WO2025253017A1 - Method of activating a carbon-based porous or nanoporous structure - Google Patents

Abstract

The present invention provides a method of activating a porous or nanoporous structure, e.g. an electrode, made of a carbon-based porous or nanoporous material, wherein the step of activating said porous or nanoporous structure is implemented in an aqueous solution characterized by a pH level that is substantially neutral. The invention further provides a carbon-based porous or nanoporous structure, said structure being activated through the above-described method. Still further, the invention provides an electrode made of a carbon- based porous or nanoporous material, said electrode being activated through the above-described method. Also, the invention provides a medical device comprising one or more electrodes as defined above. Eventually, the invention provides a method of storing and re-activating an electrode as defined above.

Description

Method of activating a carbon-based porous or nanoporous structure

The present invention belongs to the technical field of methods of activating a structure made of a carbon-based porous or nanoporous material.

In particular, said carbon-based porous or nanoporous material can be reduced graphene oxide (rGO), preferably hydrothermally-reduced graphene oxide (HT-rGO).

In particular, said structure can be an electrode made of a carbon-based porous or nanoporous material, e.g. rGO, more preferably HT-rGO.

As a non-limiting example, electrodes of this kind may be adapted for use in medical applications.

Usually, rGO or HT-rGO electrodes are electrochemically activated.

Electrochemical activation of nanoporous graphene oxide electrodes after reduction, preferably hydrothermal reduction, is a key point to increase specific capacitance and charge injection limit, while decreasing impedance.

Boosting specific capacitance of a nanoporous electrode requires an increase of the electrochemically active surface area by promoting diffusion of electrolyte ions though the pores, especially of those where the electrolyte is not, a priori, accessible at the nanometric scale (<1nm) [1],

This effect cannot be achieved by simply immersing the electrode in an electrolyte solution, since ion diffusion is constrained by the pore size [2],

The filling of such pores further has an effect on the solution resistance within the pores [3], which decreases, thereby improving charge-injection limit of the electrode.

Electrochemical activation implies the application of a given voltage to the electrode, acting as driving force for ions to enter in the smallest pores, leading to an increase of specific capacitance and charge-injection limit, and a decrease of impedance and relaxation time constant (minimum time needed to discharge all the energy) [4],

Techniques for activation, e.g. electrochemical activation, of a carbon-based porous or nanoporous material, e.g. rGO or HT-rGO, are known in the art. Chen et al. [5] describes a technique for electrochemical activation of carbon nanotubes (CNT) based on CV in acidic media (H2SO4 1M). The electrochemical activation is performed with a scan rate of 50mV/s, from 1.5 to 2.5 V to chemically functionalize the material. Such electrochemical activation protocol is based on increasing the thickness of the material to provide additional space for Polyaniline (PANI) deposition, increasing the electrochemical surface area and, as a consequence, the specific capacitance of the material.

Chang et al. [6] describes a technique for electrochemical activation of rGO-modified glassy carbon based on CV in acidic media (HNO3, 0.2 M), conducted at 50 mV/s from 1 to 2 V. During this electrochemical activation process, oxidation of the rGO occurs, increasing oxygen functional groups that impacts on the pseudocapacitive response due to Faradaic redox reaction.

Dong et al. [7] describes a technique for electrochemical activation of a graphite rod based on CV (15000 cycles) in acidic media (H2SO4, 0.5M), conducted at 50 mV/s, from -0.56 V to 0.24

V by improving the HER overpotential. This electrochemical activation technique aims at enhancing catalytic activity of the graphite rod as a consequence of nanoparticle deposition from the counter electrode.

Lyu et al. [8] describes a technique for electrochemical activation of hydrothermally reduced graphene oxide (HT-rGO) based on charge/discharge technique at 0.05 A g-1 from 4.5 to 1.5

V vs Li/Li+, respectively. This technique is based on anion intercalation to operate at high potentials for energy storage application.

The above-described, known electrochemical activation techniques are mostly focused on electrochemical performance working at high potential. Also, some of these electrochemical activation techniques involve material functionalization to enhance the electrochemical performance.

Additionally, European Patent Application Publication No. 4098317A1 discloses, inter alia, a method for preparing an electrochemically activated rGO structure, the method comprising: providing a graphene oxide (GO) structure comprising a stack of layered graphene oxide flakes; reducing the GO structure; and electrochemically activating the rGO structure in an electrolyte system, e g. an aqueous environment. Here, electrochemical activation of the rGO structure comprises at least partially sweeping cyclically an electrical potential around a potential equilibrium within a plurality of predetermined ranges.

Although effective, said known activation techniques still have some drawbacks. In particular, the results in terms of increase in specific capacitance and charge-injection limit and decrease in impedance, obtained though said known techniques, are often sub-optimal.

Further, carbon-based porous or nanoporous structures (e.g., rGO or HT-rGO electrodes) activated through said known techniques show poor stability over time.

Still further, most of these techniques require material functionalization, which may generate issues in terms of residues, especially when medical applications are concerned.

In the light of the above, it is an object of the present invention to provide a method for activating a carbon-based porous or nanoporous structure, e.g. a rGO or HT-rGO structure, preferably an electrode, allowing obtaining an activated structure having enhanced properties in terms of increased specific capacitance and charge-injection limit and decreased impedance, further characterized by an improved stability over time.

The present invention provides a method of activating a porous or nanoporous structure made of a carbon-based porous or nanoporous material, wherein the step of activating said porous or nanoporous structure is implemented in an aqueous solution characterized by a pH level that is substantially neutral.

The present invention provides a method of activating a porous or nanoporous structure, made of a carbon-based porous or nanoporous material.

In particular, according to the invention, the step of activating said porous or nanoporous structure is implemented in an aqueous solution characterized by a pH level that is substantially neutral.

In the context of the present application, the term “substantially neutral” it is to be understood as a pH level between 6 and 8.

The invention is based on the basic idea that, by activating a carbon-based porous or nanoporous structure in an aqueous solution with a pH level that is substantially neutral, results in terms of increase in specific capacitance and charge-injection limit, and decrease in impedance after activation are significantly improved. Also, activation can be effectively accomplished without requiring material functionalization.

Furthermore, experimental evidence demonstrated that carbon-based porous or nanoporous structures, activated through the method of the invention, show an improved level of stability over time. The carbon-based porous or nanoporous structure can be electrochemically activated.

In one approach, the structure is electrochemically activated by applying a plurality of cyclic voltammetry (CV) cycles with a scan rate below 100 mV/s, while maintaining said structure immersed in the aqueous solution.

Here, electrochemical activation of the structure can be advantageously implemented by applying 100 CV cycles in a potential window between -0.9 V and 0.8 V, maintaining said structure immersed in the aqueous solution.

Electrochemical activation forces ion diffusion, filling the pores or nanopores and triggering participation of all electrochemical active areas.

This allows boosting specific capacitance and lowering the impedance of the structure.

Also, CV-based electrochemical activation allows implementing simultaneous activation of a number of porous/nanoporous structures, even when characterized by different sizes.

Alternatively, the structure is electrochemically activated by applying a sequence of biphasic current pulses, while maintaining said structure immersed in the aqueous solution.

Here, electrochemical activation of the structure can be implemented by applying a sequence of 1000 biphasic current pulses at equal time intervals with increasing values of current amplitude in a potential window between -0.9 V and 0.8 V, while maintaining said structure immersed in the aqueous solution.

The maximum current that can be applied depends on the overall dimension of the structure.

Accordingly, here, the increasing values of current amplitude can be advantageously defined based on charge injection of the porous or nanoporous structure.

An advantage of this approach is that the electrode can be activated in a shorter time compared to CV-based electrochemical activation.

For example, CV-based activation may require approximately 2 hours for its full accomplishment, while pulse-based activation only requires approximately 25 seconds per each electrode.

This may represent a significant advantage especially for certain applications, e.g. neural implants. Nevertheless, the observed the maximum capacitance after electrochemical activation is lower than in case of CV-based activation, indicating that electroactive area of the structure is not fully exploited.

The invention is not limited to electrochemical activation of the carbon-based porous or nanoporous structure.

Conversely, different activation techniques are possible.

In yet another alternative, the porous or nanoporous structure is thermally activated in the aqueous solution.

Here, thermal activation of the structure can be advantageously implemented by maintaining said structure immersed in the aqueous solution at a temperature of 60°C for 5 minutes.

This approach represents a faster and simpler way for activating the carbon-based porous or nanoporous structure, without requiring use of any electrochemical tool.

Thermal activation is particularly convenient for carbon-based porous or nanoporous structures, e.g. electrodes, that are adapted for use in medical applications.

However, similar as above, this approach does not allow exploiting the ultimate capabilities of the structure in terms of increase in specific capacitance and decrease in impedance.

