Encapsulation of redox mediators

Abstract

The present invention relates to methods for the preparation of bioactive ink formulations for making printed bioelectrodes and their use in biodegradable enzymatic fuel cells.

Description

[Technical field]

[0001] The present invention relates to the use of redox mediators encapsulated in mesoporous carbon (i.e., redox mediator / mesoporous carbon nanocomposites) for the preparation of bioactive ink formulations for making printed bioelectrodes and their use in biodegradable enzymatic fuel cells.

[0002] In the following description, references in square brackets ([]) refer to the references list at the end of the text. [Background technology]

[0003] Biological fuel cells (biofuel cells) offer an attractive means of providing environmentally friendly and sustainable power to electronic devices, especially small portable devices for applications such as healthcare, environmental monitoring, and biodefense. Given that enzyme-based fuel cells can operate using abundant substrates in biological fluids and environmental exhaust (i.e., glucose and oxygen) while exhibiting power densities that are often superior to microbial fuel cells, they offer an attractive proposition for augmenting or self-powering miniaturized wearable or implantable devices [1, 2, 3]. Furthermore, paper-based devices have gained popularity as a proposition for these types of applications due to their low mass, small form factor, and flexibility, allowing them to conform to a variety of different surfaces.

[0004] The core of this technology is based on a pair of oxidoreductase enzymes that ensure the catalysis of the fuel. The oxidoreductase enzymes have a protein structure (apoenzyme) that contains an active redox cofactor capable of transferring electrons from a substrate to a cosubstrate. When the redox protein is able to exchange these electrons with an electrode and then displace the substrate or cosubstrate, this is called a direct electron transfer (DET) process (Figure 1).

[0005] Although this phenomenon has been known for more than 30 years and has been observed in many proteins, it is not always possible to observe this DET. Indeed, depending on the redox protein and electrode material being studied, direct electron exchange is not always advantageous. In order to be able to observe the bioelectrocatalysis phenomenon, it is necessary to add a redox molecule (often of low molecular weight) that acts as an electron transporter. This is mediated electron transfer (MET), and the molecules used as "shuttle" are commonly called redox mediators (Figure 1).

[0006] The electron transfer mode (DET or MET) is very often specific to the biomolecule and electrode material used.

[0007] There is a wide variety of redox mediators that are commonly used in the development of biofuel cells: among the best known are metal complexes (osmium complexes, ferrocene), quinones, azines, or viologen-type derivatives.

[0008] Therefore, to be a suitable candidate as a redox mediator, a redox probe must meet several criteria. -The potential of this mediator must be such that the biocatalysis is thermodynamically favorable, i.e., its potential must be higher than that of the enzyme at the anode and, conversely, lower than that of the enzyme at the cathode (Figure 2). -The redox system must be reversible. -Enzyme / mediator electron transfer must be efficient to allow good electrocatalysis. -The chemical structure of the redox probe must take into account a certain size so that it can "access" the active site of the enzyme, which is embedded in the protein structure, so that it can act as a mediator between the enzyme and the electrode.

[0009] Glucose oxidase (GOx) was initially widely used in the field of biotechnology, especially in biosensors. A few years later, it became part of the glucose / O2 biofuel cell conversation. Indeed, it was soon demonstrated that it was possible to create anode electrodes with this enzyme, using different electrode materials (carbon nanotubes, nanoparticles, graphene, nanofibers). However, to date, obtaining DET with this enzyme remains difficult due to its size and its inaccessible FAD / FADH2 active site.

[0010] Considering the difficulty of obtaining DET with GOx without modifying the protein, many studies have focused on electron transfer using redox mediators since the late 1980s. There is a very large variety of redox mediators that are commonly used in the production of biofuel cells. Among the best known are metal complexes (osmium complexes, ferrocene), quinones, azines, or viologen-type derivatives. It should be noted that other enzymes besides GOx may also require the use of redox mediators, such as, for example, lactate oxidase or sarcosine oxidase [4].

[0011] Mediation usually occurs with both species (enzyme and mediator) in aqueous solution.

