WO2007010313A2

WO2007010313A2 - Multi-component conducting polymer electrode and use thereof

Info

Publication number: WO2007010313A2
Application number: PCT/HU2006/000060
Authority: WO
Inventors: Visy Csaba; Kovács KORNÉL; Peintler-Kriván EMESE; Rákhely GÁBOR; Csízi JÚLIA IVETT; Varga ANDRÁS
Original assignee: University of Szeged
Current assignee: University of Szeged
Priority date: 2005-07-20
Filing date: 2006-07-20
Publication date: 2007-01-25
Anticipated expiration: 2008-01-20
Also published as: WO2007010313A3; HU0500701D0; HUP0500701A2

Abstract

The present invention relates to a multi-component electrode which is at least partially covered by a conducting polymer at least on that part of its surface which is in connection with the surrounding medium, and which contains an electron transfer mediator and counter ion, characterized by that the electron transfer mediator is a complex compound containing a transition metal with variable valency. The further object of the invention is the use of the above described conducting polymer based electrode to realize redox processes at the boundary of the electrode and its surrounding medium. The use preferably relates to a redox process catalyzed by the oxido-reductase enzyme, preferably producing hydrogen gas.

Description

A

Multi-component conducting polymer electrode and use thereof

The subject of the invention is a multi-component conducting polymer electrode containing an electron transfer mediator, characterized by that the electron transfer mediator is an organic complex compound containing a transition metal with variable valency, The electrode according to the invention is particularly suitable for the realization of redox processes at the boundary of the electrode and its surrounding medium, e.g. for the electro- biocatalytic production of hydrogen catalyzed by a hydrogenase enzyme. Background of the invention

One of the most important groups of the multi-component electrodes is the electrodes including conducting polymers. Conducting polymers are organic macromolecules having conjugation along the polymeric chain which can be altered electrochemically to develop electronically conducting material [I]. These polymers can be formed from the solution of their monomer either chemically or electrochemically on the surface of any conducting metal (especially platinum or graphite), or any (even non-conducting) surface can be coated with them by applying appropriate procedures. The conducting polymer layer formed on the surface of the conducting metal constitutes a new layer having new and different properties compared to the support, so it modifies basically the properties of the electrode. During the synthesis the conducting polymer part of the electrode is formed in oxidized state, and their positive charges are compensated by oppositely charged ions being present during the preparation [I]. One of the most promising properties of these polymers is that biologically important materials, e.g. enzymes can be immobilized in them [2]. For realization it is necessary to present a motive force to drive the materials into the polymer. With respect to the excess positive charges of the polymer, the incorporation of the materials can be facilitated by their anionic character. Consequently, the incorporation of ampholytic biological materials is possible only if the pH of the solution is higher than the isoelectronic point of the organic substance [3]. For this purpose the biological buffers seem to be suitable [4] in which the mass of the anion is usually larger than 200 D (200 atomic unit). Among the studied materials the disulfonic acid derivative of piperazine (PIPES) proved to be useful, however, the low rate of the polymerization and the limitations of the stable immobilization required that additional electrolytes (e.g. perchlorate, chloride and dodecyl sulfate) had to be present during the preparation of the polymer films [5]. Later this technique became generally applied, so the polymerization solutions buffered by PIPES generally contained the above- mentioned or similar additives. The presence of other anions, however, is not favorable in respect of the immobilization of enzymes and other biologically important materials, since the anions ab ovo decrease the proportion of the above-mentioned biologically important materials in the compensation of the positive charges of the polymeric chain. Moreover, the "competitiveness" of the biologically important materials is strongly hindered because the incorporation depends on the mobility, so the biologically important materials with relatively large molecular mass are handicapped compared to the small and mobile other anions. Hence, in case of incorporation of enzymes having zwitter ionic character [8] or other large- size entities, the exclusive use of the large-size, organic buffer molecules is advisable during the preparation of polymeric films, increasing the relative chance of the incorporation. On the basis of this principle, the immobilization of uricase enzyme in the polymer has successfully been realized in the presence of dodecyl-sulfate ions [9].

It is generally accepted among experts that a conducting polymer is suitable as electrode of enzymatic sensors because the organic matrix ensures a favorable environment to realize the electron transfer to the active center of oxido-reductase enzymes having complicated structure.. Moreover, conducting polymers are also able to transmit the electronic signal for the detection. In order to promote the electron transfer in these sensors, so-called mediators are also applied (mentioned first in [10]). The aim with these mediators is to increase the sensitivity of the electroanalytical applications. The mediators used so far in sensor applications are as follows: derivatives of ferrocene (see ref. 63 in [2], further citation: [2/63]), quinones [2/65], hexacyano-ferrate [2/68], phtalocyanins [2/56] and phorphyrins [2/35-37]. These mediators have been applied in biological fuel cells [I. Taniguchi et al., Electrochemistry, 72 (2004) 427], [N. Mano et al., J. Phys. Chem. B, 106 (2002)8842], [U. Schroder et al., Angewante Chem. int. ed., 42 (2003) 2880] and [K. H. R. Baronian et al., Appl. Microbiol. Biotechn., 60 108].