In principle, an optimized solution balancing effectiveness of the achieved results and required activation time can be achieved by combining either pulse-based electrochemical activation or thermal activation with one or more, e.g. three, CV final cycles after activation.

In particular, in case of pulse-based activation or thermal activation, the method of the invention may further comprise applying one or more final CV cycles after activation of the porous or nanoporous structure, while maintaining said structure immersed in the aqueous solution.

Accordingly, effectiveness of the activation method can be optimized.

In particular, by the application of one or more final CV cycles after activation of the structure, the required time for activation completion can be reduced, without hindering the outcome in terms of increase in specific capacitance and charge-injection limit, decrease in impedance, and improved stability over time. As a non-limiting example, said one or more final CV cycles may comprise one to three CV cycles.

For instance, said porous or nanoporous carbon-based material can be reduced graphene oxide (rGO).

Preferably, said porous or nanoporous carbon-based material can be hydrothermally-reduced graphene oxide (HT-rGO).

In particular, experimental evidence demonstrated that hydrothermal reduction allows obtaining a material that is characterized by smaller pores and higher water content. These two characteristics are crucial for having a high capacitive material after activation in an aqueous solution [17],

The aqueous solution with substantially neutral pH level can be a saline solution.

In particular, the aqueous solution can a Phosphate-buffered saline (PBS) solution.

Preferably, the aqueous solution is a PBS 150 to 500 mM, preferably a 150 mM solution.

However, the use of different aqueous solutions is also possible, provided that they show a pH level close to neutral.

For instance, the aqueous solution can be a LiCI solution.

Additionally or alternatively, the aqueous solution can be a NaCI solution.

Additionally or alternatively, the aqueous solution can be a KCI solution.

Additionally or alternatively, the aqueous solution can be a CsCI solution.

Advantageously, said carbon-based porous or nanoporous structure can be an electrode made of a porous or nanoporous carbon-based material.

Preferably, said porous or nanoporous carbon-based material is rGO.

More preferably, said porous or nanoporous carbon-based material is HT-rGO.

For instance, the electrode can be adapted for use in medical applications.

The present invention further provides a porous or nanoporous structure, said structure being activated through the above-described method. The present invention further provides an electrode made of a carbon-based porous or nanoporous material, said electrode being activated through the above-described method.

Preferably, the electrode is made of rGO.

Even more preferably, the electrode is made of HT-rGO.

For instance, the electrode can be adapted for use in medical applications.

Still further, the present invention provides a medical device comprising one or more electrodes as descried above.

Eventually, the invention provides a method of storing and re-activating an electrode, such as the electrode defined above.

The method includes a step of storing the electrode.

The electrode may be stored in a storage medium.

In particular, the storage medium may comprise an aqueous solution characterized by a pH level that is substantially neutral.

Preferably, the storage medium is a PBS 150 mM solution.

Alternatively, the electrode may be stored or under dry conditions.

In both cases, the electrode is stored for a period of time up to one year.

The method further comprises a step of re-activating the electrode.

According to the method, the electrode may be thermally re-activated.

In particular, thermal re-activation is carried out in an aqueous solution characterized by a pH level that is substantially neutral.

This is advantageous since re-activation of the electrode can be accomplished without any electrochemical tool being required.

Advantageously, the electrode can be thermally re-activated by immersion in a PBS solution for 30 minutes at a temperature of 60°C.

Preferably, said PBS solution is a PBS 500 to 150 mM solution. More preferably, said PBS solution is a PBS 150 mM solution.

Alternatively, the electrode can be electrochemically re-activated.

Here, the electrode is re-activated by applying a plurality of CV cycles while maintaining the electrode immersed in an aqueous solution characterized by a pH level that is substantially neutral.

Advantageously, one to three CV cycles can be applied to re-activate the electrode.

Further details and advantages of the invention shall now be disclosed in connection with the drawings, showing:

Fig. 1 Electrochemical activation of an HT-rGO electrode by applying CV cycles in a potential window from - 0.9 V to 0.8 V, carried out in an aqueous solution with substantially neutral pH level, here a PBS 150 mM solution. In detail: a) Electrochemical activation applying 100 cycles at 50 mV/s in PBS 150 mM, b) specific capacitance extracted from CV during the electrochemical activation, c) Bode plot to characterize the impact of electrochemical activation (PEIS at 0.2 V vs Ag/AgCI), d) modulus of the impedance from Bode plot at 0.1 Hz and 1 kHz, e) real capacitance C’ vs frequency response, and f) imaginary capacitance C” vs frequency response.

Fig. 2 Electrochemical activation of a HT-rGO microelectrode array of 25 pm diameter by applying CV cycles in an aqueous solution with substantially neutral pH level, here a PBS 150 mM solution. In detail: a) Electrochemical activation by CV at 50 mV/s, b) Bode impedance before and after activation, and c) real capacitance evolution during activation.

Fig. 3 Electrochemical activation of an HT-rGo electrode by applying a sequence of biphasic current pulses with equal time interval (here, 1 ms) and increasing values of amplitude in a potential window from - 0.9 V to 0.8 V, carried out in an aqueous solution with substantially neutral pH level, here a PBS 150 mM solution. In detail: a) Pulse activation protocol by applying different charge injection, b) Bode plot during pulse activation, and c) real capacitance evolution during the pulse activation.

Fig. 4 Thermal activation of an HT-rGO electrode, carried out by maintaining the electrode immersed in an aqueous solution with substantially neutral pH level, here a PBS 150 mM solution, at a temperature of 60°C for 5 minutes. In detail: a) Bode plot before and after activation process, and b) real capacitance evolution with the thermal activation. Measurement performed in macroelectrodes with a diameter of 1.5 mm.

Fig. 5 Stability study in a PBS 150 mM after 1 day and 1 week of activation. In detail: a) Bode plot, and b) real capacitance evolution after different time periods.

Fig. 6 Electrochemical activation of an HT-rGo electrode by applying CV cycles in a potential window from - 0.9 V to 0.8 V, carried out in different saline solutions with substantially neutral pH level. In detail: a) Electrochemical activation based on CV using different saline solutions, namely LiCI solution, NaCI solution, KCI solution, and CsCI solution, respectively, and b) Bode plot to characterize the impact of electrochemical activation (PEIS at 0.2 V vs Ag/AgCI) for each of said different saline solutions.

Fig. 7 EA of HT-rGO in PBS 150 mM. In detail: a) CV activation evolution at different EA time-points (50 mV s-1), b) specific capacitance evolution as a function of CV cycle number, during EA, c) specific capacitance as a function of the scan rate after E, d) PEIS measurement, applied potential of 0.2 V vs Ag/AgCI (inset equivalent circuit), and e) real (top) and imaginary capacitance (bottom) representation as function of frequency at different EA time-points.

Fig. 8 In-situ/operando characterization at different EA time-points of activation, in PBS 150 mM. In detail: a) Mass evolution, calculated from the frequency change of EQCM (inset first cycle), b) FTIR spectra (left) and difference spectra, referenced to spectrum before EA (right). FTIR spectra fitting curves, including n_c=o Lorentzian and n_O-H strong Gaussian fitting functions, c) Raman spectra and Lorentzian fitting functions (see Experimental Section), and d) XRD patterns.

Fig. 9 a) Schematic representation of the irreversible phenomena taking place during the EA, including chemical reduction process, interlayer distance decrease and water confinement, and b) specific capacitance, obtained from the CV experiments, and electrode mass variation, obtained from the EQCM experiments, as a function of CV cycle number.

Fig. 10 Operando characterization after EA, in PBS 150 mM. In detail: a) Electrode mass variation during a CV (50 mV s'¹), b) schematic representation of the reversible phenomena after EA, including cation electro-adsorption/desorption process and interlayer distance modulation, c) (in-situ) XRD patterns measured at different applied potential during a simulated CV, and d) interlayer distance as function of applied potential, calculated from the maximum of d₀02 peak, in Figure 10c, using Bragg’s equation (datum in red indicates the initial potential of the simulated CV).

Fig. 11 Operando Raman spectroscopy, in PBS 150 mM. In detail: a) Time scan of w_G for the last 50 CV cycles during EA (50 mV s'¹), b) average w_G (from 5a) as function of applied potential, c) intensity ratio I_D7ID as function of applied potential after EA, and d) intensity ratio I_D7IG as a function of intensity ratio, I_D/IG after EA. Linear functions corresponding to vacancies (red) and sp³ (blue) defects, according to Ref. [32], In c) and d) different symbols (squares and triangles) stand for different CV scan direction. Dashed lines are guide to the eye.

Fig. 12 Chemical and morphological characterization before and after hydrothermal reduction of GO. In detail: a) SEM cross-section of HT-rGO, b) XRD study before and after HT reduction in ambient conditions, c) XPS overview before and after HT reduction, d) C1s deconvoluted spectra of GO and rGO, and e) O1s deconvoluted spectra of GO.