[0012] In previous studies, ferrocene and derivatives [5], ferro / ferricyanides [6], and quinone derivatives [7] have been applied as redox mediators. Other known mediators include methylene blue, Prussian blue, phenazine, methyl violet, and toluidine blue [8]. However, such mediators usually have the problem of their insolubility. For example, the use of phenanthrenequinone (PQ, Figure 3) as a redox mediator for the anodic reaction is limited by the very low solubility of this molecule in water and alcohol, and therefore cannot be added in excess. Moreover, even in solution, the mediated signal is lost after a few hours due to diffusion of the mediator away from the enzyme and / or the electrode surface. Incorporating this molecule into an aqueous formulation is also problematic for printing directly on a paper substrate. On the one hand, its insolubility does not allow to obtain a good dispersion, and on the other hand, in this configuration, a large part of the amount of PQ diffuses into the paper during printing and is lost due to mediation with the enzyme. One strategy that is often described is the addition of an organic solvent such as acetonitrile (neat or mixed with water) to increase the solubility of the mediator.[9] However, this strategy is not effective for bio-based fuel cell applications.

[0013] Thus, there remains a need to provide new redox mediator systems that do not suffer from the prior art disadvantages due to the insolubility of the prior art redox mediators. Summary of the Invention

[0014] Therefore, we solved the technical problem by implementing a redox / mesoporous carbon nanocomposite (containing a redox mediator entrapped within the mesoporous carbon structure by the melt-diffusion-evaporation strategy defined herein [10,11,12]) into a bioactive ink formulation to fabricate a printed bioanode. The encapsulation of the mediator allows for the retention of the mediator in the ink. A local supersaturation of the mediator around the mesopores is maintained during the life cycle of the biobattery. This allows for (i) the nano-electrochemical properties of the mediator around the mesopores. By using the composite as a mediator reservoir, it is possible (ii) to increase the enzyme / mediator ratio by at least 150 times (compared to the use of the mediator alone in the ink formulation) and (iii) to avoid the use of organic solvents, making the solution environmentally friendly. Thus, the immediate environment of the mesoporous carbon is always saturated with mediator, and the mediator constantly migrating to the paper (i.e., the support of the electrode) is always compensated by the mediator release trapped in the mesoporous carbon. This slow and gradual release of mediator makes the bioanode more permanent (increasing its cycle life).

[0015] It is known to incorporate insoluble redox mediators into mesoporous carbon structures to provide redox mediator / mesoporous carbon nanocomposites. These nanocomposites are then used as electrode materials for the realization of bioanode sheets for glucose oxidation. The realization of this material undergoes a thermal treatment in the presence of mesoporous carbon until the mediator exceeds its melting point (T melt), which is evaluated, for example, as ((T boiling - T melting) / 4) + T melting

[11] . This process can be divided into two stages. First, the redox mediator reaches its melting point and starts to fill the space formed by the mesopores in the carbon structure, forming a thin layer on the effective surface of the mesoporous carbon through pp interactions. Second, a thin layer of the redox mediator is formed on the effective surface through pp interactions, and the excess mediator is vaporized (Figure 4). Depending on the size of the mesopores and the specific surface area of ​​the carbon, a greater or lesser amount of mediator can be incorporated into the structure. Advantageously, the redox signal of the mediator persists for a longer period of time compared to formulating it as a solution in the immobilization solution, which makes it possible to observe the catalytic current for a longer period of time than in the state of the art.

[0016] The term "redox mediator" refers to a water and potentially alcohol insoluble redox mediator selected from the group consisting of, for example, quinones (such as 9,10-phenanthrenequinone (PQ), naphthoquinone, 1-10 phenanthroline-5,6 dione), ferrocene, viologens (such as methyl viologen), azines (such as methylene blue, methylene green, azure A, tolidine blue, and thionine).

[0017] The term "mesoporous carbon" refers to materials with pores ranging from 2 to 50 nm, according to the IUPAC classification of porosity. Conventional porous carbon materials, including activated carbon and molecular sieve carbon, are generally synthesized using a pyrolysis process with suitable carbon precursors such as coal, polymers, and carbides, through activation with potassium hydroxide or selective etching of metal ions in the carbides with halogen gases

[13] . Major manufacturers include: Kuraray, BASF, Cabot Norit, Jacobi, Carbons Ingevity Corporation, Dow Corning, Toyo Tanso, Wacker Chemicals, Shin-Etsu, Momentive Performance Materials, Honeywell International, Axens, CECA (Arkema), Zeolyst, Fujian Yuanli Active Carbon, Gelest, ADA-ES, Haycarb, Clariant, CHALCO, Huber. For example, the mesoporous carbon is selected from the group consisting of carbon black (KB600JD, CAS number: 1333-86-4).