In an advantageous embodiment of the present invention the mediator molecule is the vitamin B12 [cyanocobalamin, see the Merck Index , ed. 11, p. 1577. (1989),from now on: B12]. It is known that B12 has redox activity [11-12] but this behavior was studied only at carbon and mercury electrodes. It can be applied as catalyst in redox processes, but it has been applied so far either in liquid phase [13-14] or its adsorptive ability was exploited, e.g. in the determination of NO^ -ions, which were bound coordinatively by B 12 [13].

In patent No. JP56112071 B12 is mixed with carbon powder, activated carbon, graphite or "acetylene black" powder. This mixture is spread on silver or nickel surface under high pressure. The "acetylene black" is mentioned there as a possible form of the carbon electrodes beside the carbon powder, activated carbon and graphite electrodes, but it is not identical with the conducting polyacetylene [15].

On the basis of the related literature, only one attempt has been found to incorporate B 12 into a polymer electrode, according to which a pyrrole substituent was built into the heptaester derivative of a B12-analogue (having simplified skeleton) through the hexamethylene side-chain, and the electrochemical polymerization of this complicated molecule has been attempted [16]. Although the authors reported on the formation of an electroactive layer, the electrochemical behavior of the layer proved that the oxidation of the film resulted in only a weakly conducting film. It is supposed that the redox activity of the polypyrrole film was degraded because the monomer to be polymerized was situated at a side- chain of a large organic molecule. The authors concluded from ellipsometric results that only short chains had been developed. It is obvious that the polymerization process, i.e. the layer formation was hampered by the fact that the coupling of the cation radicals formed from the monomers was hindered by the relatively large-size ring-containing part of the monomeric molecules.

In connection with the enzyme, which was found favorable in the present invention (see Examples 2 and 4) it should be mentioned that there are published results in [17] reporting the use of the enzyme originating from Tiocapsa roseopersicina (without any kind of mediator) adsorbed on the surface of a polypyrrole derivative. This polymer was synthesized in nonaqueous medium in the presence of tetrabutyl-ammonium-perchlorate salt. The quantity of the formed hydrogen, however, is small, the highest calculated results were around 20 nmol/min/cm for the hydrogen production at a potential where hydrogen formation takes place at a Pt electrode, too. The subject of the invention

At the beginning of the work resulting in the present invention, our aim was to elaborate a multi-component conducting polymer electrode that contained an immobilized mediator to increase the rate of the electron transfer at the boundary of the electrode and its surrounding medium. We found in the course of our study that the task can be realized in a surprisingly effective way, if the mediator is an organic complex of a transition metal with variable valency, moreover, some of these complexes are applicable even in vivo. The primary subject of the invention is a multi-component electrode, which is at least partially covered by a conducting polymer at least on that part of its surface that is in connection with the surrounding medium, and which contains an electron transfer mediator and counter ion, characterized by that the electron transfer mediator is a complex compound containing a transition metal with variable valency.

Components of the electrode of the invention

Conducting polymer

Any conjugated polymer can be used as conducting polymer (polypyrrole, polyaniline, polythiophene, polyacetylene, their derivatives or the copolymers built up from their monomers), where the monomers are soluble in a solvent in which the organic compound to be incorporated may exist without damage. Polypyrrole was found particularly preferable.

Counter ion

Due to the large size of the electron transfer mediator to be incorporated (and therefore its weak ability for competition), advantageously such anions are present in the solution during the preparation of the electrode which make possible - due to their size - the incorporation of the mediator molecules. The mass of the anion is preferably larger than 200 D, more preferably larger than 300 D. Preferably only the counter ion of the applied buffer having large enough molecular mass is present in the solution as other counter ion.

The charge of the counter ion depends on the charge found in the skeleton of the conducting polymer.

In case of the preferable polypyrrole polymer, the counter ion is an anion. The following buffer systems deliver the appropriately sized anions: PIPES, MES (morpholino-ethane- sulfonic acid), BES [bis(hydroxyethyl) amino-ethanesulfonic acid], MOPS (morpholino- propanesulfonic acid), TES {tris[(hydroxymethyl)methyl]amino-ethane-sulfonic acid}, HEPES (hydroxyethylpyperazine-ethanesulfonic acid). PIPES, MES and BES are recommended in case of polypyrrole [4], but PIPES is the most preferred.

Electron transfer mediator

As it was mentioned above, organic complex compounds of transition metals with variable valency are applied as electron transfer mediator. These complexes have a redox metal center, where the central metal can preferably be cobalt, iron, chromium, nickel, copper, vanadium or manganese. Especially preferable, if the given valency of the ion is an unstable oxidation state. The metal center of the complex is able to take part in redox processes on the basis of its variable valency, thus its oxidation number increases or decreases upon oxidation or reduction, respectively. Consequently, the central "metal atom" can also exist in ionic form in the complex compound. Hereafter, for the sake of simplicity, just the "metal atom" expression will be used in this description, but it embraces all possible oxidation states of the metal atom in question. Preferably, the organic complex forming part of the complex compound applied as electron transfer mediator is preferably a nitrogen atom containing heterocyclic system having more rings, which contains delocalized electrons preferably. For this purpose such compounds may be applied in which the part playing important role in the complex formation has aporphyrine (e.g. hem), corrin or analogue structure. Very good electron transfer mediator behavior is shown by such complexing molecules in case of which the central metal atom is coordinated with 4 to 6 nitrogen atoms, more preferably with 6 nitrogen atoms.