Fig. 13 Pseudocapacitance response study. In detail: a) CV study in different potential windows at 50 mV/s in PBS 150 mM. First CV cycle at positive polarization before EA (solid light-grey line); after 10 cycles at positive polarization before EA (dashed light-grey line); CV cycle after EA (solid grey line); negative polarization cycle after EA (dashed darker-grey line); positive polarization cycle after EA (dashed-dots light-grey line). The dots light-grey line is a schematic representation of the CV response of an ideal capacitor, b) Real capacitance as a function of frequency of HT-rGO after EA at different potential.

Fig. 14 Bode plot fit of electrode impedance, measured after EA, following Wo element described in Zview software.

Fig. 15 Water up-take study monitored by PEIS at 0,2 V vs Ag/AgCI during 20, 40 and 60 min and 3 and 24 hours without EA and after EA. In detail: a) Bode plot representation, and b) real and imaginary capacitance as function of frequency. Fig. 16 QCM-D measurements. In detail: a) Admittance as function of the frequency for the blank resonator and the resonator with rGO film transferred in PBS 150 mM, and b) characteristic BVD equivalent circuit to fit the admittance value.

Fig. 17 In-situ FTIR experiment in PBS 150 mM based on isotopic dilution (7% H2O and 93% D2O) in OCV after several cycles. In detail: a) FTIR spectra after different EA timepoints, and b) spectra subtraction from the OCV FTIR spectra.

Fig. 18 SEM cross-section of rGO. a) Before EA, and b) after EA.

Fig. 19 Contact angle measurement before and after EA. In detail: a) Represents the contact angle before EA with an angle of 90.4°±0.03°, and b) represents the contact angle after EA with an angle of 71.8°±0.4°.

Fig. 20 Operando FTIR experiment during CV cycling, recorded in PBS 150 mM at 20 mV/s in a rGO electrode after EA. In detail: a) Recording spectra of FTIR during the CV cycle (left) and spectra subtraction to the first recorded spectra (right), and b) intensity area of Lorentzian curve fit of carboxyl/carbonyl band (nc=o/cooH, 1759 cm^-1) as function of the applied potential.

Fig. 21 In-situ/operando XRD set up. In detail: a) rGO electrode fabrication using a SiO2 wafer (1) as substrate, deposit a PI layer (2), an e-beam layer deposition of Ti/Au (15/150 nm) (3) with a hole for the XRD measurements and transfer and hydrothermal reduction of rGO (4), b) custom-made electrochemical cell set-up, and c) Electrochemical cell coupled in the X'Pert XRD showing the electrochemical connectors (a) WE connected to the current collector, (b) Ag/AgOI as reference electrode and (c) Pt wire as counter electrode externally to a potentiostat.

In the following, a method for activating a porous or nanoporous structure made of a carbonbased porous or nanoporous material according to an embodiment of the invention will be described in detail.

In the present embodiment, the carbon-based porous or nanoporous structure is an electrode, in particular designed for use in medical applications.

However, the invention is not intended to be limited to such a solution.

Also, in the present embodiment, the carbon-based porous or nanoporous material is reduced graphene oxide (rGO), in particular hydrothermally-reduced graphene oxide (HT-rGO). However, the method of the invention may as well be implemented with different carbon-based porous and nanoporous materials, other than rGO or HT-rGO.

Activation of the electrode is implemented in an aqueous solution characterized by a pH level that is substantially neutral.

Here, for “substantially neutral” a pH level between 6 and 8 shall be understood as a neutral pH level.

In the present embodiment, said aqueous solution is a saline solution, in particular a Phosphate-buffered saline (PBS) solution.

For instance, said aqueous solution can be a PBS 150 to 500 mM solution.

In particular, in the present embodiment, said aqueous solution is a PBS 150 mM solution.

However, other aqueous solutions may as well be used in the method of the invention, provided that they are characterized by a pH level that is substantially neutral.

As a non-limiting example, the method of the invention may be carried out by using one among a LiCI solution, a NaCI solution, a KCI solution, or a CsCI solution.

The method of the invention may comprise electrochemical activation of a carbon-based porous or nanoporous structure, e.g. an electrode, in an aqueous solution characterized by a substantially neutral pH level.

In particular, one approach may comprise electrochemically activating the structure in an aqueous solution, e.g. a PBS 150 mM solution, by applying a plurality of cyclic voltammetry (CV) cycles with a scan rate below 100 mV/s.

Also, an alternative approach may comprise electrochemically activating the structure in said aqueous solution, e.g. a PBS 150 mM solution, by applying a sequence of biphasic current pulses.

However, the invention is not limited to electrochemical activation of the structure, e.g. an electrode.

As an alternative, the structure, e.g. an electrode, can be thermally activated in the aqueous solution, e.g. a PBS 150 mM solution, without requiring the use of any electrochemical tool. Cyclic voltammetry (CV)-based electrochemical activation

In one approach, the step of activating the porous or nanoporous structure, here a HT-rGO electrode, comprises electrochemically activating said structure by applying a plurality of cyclic voltammetry (CV) cycles, while maintaining said structure immersed in the aqueous solution, here a PBS 150 mM solution.

Here, the structure was electrochemically activated by applying 100 CV cycles in a potential window between -0.9 V and 0.8 V with a scan rate below 100 mV/s, while maintaining said structure immersed in the aqueous solution.

Figs. 1a-f shows experimental results referring to CV-based activation of an HT-rGo electrode in a PBS 150 mM solution with a scan rate of 50 mV/s.

In particular, macroelectrodes of 1.5 mm of diameter were tested and studied.

Here, 100 CV cycles were applied in a potential window between -0.9 V and 0.8 V vs Ag/AgCl.

Fig 1a shows the first, 10^th and last, i.e. 100^th, cycles.

Fig 1 b shows the specific capacitance calculated from CV as a function of the number of applied cycles, which increases from 41.2 mF cnr² to 81.8 mF cnr².

Fig. 1c shows a Bode plot at different stages of the activation process.

Fig. 1d shows the modulus of the impedance (|Z|) with the number of cycles, which decreases down to 3,5 kOhm at 0.1 Hz.

Eventually, Figs. 1e and 1f show frequency dependence of the real and imaginary capacitance, respectively.

It may be appreciated that real capacitance at lower frequencies increases after electrode activation (Fig. 1e).

This real capacitance improvement is due to the minimization of the relaxation time (T₀), calculated from the maximum of the imaginary capacitance (-C”) at frequency f₀, T₀= 1 /fo (Fig. 1f).

Here, CV-based electrochemical activation forces ion diffusion, filling nanometric pores of the HT-rGO electrode and triggering the participation of all the electrochemical active area, thereby boosting specific capacitance and lowering impedance. To study the impact on the scalability and validate it, the same approach was tested by the inventors in microelectrodes of 25 pm of diameter, fabricated on 4-inch wafers (Fig. 2).

Here, the activation process resulted in an increase of specific capacitance up to 156 mF cm- 2 (Fig. 2a).

Also, despite microelectrodes being more resistive compared to macroelectrodes (in particular due to the microfabrication process), further considering that electrode size has an impact on impedance response, a decrease of the modulus of the impedance could be observed (Fig. 2b).

This becomes even more evident with the increase of real capacitance (Fig. 2c), substantially following the same behavior observed in macroelectrodes (Fig. 1).

The tested electrodes showed no structural and/or functional damages after activation.

Pulse-based electrochemical activation

In one alternative approach, the step of activating the porous or nanoporous structure, here a HT-rGO electrode, comprises electrochemically activating the structure by applying a sequence of biphasic current pulses with equal time interval and increasing values of current amplitude, maintaining said structure immersed in the aqueous solution, here a PBS 150 mM solution.

Here, the structure was electrochemically activated by applying a sequence of 1000 biphasic current pulses at equal time intervals with increasing values of current amplitude in a potential window between -0.9 V and 0.8 V, maintaining said structure immersed in the aqueous solution.

Here, the time interval between one pulse and a subsequent pulse in the sequence of biphasic current pulses was set as 1 ms.

However, the invention is not intended to be limited to such a solution.

Figs. 3a-c shows experimental results referring to pulse-based activation of a HT-rGO microelectrode array of 25 mm of diameter in a PBS 150 mM solution, with a scan rate of 50 mV/s. The maximum current that can be applied depends on electrode dimensions. Accordingly, the increasing values of current amplitude were based on charge injection (here, 0.2, 0.4, 0.6, 0.8 and 1 mC cm-2) (Fig. 3a).

The method was first tested on macroelectrodes and then validated in microelectrodes arrays by scaling charge injection to the overall area.

This methodology was previously reported using DC voltage pulses to activate porous metal oxide-based electrodes, as described in Baumann et al. [9],

Following the pulse activation with impedance spectroscopy, it is possible to observe the decrease in impedance with the applied pulses (Fig. 3b), as well as the increase in real capacitance, in particular due to the filling of the pores (Fig. 3c).