[0018] The term "melt-diffusion evaporation strategy" refers to heating mixtures of different weight ratios of redox mediator (e.g., PQ) and mesoporous carbon between the melting point (T melt ) and boiling point (T boil ) of the redox mediator (i.e., between 200 °C and 360 °C for PQ) in air (e.g., at about 240 °C for PQ) for 3 h. The molten redox mediator was absorbed into the pores by capillary forces. However, the heat treatment was not effective. After heating, only the redox mediator remained adsorbed on the surface of the carbon pores due to the pp interaction between the redox mediator and the mesoporous carbon, and the redox mediator residue without pp interaction was evaporated and removed with the air flow due to the high vapor pressure of the redox mediator. Therefore, after cooling, the solidification of the redox mediator formed the redox mediator trapped within the carbon particles with intimate contact between the carbon particles.

[0019] The term "bioelectrode" refers to an electrode composed of an oxidoreductase (e.g., with a redox mediator in the form of a redox mediator / mesoporous carbon nanocomposite to allow direct electron transfer (DET) since its active site may be located in the core of the enzyme and inaccessible), in particular an oxidase or a dehydrogenase, to oxidize or dehydrogenate a fuel (e.g., glucose), thereby generating electrons, e.g., it is a bioanode.

[0020] Therefore, an object of the present invention is a method for preparing a bioactive ink formulation, said method comprising the following steps: a) mixing the redox mediator / mesoporous carbon nanocomposite with a polymer binder in water; and b) mixing an oxidoreductase with the mixture of step a) to form a bioactive ink; Includes.

[0021] According to certain embodiments of the invention, the oxidoreductase is an oxidase, such as glucose oxidase, lactate oxidase, laccase, bilirubin oxidase, and the like.

[0022] Therefore, another object of the present invention is a method for preparing a bioelectrode, said method comprising the following steps: a) Prepare a bio-ink formulation as described above; b) coating the bioactive ink of step a) onto a substrate; and c) drying the coated substrate of step b). Includes.

[0023] According to a particular embodiment of the method of the present invention, the polymeric binder is selected from the group consisting of water-soluble polymers, such as carboxymethylcellulose, polyvinyl alcohol, or polyacrylic acid.

[0024] According to a particular embodiment of the method of the invention, the mixture of step b) comprises less than 5% redox mediator / mesoporous carbon nanocomposite, preferably less than 3% redox mediator / mesoporous carbon nanocomposite.

[0025] According to a particular embodiment of the method of the present invention, the mixture of step b) comprises about 0.1-5%, 0.3-4%, 0.5-2% oxidoreductase, preferably about 1% oxidoreductase.

[0026] According to a particular embodiment of the method of the present invention, the coating step is carried out by depositing a layer of the bioactive ink formulation having a thickness of 5-40 μm onto the substrate.

[0027] According to a particular embodiment of the method of the present invention, the substrate is selected from the group consisting of carbon-based materials such as buckypaper (BP) or cellulosic materials such as paper. In particular, the substrate is The current collector ink is pre-coated on a paper substrate and dried at about 200°C, preferably at about 150°C, and even more preferably at about 120°C.

[0028] According to a particular embodiment of the method of the present invention, the drying step is carried out at a temperature between about 25°C (room temperature) and 40°C.

[0029] Another object of the present invention is a bioactive ink formulation obtained by the method of the present invention. Specifically, the bioactive ink formulation comprises a redox mediator / mesoporous carbon nanocomposite, a polymeric binder in water, and an oxidoreductase. The bioactive ink formulation can be used in electrode materials, especially bioelectrodes.

[0030] Another object of the invention is a bioelectrode obtainable by the method of the invention, which can be used in fuel cells, for example enzymatic biofuel cells or bacterial fuel cells, which can be used in devices, for example electronic devices, in particular small portable devices for applications such as healthcare, environmental monitoring, biodefense, etc. Also, the bioelectrode can be used in biosensor applications.