Preferably such mediator is chosen to be built into the electrode which can ensure the electrode potential appropriate for the redox process to be realized. As it is demonstrated in Examples 2 and 4, especially excellent result has been achieved with B 12 electron transfer mediator which contains cobalt atom as central metal atom with 6- fold nitrogen coordination and the cobalt is environed by partially saturated porphyrine skeleton with 5 double bonds. Beside the excellent mediator function of B 12, it can be regarded as a bio-conform substrate based on its known medical applications. The electron transfer mediator can be an inorganic complex, too, containing the above- mentioned transition metal with variable valency. For example: [Co(TN(H₃ ^(NC^)^]^",

[Co(NH₃)₄(NO₂)₂]⁺ , [Co(NH₃)₅NO₂]²⁺ or [Co(NH₃)₆]³⁺

Procedure of the electrode preparation

The preparation of the electrode according to the invention is realized preferably by electrochemical method, preferably at room temperature. The preferred electrochemical procedure is the chronocoulometry by which the thickness of the polymer can be controlled. As support electrode preferably noble metal or steel, more preferably platinum or stainless steel (SS) electrode is used, which previously was preferably cleaned and/or electrochemically pretreated [in case of Pt it is carried out at potential of -1.2-(-0.2) V, usually, with respect to Ag/ AgCl (3.4 M KCl) reference electrode; in case of stainless steel polishing is followed by bleaching in water then rinsing with acetone and carbon tetrachloride]. After the pretreatment of the surface, the potential was set to the polymerization value (which is +0.8 V with respect to the reference electrode mentioned above in case of the preferred pyrrole monomer).

The pH of the monomer solution was set to the appropriate value of the electron transferring mediator (if it is an ampholyte, the pH must be higher than its isoelectronic point). The applied buffer must contain an anion having molecular mass preferably larger than 200 D, more preferably 300 D. A preferred buffer system is the fully protonated PIPES and its disodium salt in appropriate ratio, adjusting the pH of the system between 4.5 to 8. As ' other similar buffers MES (morpholino-ethanesulfonic acid), BES [bis(hydroxyethyl)amino- ethanesulfonic acid), MOPS (morpholino-propanesulfonic acid), TES {tris[(hydroxymethyl)methyl]amino-ethanesulfonic acid}, HEPES (hydroxyethyl-pyperazine- ethanesulfonic acid) can be mentioned, from which MES and BES are also recommended in case of polypyrrole [4] (beside PIPES).

Salts which exert no or negligible influence on the pH of the solution can also be applied in order to increase the conductance of the solution, but they lead to the incorporation of the anions having the above-mentioned size and mass values [e.g. lauryl sulfate (also named as dodecyl sulfate) can be applied, see example 3].

After setting pH the monomer is added to the solution in a concentration of preferably 0.01 to 1 M, more preferably 0.1 to 0.6 M, then the electron transfer mediator is added in a concentration of preferably 0.0001 to 0.1 M, more preferably 0.001 to 0.01 M. Of course, more than one mediator can be applied, if it is advantageous to achieve the declared aim. The polymerization is carried out at a charge density of preferably up to 100 to 4000 mC/cm², more preferably 400 to 3000 mC/cm², even more preferably 600 to 2500 mC/cm².

The thickness of the polymer layer on the surface of the support electrode can practically be formed arbitrary within certain range (preferably from 50 nm to the μm range). The polymer electrode can be stored in the buffer solution applied during the preparation (pH=4.5 to 8) after rinsing.

It should be mentioned that the incorporation of the buffer anions, too, besides the electron transfer mediator is unavoidable during the above procedure. However, at least according to our present knowledge, the anions of the applied buffer do not play role in the electron transport catalyzed by the polymer electrode, but the application of the buffer ensures the effective incorporation of the mediator which preserves its electrochemical activity in polymer having the above additives. For example, the conductive polymer layer prepared in Example 1 still contains PIPES anions besides polypyrrole and B 12 main components, and in Example 3 lauryl sulfate anions are in the conductive polymer layer, too.

. Fields of application The multi-component electrode according to the invention can be applied in several fields since the electrode assists the electron transfer to be significantly easier (i.e. through a lower energy barrier), consequently faster between the electrode and one or more components (in the following parts: component) of the surrounding medium (preferably a buffered solution). The second object of the invention is the use of the above-detailed conducting polymer based electrode for realizing redox processes occurring at the boundary of the electrode and its surrounding medium.

The special electron transfer mediator applied in the electrode according to the invention can be used for processes when the electrode transfers the electron to the species in the medium (e.g. regeneration of co-enzymes, dehalogenization) and also for processes when the electron transfer occurs in the reversed direction (oxidation of thiols, cistein, etc.).