An advantage of this approach is that the electrode can be activated in a shorter time compared to CV-based electrochemical activation.

For example, CV-based activation may require approximately 2 hours for its full accomplishment, while pulse-based activation only requires approximately 25 seconds per each electrode.

This may represent a significant advantage especially for certain applications, e.g. neural implants.

However, the obtained maximum capacitance after activation (Fig. 4c) is lower compared to maximum capacitance obtained in case of CV-based activation, indicating that electroactive area of the material is not fully exploited.

In case of pulse-based activation, one or more final CV cycles, preferably one to three CV cycles, can be applied after activation of the porous or nanoporous structure, while maintaining said structure immersed in the aqueous solution.

Accordingly, effectiveness of the activation method can be optimized.

The tested electrodes showed no structural and/or functional damages after activation.

Thermal activation In yet another approach, the step of activating the porous or nanoporous structure, here a HT- rGO electrode, comprises thermally activating said structure in the aqueous solution, here a PBS 150 mM solution.

Here, the structure was thermally activated by maintaining said structure immersed in the aqueous solution at a temperature of 60°C for 5 minutes.

Figs. 4a-c show experimental results referring to thermal activation of a HT-rGO electrode in a PBS 150 mM solution.

Activation was accomplished by maintaining the electrode in the PBS 150 mM solution for 5 minutes at a temperature of 60°C.

A decrease in impedance (Fig. 4a) and an increase in specific capacitance (Fig. 4b) were observed after thermal treatment.

In particular, this phenomenon is due to the decrease in dynamic viscosity, which improves the ionic diffusion, as explained in Vogt et al. [10],

The present approach represents not only a faster, but also a simpler way for activating a carbon-based porous or nanoporous structure, e.g. an electrode, without the use of any electrochemical tool.

However, the as in the case of pulse-based electrochemical activation, this approach does not allow exploiting the ultimate capabilities of the electrode in terms of impedance and capacitance.

In case of thermal activation, one or more final CV cycles, preferably one to three CV cycles, can be applied after activation of the porous or nanoporous structure, while maintaining said structure immersed in the aqueous solution.

Accordingly, effectiveness of the activation method can be optimized.

The tested electrodes showed no structural and/or functional damages after activation.

Stability test - Storage and re-activation

A stability test was performed after 1 day and after 1-week of the electrochemical activation of an HT-rGO electrode, stored in a PBS 150 mM solution. The results of this stability test are shown in Fig. 5.

An increase of |Z| was observed at low frequencies (0.1 Hz) of 20% after 1-day and 25% after 1-week (Fig. 5a). Such change was observed on the real capacitance at low frequencies (0,1 Hz), more evident after 1 day of activation (Fig. 5b). The invention provides a method of storing and re-activating an electrode as defined in the foregoing.

In particular, the method comprises a step of storing the electrode for a period of time up to one year, , time after which the performance improvement due to activation is completely lost.

The electrode may be stored in a storage medium comprising an aqueous solution characterized by a pH level that is substantially neutral.

Preferably, said storage medium is a PBS 150 mM solution, as mentioned in the foregoing.

Alternatively, the electrode may be stored under dry conditions.

The method further comprises a step of re-activating said electrode.

In particular, the electrode can be thermally re-activated in an aqueous solution characterized by a pH level that is substantially neutral.

This is advantageous as no electrochemical tool is required for the purpose of re-activating the electrode.

Interestingly, experimental evidence revealed that optimized values of electrode impedance and capacitance for the electrode could be restored through simple immersion of the electrode in a PBS solution for 30 minutes at a temperature of 60°C, without the need of any electrochemical tool.

Alternatively, the electrode can be electrochemically re-activated, in particular by applying a plurality of CV cycles, preferably one to three CV cycles, while maintaining the electrode immersed in an aqueous solution characterized by a pH level that is substantially neutral.

Method implementation with different aqueous solutions

The above described experiments were carried out using a PBS 150 mM solution as the aqueous solution. However, as mentioned, different aqueous solutions may as well be used in the method of the invention, provided that they are characterized by a pH value that is substantially neutral.

In particular, experiments were carried out by the inventors using different saline solutions, namely:

- a LiCI solution,

- a NaCI solution,

- a KCI solution, and

- a CsCI solution.

The experiments involved electrochemically activating HT-rGO electrodes by applying a plurality of CV cycles in each of the above-listed saline solutions.

The results of the experiments are shown in Fig. 6.

The above-listed saline solutions allow obtaining the same results as in case a PBS 150 mM solution is used.

Electrochemical activation of the tested electrodes was successfully accomplished with each of the above-listed saline solutions.

The invention further provides a carbon-based porous or nanoporous structure, said structure being activated through the above-described method.

The present invention further provides an electrode made of a carbon-based porous or nanoporous material, preferably rGO, more preferably HT-rGO, said electrode being activated through the above-described method.

In particular, the electrode can be adapted for use in medical applications.

Accordingly, the present invention further provides a medical device, comprising one or more electrodes, preferably one or more rGo electrodes, even more preferably HT-rGO electrodes, as defined above.

As a non-limiting example, said medical device can be a neural implant.

EXPERIMENT ON rGO ELECTRODES 1. Introduction

Nanoporous reduce graphene oxide (rGO) has gained significant scientific attention due to its outstanding physicochemical properties, such as large surface area, high capacitance, conductivity and chemical stability, along with its low cost and availability [11,12]. These properties make rGO films suitable candidates for use as flexible and transparent electrodes with low sheet resistance [13,14] in energy [15] and healthcare applications [16,17], among others.

During the reduction process of graphene oxide, the restacking of its layers can result in the formation of a porous film containing micro and nanopores [18] as well as interlayer nanochannels [19,20] which together govern the electrochemical performance of the film. Moreover, hydrothermal reduction, apart from being a simple, fast and environmentally friendly process [21], results in residual oxygen-containing functional groups decorating the rGO film, which modifies its hydrophobic nature [22] and strongly impacts on its electrochemical properties.

It is well known that the electrochemical double-layer (EDL) caused by the electrostatic attraction between the polarized electrode and the counter-ions that get absorbed onto its surface is the origin of the electrochemical capacitance of these electrodes [23], Moreover, nanoporous carbon-based electrodes have been found to exhibit an extraordinary increase in capacitance when the pore dimension is below 1 nm [4], Such pore dimension presents a limitation for ion and water transport [24, 25]: ions needs to de-solvate to enter such small nanopores, reducing the minimum distance between ions and the nanopore walls [26,27], which has been described as the origin of the enhanced capacitance.

A complete understanding of the structure of nanoporous carbon materials, such as rGO films, the role of confined water, the resulting nano-interfacial phenomena, as well as the ionic transport, especially in the hydrophobic nanochannels [28,29], is missing; yet, it is crucial to exploit the electrochemical properties of rGO devices for a number of applications. In this context, advanced in-situ/operando characterization techniques are needed to understand nanoconfinement phenomena which accompany the porous nature of rGO and to reveal the origin of the EDL in nanopores under working conditions [30],

Here, electrodes based on hydrothermally reduced graphene oxide (HT-rGO) films are developed and activated by applying cyclic voltammetry (CV). The voltage-controlled electrochemical activation (EA) results in a drastic enhancement of the electrode’s electrochemical performance, measured as an increase in the specific capacitance and a decrease in the impedance. By combining different advanced in-situ/operando analytical techniques, including electrochemical quartz crystal microbalance (EQCM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and Raman spectroscopy, the inventors shed light on the chemical and structural changes that boost the electrochemical performance of nanoporous rGO electrodes. By providing detailed information about ionic electro- adsorption/desorption and charge transfer processes taking place at the electrode-electrolyte interface in the nanoporous cavities of the material, our results provide new insights into the EA of rGO-based nanoporous electrodes.

2. Experimental section

Material fabrication: A GO solution of 0.15 mg-mL’¹ was prepared from a commercial GO dispersion of 1w.% (Global Graphene Group) in MilliQ. The GO film was prepared by filtering 40ml_ of this solution, using anopore inorganic anodistic membrane (47mm diameter) with 0.02 mm pore size, for 16h. Depending on the volume and filtration conditions, the GO film thickness can be controlled. To fabricate the electrodes, from this filtered film, 4 mm GO disks are mechanically prepared, peeled off and transferred to a gold substrate by a wet transfer method. GO films were hydrothermally reduced, using a commercial autoclave, for 3h at 134°C and 2 bar. After reduction, the HT-rGO film is encapsulated with PDMS leaving an exposed central region of typically 1.5mm in diameter. The exposed area can be changed depending on the characterization method used. The standard rGO substrate was based on Si/SiO2 wafers with a layer of Ti/Au (10/100nm) deposited using an electron beam vapor deposition system. Different substrates were used when required for a specific /n-s/fu/operando characterization.

