase activity; Coche-Guerente et al.

Fig. 26. Conversion ratio versus time of H2O2 solution (10-3 M) with peroxidase membrane (membrane thickness 1 cm, flow rate 1 ml/min)

Fig. 26. Conversion ratio versus time of H2O2 solution (10-3 M) with peroxidase membrane (membrane thickness 1 cm, flow rate 1 ml/min)

1995) and 9% of free polyphenol oxidase activity (Cosnier and Innocent 1993) is maintained in the polymer matrix. This clearly illustrates the advantages offered by a soft attachment of the enzyme molecules, which preserves their conformational flexibility and hence their activity.

Dependence on pH

The optimum pH range for reactivity of the catalytic membrane was investigated with H2O2 (5 x 10-3 M) as a substrate and a circulation time of 15 min (flow rate: 2 ml/min). In the pH range of 4.7-8.3 the enzyme membrane shows a typical bell-shaped response; t. The maximum efficiency is obtained for pH 6.8 (Fig. 27). This value is in good agreement with that determined with free POD in solution (Worthington 1988). The kinetic experiments presented below were performed at pH 6.8, which corresponds to the pH of maximum efficiency for the immobilized enzymes.

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