The electrode according to the invention is applicable during any electrochemical measurement (even involving an enzyme reaction) if the component to be determined in the solution (which can be a small or a large molecule, e.g. protein with complicated structure or enzyme) is able to become either oxidized or reduced . Therefore, the electrode according to the invention is applicable either in reductive processes (reduction of C^, NO^^", NC^^", CO2 etc.) or in oxidative processes (e.g. special substances required by the physiological blood- analysis).

It is advised to apply bio-compatible electrode in cases of biological measurements, especially in vivo (e.g. diagnostics or monitoring). Such electrode includes bio-compatible mediator, like B 12.

Moreover, the electrodes in the invention can be applied excellently for electrochemical transformations when the aim is the production of a gas, especially oxygen or hydrogen [18]. Within these applications, there is an important area for utilization, when a structurally complicated organic entity (molecule) takes part in the gas production, which can be a bio- catalyst, preferably an enzyme [19].

In such electrochemical process the electrolysis can be carried out by any stationary procedure (e.g. potentiostatic or galvanostatic one) through either periodical or continuous ways. If the cathodic process is the important one, the necessary electrons can originate from any anodic process acting as the electron source (e.g. KI/In system, oxidation of water, oxidation with laccase enzyme or enzymatic oxidation of glucose). In reverse case (i.e. when the anodic process is important or interesting), any cathodic process can be applied which ensures the consumption of electrons (e.g., H reduction, Fe(III) reduction [20], reduction of coenzymes).

Of course, it would be a particularly important realization when two multi-component electrodes according to the invention (with appropriately chosen enzymes) are combined as a specially exploitable reaction (e.g. electro-biotechnological decomposition of water, since it would result parallel production of oxygen and hydrogen gases [21]).

The most emphasized application area of the electrode according to the invention is its use to produce hydrogen gas, since it is regarded as an important energy source all over the world. The production of hydrogen is possible from water or organic materials (e.g. natural gas) by using external, primary energy source. But hydrogen can already be produced from renewable primary energy sources with reasonable efficiency, too. The fast evolution of biotechnological devices and systems is making more and more realistic the extensive application of hydrogen.

A main type of hydrogen production is based on direct biological splitting of water, e.g., by algae. In these processes the solar energy reaching the Earth's surface is tried to apply directly for energy production. However, due to the fact that the light conversion efficacy of energy transformation by photosynthesis hardly exceeds 10 % , this approach would require the construction of huge bioreactors. In addition, the hydrogen production step is extremely oxygen sensitive in algae. Consequently, any system based on biological splitting of water workable in practice, too, primarily needs an oxygen resistant hydrogenase. Up to now no hydrogenase has been found with oxygen resistant feature in microorganisms capable of splitting of water [22].

An other type of biohydrogen production is embodied by systems based on indirect splitting of water. ,,Indirect" means that the steps of the process are separated in space or in time. In the first step chemical energy is produced with the help of photosynthesis and bound in a biomass and later this biomass is converted into H₂.

The fermentation processes provide another type of implementation. Some microbes possess the capability to obtain the energy for growth from fermentative transformation of organic substances. Fermentation is not the most efficient process energetically, but it is a reliable form of metabolism where the cells get rid of their excess energy relatively easily in the form of hydrogen gas [23].

As in most if the biochemical reactions, hydrogen generation is catalyzed by an enzyme, called hydrogenase, according to reaction (I): 2 H⁺ + 2 e^" ^ H₂ (I)

Hydrogenases can be found primarily in bacteria and algae. Since the hydrogenase catalyzes oxidation-reduction processes, this enzyme is in connection with the processes of the electron transport chain of bacteria, acting either as an electron donor or an electron acceptor. The simple looking task is solved by the enzyme through a complex molecular mechanism. Most of the known hydrogenases are redox metalloenzymes containing Ni or Fe atoms. In living cells the specific and complex protein molecule, surrounding the metals, and the interaction between the protein and the metal atoms make capable the Ni and Fe atoms buried in the molecule to catalyze the elementary steps of either the hydrogen synthesis or hydrogen decomposition.

Hydrogenases are spread widely in the Nature, for example the most thoroughly studied bacterium, the Escherichia coli can synthetize, depending on the growth conditions, 4 NiFe hydrogenases having different functions. In order to utilize enzymes in biotechnological applications they have to be stable enough. Similarly to most of the redox metalloenzymes, the majority of hydrogenases looses their activity easily when they are exposed to atmospheric oxygen or high temperature. This is due to the sensitive interaction between the metal atoms and the surrounding protein. Nevertheless exceptions exist, since a few microorganisms are known to contain hydrogenase with outstanding stability properties. An example is the photosynthetizing sulfur-dependent purple bacterium Thiocapsa roseopersicina. One of its native enzymes is stable against proteolytic digesting, moreover, this hydrogenase is thermophilic having an optimal working temperature beyond 70 ⁰C. This hydrogenase is hardly sensitive to oxygen inactivation [24].