Electrochemical characterization: A SP-200 Biologic potentiostat was used for the electrochemical characterization using a three-electrode configuration, with rGO, Pt wire and an Ag/AgCI flexible electrode as the working, counter and reference electrode, respectively. The working solution was PBS 150 mM (10 mM PBS, 137 mM NaCI and 3 mM KOI) tablets (Sigma Aldrich) diluted in MilliQ water. CV was performed with a scan rate (v) of 50 mV s'¹ in a potential window (AV) from -0.9 to 0.8 V vs Ag/AgCI, for 100 cycles. Specific capacitance (C_Sp) was calculated from the final CV following eq. 1 : where I (A) is the current response during CV and A is the geometric area of the electrode [31],

Potentiostatic electrochemical impedance spectroscopy (PEIS) was performed at 0.2 Vvs Ag/AgCI, with an amplitude of 10 mV and in a frequency range from 10 kHz to 10 mHz. Bode plot representation is used to evaluate the module |Z| and the phase(Z) changes during EA. A PEIS voltage scan was performed modifying the applied potential in steps of 0.2V. Bode plot was fitted using an equivalent circuit represented in Fig. 7d, using Zview software. The used model includes a solution resistance (R_s) followed by a circuit element Finite Length Warburg- Open Circuit Terminus (Wo), which is analogous to a wave transmission line for porous materials (See supplementary information).

SEM: Film thickness was measured using a scanning electron microscopy (SEM). Samples were prepared by cutting the HT-rGO electrode with a diamond tip. The measurements were performed in a FEI Quanta 650F ESEM microscope with an accelerating voltage of 10 kV. The thickness of rGO was analyzed using an Imaged software.

XPS: chemical composition of GO/HT-rGO electrodes before and after hydrothermal reduction was investigated by X-ray photoelectron spectroscopy (XPS) PHOIBOS 150 hemispherical analyzer (SPECS) in ultrahigh vacuum (5x10'¹° mbar) using a monochromatic Al Ka radiation with an energy of 1486.6 eV. The analysis and deconvolution of the C1s and O1s curves were performed by casaXPS software (See S.I.).

Contact angle: contact angle measurement was conducted using the DSA25S equipment, featuring a high-speed camera with a resolution of 1920 x 1200 px. An automatic software- controlled dispenser and tilting stage to ensure accuracy in the measurements.

/n-sftu/operando XRD: A custom-made electrochemical cell was designed to be coupled in the XRD equipment (See SI for cell design and set up). XRD measurements was performed in ambient conditions with a PANanalytical X’Pert Pro diffractometer in reflection geometry, coupled with a ceramic X-ray tube of Cu Ka radiation (A = 1.540598 A). The interlayer distance (d) between HT-rGO flakes was calculated following Bragg’s law equation (eq. 2):

A — 2dsin(0) (2) where A is 1.540598 A and 0 is the registered diffraction angle.

Operando XRD spectra were recorded with 4 minutes acquisition time, at each applied potential, starting from the most positive potential (0.8 V vs Ag/AgCI) to the most negative (- 0.9V vs Ag/AgCI) and reversibly finishing to the initial potential, in steps of 0.2 V. Peak d002 was fitted with Gaussian amplitude fitting functions.

In-situloperando FTIR: FTIR measurements were performed in the attenuated total reflectance (ATR) mode using a Bruker 70 v spectrometer and a liquid nitrogen cooled MCT detector. The spectrometer operated under vacuum conditions. A deposition of Ti/Au (4/8 nm) to the ATR crystal based on silicon wafer (I rubis) was used to ensure the conductivity and rGO adhesion. The spectra were recorded from 4500 to 750 cm^-1 using a frequency rate of 40 kHz after 1 h in vacuum to remove the humidity. The spectra were referenced to a background spectrum of a bare ATR-Ti/Au substrate. The ATR/rGO was mounted in a custom-built spectroelectrochemical cell externally connected to a potentiostat (SP-200 Biologic). In situ FTIR spectra consisting of 128 scans each were recorded at open circuit voltage (OCV) after several CV cycles during EA. Operando FTIR measurements were recorded following the CV cycles. Each spectrum consisted of 14 scans and was recorded with a waiting time of 4 s to obtain a spectrum each 100 mV while CV cycling at 20 mV-s^-1.

/n-s/tu/operando Raman spectroscopy: Raman spectra were acquired using a Witec spectrograph Alpha300R equipped with a 488 nm excitation laser. The laser power was kept below 0.1 mW. Operando measurements were performed using a custom-made electrochemical cell coupled to a Biologic SP-200 potentiostat, using a three-electrode configuration. Sample was prepared using the standard substrate (SiOz/Ti/Au/rGO) and encapsulated with PDMS with an expose central region of 1.5 mm of diameter. An 63x immersion objective was used. In-situ measurements were performed mapping a 10x10 mm area of the rGO electrode, with 1 mm spatial resolution and an integration time of 3 s. Real time spectra of 5 s acquisition time (10 Raman spectra per CV cycle), were performed to follow EA during the overall 100 CV cycles.

Witec PROJECT 5.0 software was used to fit the Raman bands using Lorentzian fitting functions. The Raman band intensity was calculated as the amplitude of the peak, with exception of Fig.10d, where the intensity was calculated as the area of the peak, in accordance with Eckman et al. [32] Raman spectrum deconvolution includes the characteristic contributions of graphene: i)the G (1582 cm^-1) and 2D (2700 cm^-1) bands [33], ii) the bands attributed to defects D (1350 cm^-1), D’ (1625 cm^-1) and D+G (2900 cm^-1) [33], and iii) the bands correlated with the oxygen content and crystallinity D*(1150-1200 cm^-1) and D” (1500-1550 erm ¹) [34],

EQCM and QCM-D: EQCM measurements were performed with a custom-made QCM, coupled to an Autolab potentiostat (PGSTAT12) [31], GO was transferred on a gold electrode over the quartz crystal resonators (9MHz-AWS, Valencia, Spain), with a surface area of 0.2 cm^-2 and hydrothermally reduced. Before EQCM measurements, the gravimetric regime was assured by measuring the film acoustically, monitoring the QCM dissipation (QCM-D), using an Agilent 429A impedance analyzer (See SI). The EQCM measurement were performed in a three-electrode configuration by recording the frequency changes with the QCM while EA at 50 mV/s is performed. To provide information about the mass change during EA, the frequency change (Df_m) of the QCM resonator was converted to mass change (Dm), following Sauerbrey equation (eq. 3) [35]:

Am = —k_s ■ f_m (3) where k_s is the theorical sensitivity factor (experimental factor is 1.09 ng Hz^-1 for 9MHz quartz resonator) and Df_m is the frequency change of the resonator with respect to the initial frequency value in OCV.

3. Results and discussion

Electrode fabrication and morphological characterization

Porous rGO thin films were prepared as reported previously [7], In brief, a known volume of an aqueous solution of commercial GO flakes is filtered through an inorganic porous membrane. Here, 1.5 mm thick GO samples were prepared. A GO membrane is transferred to a gold substrate and hydrothermally reduced (HT-rGO). More details about the HT-rGO electrode preparation can be found in the Methods section.

Cross-sectional scanning electron microscopy (SEM) of the HT-rGO is shown in Fig. 7a, revealing the characteristic stacked microstructure and the nanoporous morphology. The GO was analyzed by XRD before and after reduction. Fig. 7b shows the characteristic diffraction peak of the (002) plane at 2© angles of 11.4° and 24. T for GO and HT-rGO films, respectively, indicating a reduction of the flake interlayer distance, from 7.8 A to 3.7 A. HT-rGO shows an additional peak at 2© ~18.9° revealing stacking inhomogeneities due to the presence of remaining oxygenated functional groups [36], The chemical composition of GO and HT-rGO films was analyzed using X-ray photoelectron spectroscopy (XPS). Fig. 7d reveals that upon reduction of the GO film, the C/O ratio increases from 2.5 to 6, corresponding to a higher content of oxygen for GO (~28%) compared to HT-rGO (~14%). The evolution of chemical functional groups after reduction process was analyzed by the deconvolution of C1s peak, revealing that the main effect of the reduction process is associated with the decrease in the C-O/C-OH content, from 25% down to 19%, followed by the C=O group, decreasing from 19% down to 5% (see Fig. 7d).