[0031] Another object of the present invention is therefore a biodegradable enzymatic fuel cell comprising the bioelectrode of the present invention, and an electronic device comprising said biodegradable enzymatic fuel cell. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 is a schematic diagram of a simple arrangement for (A) direct electron transfer (DET) and (B) mediated electron transfer (MET) between the active site of an electrochemically active enzyme and a solid electrode during the oxidation of a substrate. [Diagram 2] 1 represents the potential window of the redox mediator. [Diagram 3] Represents the 9,10-phenanthrenequinone structure. [Figure 4] Represents heat treatment. [Figure 5-1] (A) Cyclic voltammograms showing the redox response of a fresh electrode for cycle 2. (squares) Anode with PQ in KB600JD (circles) Anode without PQ in KB600JD (triangles) Anode without PQ in KB600JD but with PQ saturated in the activation solution. [Figure 5-2] (B) Cyclic voltammograms showing the redox response of the aged electrodes at cycle 92. (squares) Anode with PQ in KB600JD (circles) Anode without PQ in KB600JD (triangles) Anode without PQ in KB600JD but with PQ saturated in the activation solution. [Figure 6]Figure 1 depicts cyclic voltammograms showing the redox response of electrodes containing different PQ / KB600JD ratios: (crosses) anode with 20% PQ in KB600JD, (circles) anode with 70% PQ in KB600JD, (squares) anode with 120% PQ in KB600JD. [Figure 7-1] (A) Cyclic voltamperometry from cycles 1 to 20. [Figure 7-2] (B) Cyclic voltamperometry from 20 to 50 cycles. [Figure 7-3] (C) Cyclic voltamperometry after 50 cycles. [Figure 8-1] (A) Cyclic voltammograms showing the redox response of electrodes F62D / F64D with buckypaper (BP) referring to cycle 2. (Circles) F64D (Crosses) BP (Triangles) F62D [Figure 8-2] (B) Cyclic voltammograms showing the redox response of electrodes F62D / F64D with buckypaper (BP) referring to cycle 20. (Circles) F64D (Crosses) BP (Triangles) F62D EXAMPLES

[0033] Example 1: Preparation of redox mediator / mesoporous carbon nanocomposite [9] The protocol for the selected mediator 9,10-Phenanthrenequinone (PQ) / mesoporous carbon (carbon black (KB600JD)) nanocomposites were prepared according to the melt-diffusion-vaporization method. The process temperature was set between the melting point and boiling point of the mediator. Experimentally, higher temperatures of the two reduced the efficiency and caused the loss of material in vapor form.

[0034] [Table 1]

[0035] First, carbon black (KB600JD) and 9,10-phenanthrenequinone (1 g each for a 1:1 ratio) were ground together, either manually at lab scale or using a 3D mixer for larger quantities, pelletized, either manually at lab scale or using a hydraulic press for larger quantities, and then heated in an oven at 240 °C in air for 3 hours. When 9,10-phenanthrenequinone reached its melting point, it began to fill the space formed by the mesopores in the KB600JD carbon structure. Then, through pp interactions, the effective surface (measurement of specific surface area by BET method (m 2 A thin layer of 9,10-phenanthrenequinone was formed on the catalyst (A) and excess mediator was allowed to evaporate.

[0036] Other redox mediators (eg, ferrocene) can be loaded in the same manner as long as their decomposition temperatures are higher than their melting and boiling points.

[0037] Different mesoporous carbons Four different carbons were evaluated, all loaded with the same ratio of 9,10-phenanthrenequinone (PQ).

[0038] A mixture of PQ and mesoporous carbon with a weight ratio (1:1) was heated in air at 240 °C for 3 h, between the melting point (200 °C) and boiling point (360 °C) of PQ. The molten PQ was absorbed into the pores by capillary forces. However, after heating, only PQ adsorbed on the surface of the carbon pores remained due to the pp interactions between PQ and mesoporous carbon, and the PQ residue without pp interactions was evaporated and removed with the air flow due to the high vapor pressure of PS. Thus, after cooling, the solidification of PQ formed PQ trapped within the carbon particles with intimate contact between the carbon particles.

[0039] [Table 2]

[0040] Mass loss versus mesopore size and temperature The same procedure as above was applied and weight comparison was made between initial and post-treatment.

[0041] [Table 3]

[0042] The mass loss of 9,10-phenanthrenequinone (PQ) in mesoporous carbon appears to correlate with surface area: the less surface, the more of the PQ lost. Similarly, when the temperature was too high or away from the melting point, the mass loss increased dramatically and most of the mediator was lost, escaping the structure in the form of vapor.

[0043] Example 2: Results using redox mediator / mesoporous carbon nanocomposite Compounds containing and not containing 9,10-phenanthrenequinone (PQ) in mesoporous carbon (black carbon KB600JD) To test the redox mediator / mesoporous carbon nanocomposite in a bioanode configuration, an electrode was fabricated by directly coating a slurry containing the nanocomposite onto a paper substrate.