An other gene family, called hυpSL, has been found in the same bacterium, which shows a high degree homology with genes of other NiFe hydrogenases. The hupSL protein is labile and impractical enzyme like most of the other NiFe hydrogenases known from the literature. The hynSL genes were identified as another type encoding a stable hydrogenase, which appears particularly suitable for biotechnological applications. Recently a third hydrogenase (HoxYH), also containing a NiFe metals in its active centre, has been discovered in T. roseopersicina, but it is dissolved in the cytoplasma. Hox hydrogenase is especially interesting because, unlike the stable (Hyn) and unstable (Hup) membrane-associated hydrogenases (which are primarily responsible for the H₂ uptake and decomposition in living cells), Hox is able to produce in vivo a remarkable amount of hydrogen. Finally, genes encoding a fourth hydrogenase QnipUV genes) have been identified in T. roseopersicina, containing structural elements similar to H₂-sensing, so-called sensor hydrogenase enzymes which are known from other bacteria. It means that T. roseopersicina, on the one hand, is rich in hydrogenases like e.g. E. coli and, on the other hand, is non-pathogenic and photosynthetic organism, so T. roseopersicina can be applied advantageously in biological hydrogen- producing systems [25],

In a preferred embodiment of the present invention the HynSL protein is a membrane- bound NiFe hydrogenase, which consists of a large (64 kDa) subunit (HynL) and a small (34 kDa) subunit (HynS). The small subunit contains 3 F4S4 cubane structures that serve as an electron channel between the outer surface of the protein and the active NiFe center buried inside the protein. The large subunit contains the NiFe heterobinuclear center playing a key role in the catalysis. This hydrogenase carries most of the cellular hydrogenase activity, both in H₂ uptake and decomposition, moreover, it is thermostable and resistant to proteolytic digestion. These properties make it is an ideal candidate for biotechnological applications [26]. An additional beneficial property of the enzyme is that its specific activity depends on the concentration of the hydrogenases being in the solution, i.e., the more diluted is the Hyn hydrogenase solution, the higher is the effectivity of hydrogenases being in the solution. Therefore, the specific activity of Hyn hydrogenase may vary in a wide range, between 100 and 1000 micromol H₂/hr/mg protein depending on the conditions used in the measurement, determined by standard activity measurement methods carried out in solution. It is to be emphasized that excellent hydrogen production results were achieved in that embodiment of the present invention where polypirrole was applied as conducting polymer, vitamin B 12 was applied as electron transfer mediator and HynSL type hydrogenase of T. roseopersicina was applied as enzyme. This preferred embodiment is disclosed more precisely in Examples 2 and 4, using the electrode of Example 1 and 3, respectively, as cathode.

According to the scientific literature, some efforts were made to develop in vitro systems. The advantage of an in vitro system is that the regulatory molecular mechanisms of living systems can be eliminated, which work against the targeted purpose, but the disadvantage is hidden in the quick loss of functional stability of the components. Development of hydrogenase containing electrodes is an especially important field. It has been published in the literature that polypirrole derivatives were studied on the surface of graphite electrode and on carbon electrode [17]. The highest calculated H₂ production values were around 20 nmol H₂/min/cm² at -0.5 V/SHE potential, although under these conditions weak H₂ production takes place on Pt electrode.

Evidently, there is a need for the development of cheap (free of nobel metal) hydrogen gas electrodes which can be applied for efficient hydrogen production.

It is to be noted that if the hydrogen production is supplemented with bioelectrochemical reaction ensuring a proper anodic process, then bioelectrochemical water splitting reaction can be implemented by the combination of two multi-component electrodes.

When used in a fuel cell type application, the B 12 behaves as a catalyst on the oxygen electrode. Oxygen is being reduced on this electrode while under properly selected conditions the hydrogenase may work in the hydrogen consumption direction and thus current can be generated in the fuel cell.

Without binding us to the theory, discussed as follows, we suppose that the excellent electron transferring properties of the electrodes according to the invention is in connection with an efficient interaction between the surface of the electrode according to the invention and the components of the solution which take part in the electron transferring process. So, those electrodes mean a specific sub-class of the electrodes according to invention where the surface of the electrode is covered, at least partially, with one or more material(s) which take part in the electron transfer process. In a preferred embodiment the one or more material(s) participating in the electron transfer process is deposited onto the electrode surface from the liquid phase contacting with the electrode. The one or more materials participating in the electron transfer process can be any material having the properties discussed in the description, preferably a biocatalyst, most preferably an enzyme.

Concerning the materials which are necessary to the elaboration of the above sub-class of the electrodes, a reference is made to the above teaching. Summary description of the figures

Fig. 1 shows the cyclic voltammetric curves of the electrode containing no mediator (PPy/PIPES) and the platinum based composite electrode, prepared according to Example 1. Curve (1) shows the current values on a PPy/PIPES electrode measured at decreasing and curve (V) at increasing potential scans. Curve (2) shows the current values on PPy/PIPES/B12 electrode measured at decreasing and curve (2') at increasing potential values. In Figure 2 the amount of the produced hydrogen, using the electrode of example 1, is presented as a function of the applied potential (but using the opposite sign) during the electrolysis according to procedure detailed in example 2. The applicability of the electrode in the invention is shown by the curve with rapidly increasing slope. Further details can be found in the evaluation part following example 2. Figures 3a - 3c show the comparative curves of voltammetric behavior obtained with electrodes formed on stainless steel, with and without B 12. The differences at different scan rates in the various cases are the consequences of whether there has been a pretreatment or not, where the pretreatment is applied in order to oxidize the metallic center of B12 incorporated in the layer. The comparison of the figures shows that the pretreatment causes a reduction current surplus at the electrodes prepared with B 12. Further details can be found in the evaluation part after example 4.