Electrochemical activation of rGO electrodes

As previously reported for similar carbon-based porous materials [5,7], an electrochemical process is necessary to activate the HT-rGO electrodes so that they can reach their maximum electrochemical performance. In our case, the EA consists of a series of CV’s. Fig. 7a shows 1^st, 10^th and 100^th CV curves during EA of the HT-rGO electrodes, revealing a significant evolution of the voltammogram shape along the EA process, in particular an increase of the current. Fig. 7b depicts the specific capacitance C_sp, calculated from the integration of the voltammogram [31], as a function of the cycle number, showing that it increases with the number of cycles, starting from 37.8 mF crrr² and reaching quasi-stationary state at about 75 mF'cm’². It is interesting to note that the value of the specific capacitance of HT-rGO is several orders of magnitude higher than that of non-porous graphene electrodes. For instance, the area-normalized specific capacitance of single layer graphene is in the range of 2 pF cnr² [37], Such phenomenon is due to high electroactive surface area of HT-rGO that enhances the real specific capacitance. Fig. 7c represents C_sp as a function of the scan rate; the observed decrease of C_sp with increasing scan rate is characteristic of nanoporous electrodes, and results from the suboptimal use of the available electroactive area due to the more sluggish ion dynamics in the nanopores at high scan rates [38],

The shape of the CV curves after the EA process (defined after CV cycle number 100) suggests that the HT-rGO electrodes present a characteristic pseudocapacitive behaviour [39], with a deviation from the ideal capacitive response mainly at negative potentials. It is found that in order to reach such pseudocapacitive response, leading to the maximum electrode performance, it is necessary to activate the electrode by means of cycling at negative potentials (see Fig. 8a). It is speculated that the negative potential triggers an electrochemical activation mechanism, the origin of which will be discussed in the following. In addition, the CV’s reveal two related redox peaks at -0.1 and 0.1 V vs Ag/AgCI which, according to the literature, are attributed to the reversible protonation of remaining oxygenated functional groups [4], This redox activity thus contributes to the overall electrochemical response of the rGO electrodes.

In order to study changes in the electrical response of the material during EA, PEIS was performed. Fig. 7d shows the PEIS of the HT-rGO electrode before and after the 1^st, 10^th and 100^th CV cycles of the EA process. PEIS is displayed in a Bode plot, which represents the module and phase of the electrochemical impedance, Z, as a function of the frequency. The EA induces a significant drop of the impedance of the HT-rGO electrodes: at low frequency (0.1 Hz) the impedance module decreases from 68.7 kQ to 3.5 kO, and at high frequency (1kHz) it decreases from 665 Q to 230 Q. The impedance decrease is attributed to the increase in the electrochemical active area, which is hypothesized to result from the ingress of the electrolyte solution within the nanoporous electrode (as confirmed by the water up-take studies shown in the SI, see Fig. 10). Additionally, as shown in Fig. 7d, the EA process also impacts the phase of the impedance; at low frequency the phase changes from around -45° to -90° with the EA. It has been observed that in nanoporous electrodes the phase of the impedance is dominated by diffusion, only reaching values close to -90° when the electrolyte solution ingress in the volumetric electrode is complete and all the pores in the material contribute to the signal [40],

PEIS data can be fitted using an equivalent electrical circuit (see Fig. 9) governed by a Warburg diffusion element, in particular by an open-ended, finite-length transmission line element (see details in Methods section), which can be used to model the effect of the electrolyte diffusion in porous electrodes [40,41], From the analysis of the fitting parameters (see Table 1 , further discussed in the supplementary information section), it can be concluded that the diffusion resistance component of the Warburg element, corresponding to the resistance of ionic species to diffuse through the electrode pores [42], decreases strongly with the number of CV cycles during the EA. Further analysis (see SI) reveals the decrease of the relaxation time constant of the Warburg element during the EA; being related with the porous length and diffusion coefficient, the decrease of the relaxation time constant suggests a more optimal utilization of the material’s porosity as diffusion is increased.

A key performance parameter when considering nanoporous graphene electrodes for applications is the frequency response of the capacitance. To evaluate this, a complex representation of the PEIS is shown in Fig. 7e, with the real capacitance (C’) and the imaginary capacitance (-C”) plotted as a function of frequency, for different cycles during the EA (see SI for the definition and calculation of the real and imaginary capacitance). It is observed that C' increases with EA (from cycle 1 to 10) and shows a plateau at low frequency. This result is consistent with the hypothesis that EA promotes the electrolyte diffusion into the HT-rGO pores. With further CV cycling (cycle 100^th) a second plateau starts to be observed, which is attributed to the existence of additional pores of different (lower) size that becomes accessible to the electrolyte [43], Fig. 8b shows the C’ as function of frequency at different applied potential once the HT-rGO is activated, revealing a potential dependence of the electrode capacitance consistent with the CV experiments which is discussed here below in terms of the pseudocapacitive nature of the rGO electrodes.

Irreversible morphological and chemical evolution

To gain insight into EA mechanisms, advanced in-situ and operando characterization tools were used. Electrochemical quartz crystal microbalance (EQCM) measurements were applied to investigate mass changes in the rGO electrodes during the EA process. To this end, the GO film was transferred on the gold electrode of a quartz crystal resonator, this system allows us to monitor the dynamic response of reactions at the electrode/solution interface (see Methods section for additional information of our setup and mass change calculation, and SI for details on EQCM measurement validation). Fig. 8a shows the mass evolution during the whole EA process, revealing the following phenomena: 1) an irreversible mass increase during the 1^st cycle (pink curve), 2) a progressive mass decrease during the next tens of cycles, and 3) a stationary behaviour after 50 cycles. Very importantly, a reversible voltage-dependent mass change is observed during the whole EA process.

The observed initial irreversible mass change after the first cycle (inset in Fig. 8a) corresponds to a mass increase of about 0.2 mg cm^-2, which is attributed to the process of filling of the nanopores with the aqueous electrolyte, as previously reported [44], To monitor this process of water uptake, in situ FTIR measurements were performed during EA. Fig. 8b shows FTIR spectra of an HT-rGO electrode recorded before activation (OCV) and after 1^st, 25^th, and 50^th cycle. The in situ FTIR experiments reveal that, with the increasing number of cycles, there is an increase in the OH stretching band (n_O-H), between 3100 and 3600 cm^-1, and in the water bending mode (d_H-o-H), around 1685 cm^-1, which supports the hypothesis of the water uptake induced during the EA. The assignment of the bending and stretching modes related to water were confirmed by conducting the same experiments using an isotopic dilution of DI water and D2O (see Fig. 17, further discussed in the supplementary information section). The no-H band is usually divided into three contributions according to the O-H bonding strength, from weaker (free water) to stronger (confined water), and three peaks at about 3600 cm^-1, 3450 cm^-1 and 3250 cm^-1 are usually identified [45], It is interesting to note that the FTIR peak mainly contributing to the observed no-H increase in intensity is the one attributed to confined water.

FTIR reveals that the water uptake process occurs during the whole EA process, although in EQCM measurements this phenomenon is only observed in the 1^st cycle, as a clear mass increase. From the 2^nd cycle onwards the EQCM reveals a progressive mass loss indicating an additional phenomenon along with the water uptake process. From the FTIR data, it can be concluded that the electrochemical process induces further chemical reduction of the HT-rGO film [46], which can explain the progressive mass decrease observed in the EQCM experiment. During EA, a reduction of the carbonyl related mode (n_c=o), at 1749 cm^-1, is observed, along with an increase of the aromatic C=C stretching mode (n_c=c) [47], Moreover, there is a slight increase of the band at 1010 cm^-1 corresponding to C-0 stretches (n_O-c), which suggests the formation of organic intermediates such as lactones and hydroquinones during the reduction process [48],

In situ Raman spectroscopy can also provide complementary information on the impact of the reduction process on the properties of the rGO film [33], Fig. 8c shows in situ HT-rGO Raman spectra at different timepoints during activation: 0^th, 25^th and 50^th cycle. A narrowing of the so- called D band during the initial CV cycles of the EA is observed, as a clear indicator of material reduction.⁴² Additionally, a decrease in the intensity ratio I_D+G/ID is observed. This parameter is typically associated with a decrease in the graphene resistivity due to the rGO reduction [50], and is in good agreement with our PEIS results. Finally, the spectra evolution also indicates that the reduction process leads to an increase of the material order and crystallinity, revealed by the observed increase in the intensity ratio I_D/IG and the decrease in the intensity ratio l_D /IG [51]. The evolution of the different Raman parameters is presented in Table 3 (further discussed in the supplementary information section).

The chemical reduction of the nanoporous HT-rGO film induced by the EA is expected to influence the structural properties of the film, which is investigated by in-situ XRD. Fig. 8d depicts the XRD patterns obtained at different times during the EA process, showing the characteristic diffraction peak (d002) associated with the distance between the rGO flakes [52], It is observed that EA induces an irreversible shift of the d002 peak towards larger 20 angles, thus implying a lowering of the interlayer distance with EA from 3.66 A down to 3.5 A. This lowering of the interlayer distance is consistent with the removal of oxygen-containing functional groups, which have a marked out-of-plane structure [53], Not only the position of the d002 changes with EA, but also its full-width half maximum (FWHM), which decreases from 8.7 down to 8.1; in good agreement with previous observations, it indicates a transition towards a film with a more homogeneous stacking of the rGO flakes due to the removal of oxygen-containing groups [20], The decrease of the interlayer distance has an effect on the HT-rGO film thickness. In Fig. 18 (further discussed in the supplementary information section) SEM cross-section micrographs of the HT-rGO are presented before and after EA, revealing a thickness decrease.