[0044] The slurry was made by mixing the redox mediator / mesoporous carbon nanocomposite or mesoporous carbon alone with a polymer binder in water. The binder could be carboxymethyl cellulose, polyvinyl alcohol, or polyacrylic acid (a water-soluble polymer). The slurry contained 3% redox mediator / mesoporous carbon nanocomposite (or 3% mesoporous carbon alone), 0.3% polymer, 1% Gox enzyme, and 95.7% water, thus forming a bioactive ink formulation. The dispersion was coated on the substrate with a doctor blade to obtain a homogenous layer that constitutes the electrode after drying at 40°C.

[0045] Electrochemical characterization was performed using a potentiostat in a three electrode configuration: working electrode = bioanode, counter electrode = platinum wire, and Ag / AgCl reference electrode. The catalytic current was evaluated at 0.2 V in each voltammogram.

[0046] The results are shown in Figures 5A and 5B.

[0047] Figure 5A represented the activity of the bioanode with a new electrode (at cycle 2). Cyclic voltammograms showed the redox response of the electrode with or without PQ. The cyclic voltammograms shown were obtained at steady state operation (scan of 2 mV.s-1). Performance metrics were as follows: -Circle: Anode with 0% PQ in KB600JD → Catalytic peak current at cycle 2 = 0.22 mA - Squares: Anode with PQ in KB600JD → catalytic peak current in cycle 2 = 0.27 mA - Triangles: anode with 0% PQ in KB600JD but saturated PQ (3.6×10−5 M) in the activation solution → catalytic peak current in cycle 2 = 0.11 mA.

[0048] The results showed that the mesoporous containing PQ exhibited higher catalytic activity than mesoporous without PQ or direct PQ in the activation solution.

[0049] Figure 5B represented the activity of the bioanode with a degraded electrode (at cycle 92). Cyclic voltammograms showed the redox response of the electrode with or without PQ. The cyclic voltammograms shown were obtained at steady state operation (scans of 2 mV.s-1). Performance metrics were as follows: -Circular: Anode with 0% PQ in KB600JD. Mediator peak current at cycle 92 = 0.1 mA. - Squares: anode with PQ in KB600JD. Mediator peak current at cycle 92 = 0.15 mA. - Triangles: anode with 0% PQ in KB600JD but saturated PQ (3.6×10−5 M) in the activation solution. Mediator peak current at cycle 92=0.10 mA.

[0050] The results showed that the mesoporous containing PQ still exhibited higher catalytic activity after 33 h of operation time than mesoporous without PQ or directly PQ in the activation solution.

[0051] KB / PQ ratio Several carbon to mediator ratios were also performed to evaluate the effect of partial / complete filling of the carbon structure.

[0052] The same preparations as above were prepared, but with different ratios of PQ and KB600JD as follows:

[0053] [Table 4]

[0054] Electrochemical characterization was carried out as described above.

[0055] The results are presented in FIG.

[0056] Figure 6 presents cyclic voltammograms showing the redox response of electrodes containing different PQ / KB ratios. The cyclic voltammograms shown were obtained in steady-state operation (scans of 2 mV.s-1). The performance metrics were as follows: - Cross: Anode with 20% PQ in KB600JD → mediator peak current at cycle 9 = 0.12 mA -Circular: Anode with 70% PQ in KB600JD → mediator peak current at cycle 9 = 0.18 mA - Squares: Anode with 120% PQ in KB600JD → mediator peak current at cycle 9 = 0.20 mA

[0057] As expected, higher concentrations of PQ gave higher current responses from the mediator.

[0058] Example 3: Persistence of redox signals from redox mediator / mesoporous carbon nanocomposites Comparison example: F64D and F62D The same protocol as described above was followed, but in this case the evolution of both the catalytic and mediator currents was followed for both formulations F64D and F62D defined above.

[0059] Between cycles 1 and 20: Decreased The results are presented in FIG. 7A.

[0060] Between cycles 20 and 50: Increase The results are depicted in FIG. 7B.

[0061] After cycle 50: Stabilization The results are depicted in FIG. 7C.

[0062] Advantageously, the mediator signal and catalytic current were stable after several hours of operation for both mediator concentrations. Furthermore, the F62D formulation with a KB / PQ ratio of 1 showed a higher catalytic current than the F64D formulation (KB / PQ of 0.66) in the first cycle. From the 50th cycle, both formulations showed the same catalytic current.

[0063] Comparison of F62D / F64D and BP (Bucky Paper) Buckypaper (BP) electrodes are considered as a reference according to the state of the art.