Curves 3 and 4 in Figure 4 show the applicability of the electrode according to example 3 for hydrogen production. Curves 1 and 2 serve for comparison. Each curve in the figure shows the formation rate of hydrogen during electrolysis carried out under electrochemically identical circumstances in the following cases: 1: S S/PPy electrode only 2: SS/PPy/B12 electrode

3: SS/PPy/B12 electrode in a solution including the enzyme, when the anodic process is the oxidation of KI 4: SS/PPy/B12 electrode in a solution including the enzyme, when the anodic process is the oxidation OfK₄[Fe(CN)₆].

The invention is presented in more details through the examples below. It should be mentioned that the chemicals given without manufacturer or reseller are known and used widely, they can be obtained from the usual commercial sources. Example 1: Preparation of the polypyrrole/PIPES based electrode with B12 electron transfer mediator (PPy/PIPES/B12 electrode)

The Pt electrode is cleaned in the mixture of hydrogen peroxide and sulfuric acid (piranha solution) and it is rinsed with water. The electrode surface is pretreated electrochemically in the polymerization solution for 1 s at -0.7 V with respect to Ag/ AgCl (3.4 M KCl) reference electrode. (The potential of the reference electrode is +0.2 V with respect to the standard hydrogen electrode (SHE), the potential values given here are referred to the Ag/ AgCl electrode.) Right after the pretreatment the potential is immediately switched to the polymerization value, which is +0.8 V in case of pyrrole monomer. The polymerization has been carried out in 10 ml buffer solution of pH=6.87 and the solution contains 0.033 M PEPES-acid and 0.1 M PIPES disodium salt. Pyrrole (as monomer) is added first to the polymerization solution up to 0.5 M concentration, followed by the addition of B 12 (as electron transfer mediator, Supelco) up to 0.003 M concentration. The polymerization made up to charge density of 600 mC/cm^ results in an approximately 100 nm thick polymer layer on the surface of the support electrode. The prepared polymer electrode is taken out from the solution and rinsed with water several times and it is stored before use in a buffer having the same pH as the solution applied in the preparation (pH=6.87).

Example 2: Application of the electrode according to example 1 for electro- biotechnological production of hydrogen

The electrode is placed into a three electrodes containing electrolysis cell which has closed airspace where the counter electrode is a Pt electrode of a surface area of approx. 5 cm². After placing the counter and reference electrodes in the cell, it is deoxygenized by bubbling of high-purity nitrogen gas. The cell is filled with PIPES solution where the pH of the solution should be adjusted to optimal value for keeping the hydrogenase enzyme in active form [27]. In the present case, this value is 6.87, practically the neutral pH. Hydrogenase enzyme kept in PIPES solution is added to the working compartment of the cell. The activity η of the enzyme in the cell is 1x10 mol/min. An easily oxidizable species, preferably KI is added in large excess (1.5 M) into the counter electrode part of the cell in order to ensure the counter oxidation process which runs at less positive potential than the oxygen evolution. The electrolysis is carried out at room temperature, preferably potentiostatically in a potential range between -0.68 and -0,74 V which equals to -0.48 and-0.54 V in SHE scale. The process is carried out in sequential steps, by restarting the electrolysis again and again. The amount of the evolved hydrogen gas is measured by GC (gas chromatograph) using heat conductivity detector and column filled with 5 A molecule sieves (Aldrich).

Study of the properties of the electrode prepared in example 1 Figure 1 presents cyclic voltammetric curves registered at the electrodes prepared according to Example 1, in the absence of the mediator (PPy/PIPES) and in its presence (PPy/PIPES/B12).

Curve (1) shows the current values on PPy/PIPES electrode measured at decreasing and curve (I¹) at increasing potential. Curve (2) shows the current values on PPy/PIPES/B12 electrode measured at decreasing and curve (2¹) at increasing potential. The voltammograms were performed in PIPES solutions.

The curves prove the incorporation of the mediator since a surplus reduction current can be seen in the negative potential range originating from the mediator being in the conductive layer of the PPy/PIPES/B12 electrode. The oxidation of the mediator gives a surplus oxidation current in the positive potential range. This oxidation is realized at +0.2 V.

In Figure 2 the production rate of hydrogen from different polymerizing solutions during the electrolysis at an electrode prepared according to Example 1 is plotted as a function of the potential applied, more precisely, of the opposite of the potential applied. Evaluation of Figure 2: In the investigated region the rate of hydrogen production at the application of the complex electrode increases significantly, seemingly exponentially as a function of the increase in the cathodic potential, and an approximately 15-fold rate increase has been experienced in the studied 60 mV potential range. We will return to the exploitation of this promising effect in examples 3 and 4 by the application of a cheaper support electrode and by the enhancement of the B 12 incorporation.