In the light of the above results, a strong connection between the reduction process and the increase of the electrode capacitance is found, as summarized in Fig. 9, where the electrode mass change and capacitance are plotted together as a function of the cycle number during EA.

The formation of intermediate hydroquinones during the electrochemical reduction results in an increase of the electrode capacitance. In addition, the reduction process, especially that taking place at the edges of the GO flakes, modifies the functionality and wettability of the pores in the rGO film. This enhances the hydrophilicity of the nanochannels [54] which in turns improves the diffusion process. To assess this possibility, contact angle measurements (see Fig. 19, further discussed in the supplementary information section) were conducted and confirmed that after EA, the rGO films exhibited a more hydrophilic nature, which is consistent with an enhanced wettability after reduction, facilitating the ingress of water of confined nature in nanochannels [55],

Dynamics at the nanoporous rGO electrode/electrolyte interface

Along with the EA process, it was also observed reversible electrochemical phenomena related to the pseudocapacitive behaviour of the electrode.

One mechanism contributing to the pseudocapacitance, already observed in the CV measurements, is the protonation/deprotonation of remaining oxygenated groups in the rGO electrodes. To confirm this reversible redox reaction, operando FTIR was performed by controlling the electrochemical potential at the electrode/electrolyte interface during FTIR spectra acquisition. While cycling to negative polarization, a reversible intensity decrease in the carboxyl peak (n_c=o/cooH at 1759 cm^-1) is observed, along with an increase in the stretching mode of carboxylate peak (asymmetric n_Coo- at 1584 cm^-1 and symmetric n_Coo- ata1430 cm^-1) [48]; see Fig. 20 (further discussed in the supplementary information section) for a more detailed description.

The EQCM experiments depicted in Fig. 8a offer a hint on the origin of the pseudocapacitance, since they reveal a reversible, polarization-dependent mass modulation that occurs during and after the EA process. Fig. 10a depicts the EQCM mass evolution after the EA. This experiment shows a reversible variation of the electrode mass at negative polarization, of approximately ±0.5 mg crrr², and an almost negligible mass change at positive polarization. This observation is tentatively attributed to a reversible electro- adsorption/desorption process dominated by cationic species of the solution, as sketched in Fig. 10a, which enhances the electrode's charge storage capability [56], To confirm this hypothesis, operando XRD and Raman experiments were conducted. Fig. 10c depicts XRD spectra registered at applied potentials from 0.8 V to -0.9 V and back to 0.8 V (in 0.2 V steps). Fig. 10d shows the HT-rGO interlayer distance, calculated from the position of the d002 peak, as a function of the applied potential. While d002 does not change significantly during positive polarization, it does decrease when negative polarization is applied, returning to the initial value once the bias is swept back. This reversible potential dependence of the interlayer distance is consistent with the electrostatic adsorption of cations at negative polarization, which leads to an enhanced staking of the rGO flakes (lowering the interlayer distance) [57], Operando FTIR experiments are also compatible with this cation electro-adsorption process (see Fig. 20): the observed increase of the intensity of the no-H band at 3630 cm^-1, corresponding to free water, is attributed to the de-solvation process of the cationic species.

Using operando Raman spectroscopy, in which the energy of the G band can be used to monitor charge modulation in graphene materials [49], the polarization dynamics of the rGO electrodes was investigated. Fig. 11a shows the time scan of the G band frequency (w_G) during multiple polarization cycles after EA, revealing a reversible modulation of w_G with the applied potential. Fig. 11b depicts the average potential-dependent modulation of w_G for the HT-rGO electrode after EA, in good agreement with the observed potential dependence of the mass evolution shown in Fig. 8a. While w_G remains almost constant under positive polarization, it downshifts with negative polarization, shifting by about 6 cm^-1 at the most negative bias. It is suggested that the polarization-induced cation electro-adsorption leads to a weakening of the C-C bonds in rGO, which then results in the observed w_G downshift.

To further explore the cation electro-adsorption phenomenon, the bias dependence of the defect-related Raman bands (D and D') was analyzed. The intensity in both bands is related to the density of defects and the lattice disorder of graphene. Furthermore, the intensity ratio between them, I_D/ID, slightly varies depending on the type of defect (vacancies, sp³ and boundaries) [32], Fig. 11c depicts the intensity ratio I_D7ID of the HT-rGO electrode after EA as a function of the applied potential (bottom axis) and the mass variation calculated from EQCM experiments (top axis). It can be observed that I_D/ID remains relatively unaltered at positive potential and decreases significantly when negative potential is applied. These results indicate that when a negative potential is applied to the nanoporous HT-rGO electrode, the type of defects in the graphene lattice evolves; furthermore, this defect evolution is reversible. To elucidate the kind of defects and their polarization dependence, Fig. 11 d presents the intensity ratio ID/IG as function of the intensity ratio I_D/IG, following the presentation by Eckman et al. [32], Linear functions with different slope are plotted, corresponding to vacancies (I_D7ID ® 0.14 in red) and sp³ (I_D7ID ® 0.08 in blue) defects [32], During positive polarization, it is observed that ID/ID is larger (darker blue data closer to the red line), indicating larger amount of vacancytype of defects. Meanwhile, when negative polarization is applied I_D ID decreases (lighter blue data closer to the blue line) indicating an evolution of the defect type towards sp³. The presence of sp³ defects during negative polarization, when cationic electro-adsorption occurs, suggests an electrostatic interaction between the adsorbed cations and the electron-rich pi system of the vacancies in the graphene's basal plane.

4. Conclusions

In the described experiment, porous electrodes based on HT-rGO were activated following an electrochemical protocol enhancing their electrochemical performance. With EA an increase of the specific capacitance and of the pseudocapacitive behaviour of the electrode is observed. PEIS characterization concludes that the EA of the rGO based electrode promotes ionic diffusion, allowing the electrolyte to reach also the smaller, sub-nanometric, cavities of the nanoporous carbon material, leading to an enhancement of the electroactive area and thus an increase of the electrode's specific capacitance.

The combination of advanced in situ/operando characterization tools reveals that during the EA there is a further irreversible reduction of HT-rGO electrode. The reduction of the residual oxygenated groups of the nanopores reduces the interlayer spacing, inducing de-solvation of cations and change of hydrogen bonding of confined water entering these smaller cavities boosting the electrode specific capacitance.

Once activated, the electrodes exhibit a polarization dependent dynamic response. EQCM reveals a reversible mass modulation at negative potential, attributed to the electro- adsorption/desorption process of cationic species. This cation absorption is also reflected in a reversible modulation of the interlayer distance obtained by XRD. The absorption of positive ions favours the inter-flake stacking due to the electrostatic interaction between the cation and the carbon lattice. Given the small dimensions of the rGO pores, the cations must de-solvate to diffuse into the pores, as indicated by the operando FTIR results. The cationic adsorption event is further explored with operando Raman spectroscopy. A reversible evolution of the nature of the rGO defects, from vacancies to sp³, confirms the electrostatic interaction between cations and the graphene's basal plane.

By using in situ/operando analytical techniques this study helps to elucidate the complex interplay between surface chemistry, ion confinement and desolvation processes occurring during electrochemical cycling in rGO films, a knowledge that is key to engineer rGO-based electrodes with enhanced electrochemical performance.

SUPPLEMENTARY INFORMATION

Chemical and morphological characterization

Before and after hydrothermally reduction, GO and rGO electrodes were morphologically and chemically characterized (Fig. 12). Morphological characterization was performed using SEM cross-section (Fig. 12a) for HT-rGO in which reveals the characteristic staking microstructure and XRD (Fig. 12b) in which we observe a decrease in the interlayer distance from 7.8 to 3.7 A after hydrothermal reduction. A distinctive peak with an angle of 38.2°C, which corresponds to Au, is observed in both samples. Chemical composition was studied by XPS spectroscopy. Fig. 12c shows the XPS survey spectra of GO and rGO, and the calculated atomic percentages of carbon and oxygen in each sample. It is clear from the survey spectra that the reduction process lowers the oxygen content in the sample. To further assess the chemical differences in the two samples, core level spectra in the C1s and O1s regions were measured (Fig. 12d-e). The deconvoluted C1s spectra show peaks associated with sp2/sp3 carbon (284.8 eV), hydroxy l/epoxy C-OH/C-O (286.4 eV), carbonyl C=0 (287.3 eV) and carboxyl COOH (288.8 eV) [36]. The contributions of the oxygen functionalities are significantly reduced in rGO compared to GO. Fig. 12e shows the deconvoluted 01s spectrum with peaks corresponding to -COOH (530.5 eV), C=0 (531.6 eV), C-0 (532.5 eV) and 0-H (533.6 eV).