[0064] Conventionally, the mediator was deposited directly on the surface of the carbon electrode by drop casting. After drying, a solution containing the enzyme (glucose oxidase from Aspergillus niger, Sigma Aldrich) was also deposited by the same method and dried. The suspension used in the reference BP contained 0.25 mM PQ, approximately 4.16 μg / cm 2This corresponds to a dry deposit of

[0065] The enzyme solution used was approximately 1.25 mg / cm 2 The concentration was 15.625 mg / ml, which corresponds to a dry sediment of 1.

[0066] The enzyme to mediator ratio was then 300.

[0067] The F62D formulation consisted of 3% KB carbon functionalized with PQ (1:1) and 1% enzyme, resulting in approximately 1 mg / cm after deposition. 2 and about 1.5 mg / cm 2 The amount of PQ obtained was 1.01 mg / kg.

[0068] The enzyme to mediator ratio was then 0.66.

[0069] For the F64D formulation, the enzyme to mediator ratio was 1.

[0070] The results are presented in Figures 8A and 8B.

[0071] In Figure 8A, the two ratios of functionalized mesoporous carbon were compared with a buckypaper electrode at equivalent enzyme loading. Figure 8A shows the catalytic response of the new electrode. The cyclic voltammograms shown were obtained at steady-state operation (scan of 2 mV.s-1). The performance metrics were: -F64D (ratio 0.66): Catalytic current cycle #2 = 0.07mA -F62D (Ratio 1): Catalytic current cycle #2 = 0.27mA -Anode Buckypaper (ratio 300): Catalytic current cycle #2 = 0.07mA

[0072] In Figure 8B, we compared the two ratios of functionalized mesoporous carbon with a buckypaper electrode at equivalent enzyme loading. Figure 8B shows the catalytic response of the aged electrode (7 hours of activity). The performance metrics were as follows: -F64D (ratio 0.66): Catalyst current cycle #20 = 0.09mA -F62D (ratio 1): Catalytic current cycle #20 = 0.17mA -Anode buckypaper (ratio 300): Catalytic current cycle #20 = 0.05mA

[0073] Unlike the printed anode, the electrode made by drop casting onto buckypaper did not have a growth or stabilization phase: the catalytic signal steadily decreased.

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Claims

1. 1. A method for preparing a bioactive ink formulation, the method comprising the steps of: a) mixing the redox mediator / mesoporous carbon nanocomposite with a polymer binder in water; and b) mixing an oxidoreductase with the mixture of step a) to form the bioactive ink; The method includes:

2. 1. A method for preparing a bioelectrode, the method comprising the steps of: a) preparing a bio-ink formulation according to the method of claim 1; b) coating the bioactive ink of step a) onto a substrate; and c) drying the coated substrate of step b). The method includes:

3. The method of claim 1 or 2, wherein the polymeric binder is selected from the group consisting of carboxymethyl cellulose, polyvinyl alcohol, or polyacrylic acid.

4. The method of any one of claims 1 to 3, wherein the redox mediator of the redox mediator / mesoporous carbon nanocomposite is selected from the group consisting of quinones, ferrocene, viologens, and azines.

5. The method of any one of claims 1 to 4, wherein the mixture of step b) comprises less than 3% of redox mediator / mesoporous carbon nanocomposite.

6. 6. The method according to any one of claims 1 to 5, wherein the mixture of step b) comprises 0.1 to 5% oxidoreductase.

7. The method according to any one of claims 2 to 6, wherein the coating step is performed by depositing a layer of the bioactive ink formulation on the substrate with a thickness of 5 to 40 μm.

8. The method according to any one of claims 2 to 7, wherein the substrate is selected from the group consisting of carbon-based materials or cellulosic materials.

9. 9. The method of claim 8, wherein the substrate is a current collector ink pre-coated on a paper substrate and dried at 120°C.

10. The method according to any one of claims 2 to 9, wherein the drying step is carried out at 25°C to 40°C.

11. A bioactive ink formulation obtainable by the method of claim 1.

12. 12. The bioactive ink formulation of claim 11 comprising a redox mediator / mesoporous carbon nanocomposite, a polymeric binder in water, and an oxidoreductase.

13. 13. Use of a bioactive ink formulation according to claim 11 or 12 as an electrode material, in particular a bioelectrode.

14. The bioelectrode comprises a bioactive ink formulation according to any of claims 11 or 12. A bioelectrode obtained by the method according to claim 2.

15. A biodegradable enzymatic fuel cell comprising the bioelectrode according to claim 14.

16. 16. An electronic device comprising the biodegradable enzymatic fuel cell of claim 15.

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