Example 3: Making PPy/B12 electrode on stainless steel (SS/PPy/B12)

The steel electrode must be polished then rinsed with water, acetone and carbon tetrachloride. After it the polymerization is carried out by applying a chronocoulometric procedure at a potential of +0.8 V. The polymerization has been carried out in 10 ml buffer solution, which contains 0.033 M PIPES-acid, 0.1 M PIPES disodium salt and 0.15 M sodium lauryl sulfate. To this polymerization solution pyrrole is added up to 0.5 M and B12 is added up to 0.003 M. The polymerization made up to charge density of 2500 mClcvcr results in an approximately 400 nm thick polymer layer on the surface of the support electrode. The prepared polymer electrode is taken out from the solution and rinsed with water several times and it is stored before use in a buffer having the same pH as the solution applied in the preparation (pH=6.87). The properties of the electrode are presented in Figure 3, in comparison with a similar electrode prepared identically except incorporating B 12. Example 4: The application of the electrode according to Example 3 for reduction process (e.g. electro-biotechnological hydrogen gas production)

The electrode is placed into a three electrodes containing electrolysis cell which has closed airspace where the parts of it are separated by Nafion 117 membrane. The counter electrode is a two times larger Pt electrode (approx. 5 cm ) than the working electrode of Example 3. After placing the counter and reference electrodes in the cell, it is filled with PIPES solution and deoxygenized by bubbling nitrogen gas. The pH of the solution should be adjusted to optimal value for keeping the hydrogenase enzyme in active form [27]. In the present case, this value is 6.87, practically the neutral pH. Hydrogenase enzyme kept in PIPES solution is added to the working compartment of the cell. The activity of the enzyme in η the cell is 4.5x10 mol/min. An easily oxidizable species, preferably KI in large excess (1.5 M) or K₄[Fe(CN)₆] (c=0.1 M) is added into the counter electrode part of the cell in order to insure a counter oxidation process which runs at less positive potential than the oxygen evolution. The electrolysis is carried out at room temperature, preferably potentiostatically at - 0.75 V (which equals to -0.55 V in SHE scale). The overall electrolysis is carried out in sequential steps, by restarting the electrolysis again and again . The amount of the evolved hydrogen gas is measured by GC (gas chromatograph) using heat conductivity detector and column filled with 5 A molecule sieves (Aldrich).

Study of the properties of the electrode prepared in example 3 Figures 3a - 3c show the comparative curves of voltammetric behavior obtained with electrodes formed on stainless steel, with and without B 12. In Figures 3a, 3b and 3c the differences at different sweep rates in the various cases are the consequences of whether there has been a pretreatment or not, where the pretreatment is applied in order to oxidize the metallic center of B 12 incorporated in the layer. In all sub-cases, the electrodes with and without B 12 are compared from the aspect of their behavior at a given sweep rate depending on the use of this pretreatment at a potential of +0.2V.

Figure 3a compares the voltammetric curves at a sweep rate of 10 mV/s. The first two curves run together indicating that there is no difference in currents at the electrodes in presence and absence of B12 when no pretreatment is applied. In case of a 6 s long pretreatment at +0.2 V, however, there is a significant current surplus at the electrode incorporating B 12.

Figures 3b and 3c present the voltammetric curves at sweep rates of 25 and 50 mV/s, respectively. In all cases, a significant current surplus can be measured at the electrodes containing B 12, provided a 6 s long pretreatment was applied at +0.2 V before the measurements. The reason of the reduction current surplus can be explained as the polarization at +0.2 V results in the oxidation of the metallic center of B12, thus the subsequent reduction involves an extra current, too. In the lack of this pretreatment, B 12 is not in oxidized stage in the layer, thus reduction surplus current cannot be measured. If B12 is not built into the layer, the pretreatment does not result in surplus current.

Curves 3 and 4 in Figure 4 show the rates of electro-biotechnological hydrogen gas production at the SS/PPy/B12 electrode according to Example 3 in comparison with some other cases (curves 1 and 2): 1 : S S/PPy electrode only

2: SS/PPy/B12 electrode

3: SS/PPy/B12 electrode in a solution including the enzyme, when the anodic process is the oxidation of KI

4: SS/PPy/B12 electrode in a solution including the enzyme, when the anodic process is the oxidation OfK₄[Fe(CN)₆] .

Figure 4 shows that the rate of the hydrogen production during the studied section of the electrolysis (approx. the first 6 hours) slightly decreases in the given cases, then it becomes closely steady. The value of the rates at the SS/PPy/B12 electrodes compared to the case 1 is 2-3 -fold larger under otherwise identical circumstances. Besides, a significant difference has been experienced when the current efficiencies are compared. In case of electrochemical hydrogen production this value is only 0.47 - 0.53 during the electrolysis at electrode 1, it changes between 0.59 - 0.74 at electrodes 2 and 3, while values between 0.87 - 1.04 are obtained at electrode 4. These data prove that by selecting a proper anodic process, a hydrogen production with 100 % efficiency can be achieved. This observation can certainly be correlated to the ion distribution at the two sides of the membrane, and to the extent of a side process originating from the occasional transport. References

[I] A. F. Diaz, J. Bargon, in Handbook of Conducting Polymers, ed. T. A. Skotheim, vol.l, 81-116, Marcel Dekker, New York (1986) [2] A. Giuseppi-Elie, G. G. Wallace, T. Matsue, in Handbook of Conducting

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[10] P. N. Bartlett, R. G. Whitaker, J. Electroanal. Chem., 224, 37 (1987)

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[17] S. V. Morozov et al.: Bioeletrocatalytic hydrogen production by hydrogenase electrodes, Int. J. Hydrogen Energy, 27, 1501-1505 (2002)

[18] K. Sasikala, Ch. V. Ramana, P. R. Rao, K. L. Kovacs, Adv. Applied Microbiol. 68, 211-295 (1993)

[19] K. L. Kovacs, Cs. Bagyinka, FEMS Microbiol. Rev., 87, 407-412 (1990)

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Claims

1. A multi-component electrode which is at least partially covered by a conducting polymer at least on that part of its surface which is in connection with the surrounding medium, and which contains an electron transfer mediator and counter ion, characterized by that the electron transfer mediator is a complex compound containing a transition metal with variable valency.

2. An electrode according to Claim 1, characterized by that the conducting polymer is any of the followings: polypyrrole, polyaniline, polythiophene, polyacetylene and their derivatives or copolymers built from their monomers, preferably a pyrrole-based polymer.

3. An electrode according to Claim 1 or Claim 2, characterized by that the support electrode is a nobel metal or steel based, preferably platinum or stainless steel based.

4. An electrode according to any of Claims 1 to 3, characterized by that the electron transfer mediator is an organic molecule complex of a transition metal with variable valency.

5. An electrode according to Claim 4, characterized by that the organic molecule complex contains π-electron system.

6. An electrode according to Claim 4 or Claim 5, characterized by that the organic complex contains one or more N-containing heterocycle(s), preferably pyrrole cycle.

7. An electrode according to Claim 6, characterized by that the transition metal in the organic molecule complex is coordinated by at least one, preferably by 4 to 6 nitrogen atoms, where preferably one, two, three or four of them are part of a heterocycle.

8. An electrode according to Claim 7, characterized by that the organic molecule complex contains a part having porphyrine or corrin structure.

9. An electrode according to any of Claims 1 to 8, characterized by that the transition metal with variable valency is any of the followings: cobalt, iron, chromium, nickel or copper, preferably cobalt.

10. An electrode according to any of Claims 1 to 9, characterized by that the electron transfer mediator is B 12.

11. An electrode according to any of Claims 1 to 3, characterized by that the electron transfer mediator is an inorganic complex.

12. An electrode according to Claim 11, characterized by that the transition metal with variable valency is selected from any of the followings: cobalt, iron, chromium, nickel or copper, preferably cobalt.

13. An electrode according to Claim 12, characterized by that the electron transfer mediator is [Co(NH₃)₂(NO₂)4r, [Co(MH₃ )₄(NO₂)₂] ⁺, [Co(NH₃)₅(NO₂)]²⁺ or

[Co(NH₃)₆]³⁺

14. An electrode according to any of Claims 1 to 13, characterized by that the counter ion is an organic ion and its mass is higher than 200 D (atomic unit), preferably higher than 300 D.

15. An electrode according to any of Claims 1 to 14, characterized by that the counter ion is an anion deriving from any of the following compounds: PIPES, MES, BES, MOPS,

TES and HEPES.

16. An electrode according to Claim 15, characterized by that the counter ion derives from PIPES.

17. An conducting polymer based electrode according to any of Claims 1 to 16, characterized by that the surface of the electrode is, at least partially, covered by one or more material(s) taking part in the electron transfer process.

18. An electrode according to Claim 17, characterized by that the one or more material(s) taking part in the electron transfer process is deposited on the surface of the electrode from the surrounding solution contacting with the electrode.

19. An electrode according to Claim 18, characterized by that the material taking part in the electron transfer process is an oxido-reductase enzyme, preferably hydrogenase enzyme.

20. An electrode according to Claim 19, characterized by that the hydrogenase enzyme derives from bacterium Thiocapsa roseopersicina, preferably it is the HynSL enzyme.

21. Use of a conducting polymer based electrode according to any of Claims 1 to 20 for carrying out a redox process running at the boundary of the electrode and its surrounding medium.

22. Use according to Claim 21, where the redox process is a reaction catalyzed by an oxido-reductase enzyme.

23. Use according to Claim 21 or 22, where the electrode is applied as an anode.

24. Use according to Claim 23, where hydrogen gas is oxidized during the redox process.

25. Use according to Claim 21 or 22, where the electrode is applied as a cathode.

26. Use according to Claim 25, where hydrogen gas is produced during the redox process.

27. Use according to Claim 26, where the redox process is an electro-biocatalytic hydrogen gas production catalyzed by hydrogenase enzyme.

28. Use according to Claim 27, where the hydrogenase derives from bacterium Thiocapsa roseopersicina, preferably it is the HynSL enzyme.

29. Use according to Claim 21 or 22, where the enzyme-catalyzed redox process is the electrode process of an oxygen electrode.

30. Use of a conducting polymer based electrode according to any of Claims 1 to 20 for the purpose of qualitative and/or quantitative analytical determination.