Pseudocapacitive response revealed by cyclic voltammetry

To assess the pseudocapacitive behaviour depicted in Fig. 7, a CV study was conducted by cycling the rGO within various potential windows before and after electrochemical activation (Fig. 13a). Prior to EA, 10 cycles are applied in the potential window from 0 to 0.8 V vs Ag/AgCI (1^st cycle solid light-grey line and 10^th cycle dashed light-grey line). It was observed that there is no significant increase of the specific capacitance while cycling in the positive potential window. After EA in the standard potential window (solid grey line), a CV study is performed in different potential window: the positive polarization (from 0 to 0.8 V vs Ag/AgCI, dashed lightgrey line) and negative polarization (from 0 to -0.9 V vs Ag/AgCI, dashed darker-grey lines). Here, it was observed that, after EA, there is an increase of the specific capacitance at positive polarization (light-grey dash-dot line) which confirms that negative polarization cycling is needed to increase the overall specific capacitance. In addition, it was observed that the final CV shape does not resemble that of a pure capacitor as represented by the dot light-grey lines; instead, the CV exhibits is a characteristic pseudocapacitive response, as previously reported [58], In Fig. 13b, C’ was calculated as function of frequencies for different applied potential once the HT-rGO is activated. This result reveals a potential dependence with the electrode capacitance consistent with the CV response (Fig. 13a).

Potentiostatic Electrochemical Impedance Spectroscopy (PEIS)

PEIS fitting of the results shown in Fig. 7e used Zview software. The model used is a electrolyte resistance (R_s) followed by an open Warburg element (Z_Wo), which is a simplification of a transmission line for porous material [59], described by the following equation (eq. S.1):

> coth[Q' • T ■ d))P] (S.1)

^{Zwo R} ' (j - T - where R is the diffusion resistance related to the resistance of the ionic species to penetrate the nano-pores; T is the time constant related to the diffusion, the length and interconnection between pores; p is the characteristic exponent of the diffusion process, with a value close to 0.5; and w is the angular frequency. The diffusion interpretation is based on time constant (Two), calculated as Two=L²/D, where L is the effective diffusion thickness, and D is the effective diffusion coefficient [42]

Fig. 14 shows the PEIS fitting of electrodes after EA with the equivalent circuit described above. The results are summarized in Table 1 .

Water up-take study

A water up-take study was performed to confirm that EA is needed to increase/promote diffusion through the nanopores [56], The electrode was submerged into the solution for 24 hour and monitored by PEIS after 20, 40 and 60 min and 1 , 3 and 24h. The PEIS results were analysed in terms of real and imaginary capacitance following the equation described for the complex capacitance (eq. S.2): where C’( >) is the real part of the complex capacitance and C”( J) is the imaginary part of the complex capacitance [43], The real and imaginary parts of the complex capacitance were calculated from the real (Z’) and imaginary impedance (-Z”) following the equations (eq. S.3 and eq. S.4): where |Z(o>)| is the modulus of the impedance, Z’(<D) is the real part of the complex impedance, Z”(aj) is the imaginary part of the complex capacitance, and is the angular frequency in rad/s.

The study suggests that, before EA, water is not able to diffuse through the pores due to the hydrophobic character of the material, as indicated by the negligible decrease in the |Z| (Fig. 15a). Only after EA, a significant decrease in |Z| and an increase in C’ and -C” (Fig. 15b) is observed.

Quartz crystal microbalance

Quartz crystal microbalance with dissipation monitoring (QCM-D) was performed to ensure the gravimetric regime of the rGO. Fig. 16 shows the QCM-D measurements performed using the quartz resonator coated with gold in air and in PBS 150 mM, with and without rGO. Fig. 16a shows the admittance response as function of the resonance frequency. This measurement assesses the gravimetric regime and the stability of the film in solution. The gravimetric regime is ensured when the difference between half-bandwidth (r) of the electroacoustic admittance is lower than the resonance frequency at the maximum admittance value (Ar « -Af_s) for the quartz resonator with the rGO compared to the quartz resonator without rGO. T was calculate from Butterworth-Van Dyke (BVD) equivalent circuit (see Fig. 16b, further discussed in the supplementary information section) that fits the admittance to extract the motional resistance (R_m) and motional inductance (L_m) to apply in equation D = R_m/4TTL_m [31]. The fitting parameters (see Table 2) show that Dr«-Df_s, confirming that it is possible to perform the measurements with the EQCM.

In-situ FTIR measurements

In-situ FTIR experiments with a working solution of PBS prepared using an isotopic dilution of 7% of MilliQ water and 93% of D₂O were conducted to confirm that the O-H stretching response observed in PBS based on 100% of MilliQ water is due to water. Such isotopic dilution of water shifts the n_O-H to n_O-D stretching mode from 3500 cm^-1 to 2300-2700 cm^-1 by the ratio of the square root of reduced mass [54], Fig. 17a shows the in-situ experiments performed with 7% of MilliQ water and 93% of D₂O in OCV after different EA timepoints. It was observed the contribution of MilliQ water with the stretching bands of n_O-H in the region of 3500 cm^-1 and the contribution of the D₂O with stretching bands regarding n_O-D mode in the region of 2500 cm^-1. Fig. 17b represents the spectra, after subtraction of the spectrum before EA. We observe an increase of the bending mode d_D-o-H at 1440 cm^-1 and the bending mode d_D-o-D at 1200 cm^-1 with the number of EA cycles which confirms the increase of the water content in the rGO film during EA.

Table 3 compiles all the parameters extracted from the peak fit shown in Fig. 8. FTIR fit analysis was perform using Lorentzian curves to calculate the area of the peaks assigned to the stretching bond of the carboxyl/carboxyl functional groups (ncooH/c=o, 1749 cm^-1) and stretching mode of confined water (n_O-H, 3240 cm^-1) (see Fig. 8b). Raman spectroscopy peaks were fitted using Lorentzian curves using Projects software and we extract the intensity ratio ID/IG, ID+G/ID and I_D”/IG and the FWHM of D band (Fig. 8c). A Gaussian fit was performed to the peak d002 from XRD results (Fig. 8d). Using Bragg’s law, we extract the interlayer distance related to d₀₀₂ peak.

SEM cross-section

The impact of EA on the rGO thickness was evaluated by SEM. For this purpose, a crosssection of the Au/rGO electrode was mechanically performed using a diamond tip. Fig. 18 a-b shows the cross-section SEM images. The SEM images show a decrease of the rGO thickness from 1.68±0.05 to 1.60±0.01 mm due to the experimentally confirmed further reduction and decrease of interlayer distance. Contact angle measurements

Changes in the hydrophobicity of rGO films were evaluated by contact angle measurements before and after EA. Fig. 19a-b shows a decrease in the contact angle from 90.4°±0.03 to 71.8°±0.4° for rGO and activated-rGO, respectively. This result confirms that rGO becomes more hydrophilic after EA treatment.

Operando FTIR measurements

Operando FTIR measurements were performed by recording FTIR spectra at 100 mV intervals during CV cycling. Fig. 20a shows the FTIR spectra referenced to the bare gold-coated Si substrate and the difference spectra referenced to +0.2V to evaluate reversible changes (right). Polarization induces protonation of the oxygen functional groups. A reversible decrease in the carboxyl peak (nc=o/cooH, 1759 cm^-1) is observed while cycling to negative polarization, which is confirmed by the increase of asymmetric ncoo- (1584 cm^-1) and symmetric ncoo- (1430 cm- ¹), as shown in Fig. 20b, where we plot the area of the nc=o/cooH peak fitted by a Lorentzian curve [48], There is an increase in the band located at 1010cm'¹ suggesting a reduction of the carbonyl groups in contrast to the nc-o vibrations of organic intermediates, such a lactones and hydroquinones [48], Regarding the stretching and bending modes of water, when negative polarization is applied, there is an increase in the non-hydrogen-bonded O-H stretching mode of water at 3631 cm^-1 due to the de-solvation process of cationic species. There is a decrease in the nanoconfined water as evidenced by the reduction of the stretching band of O-H (n_O-H 3200 cm^-1) and bending mode of water (d_O-H 1664 cm'¹). Such decrease in the amount of water present in the nanopores could be due to i) the expulsion of water when the interlayer distance decreases and ii) the expelled water due to cation de-solvation.

X-ray diffraction electrochemical cell

A custom-made electrochemical cell was developed to carried out the /n-s/fu/operando XRD measurements. Fig. 21a shows the electrode fabrication to perform these experiments. XRD spectra were obtained by measuring the bottom side electrode through a transparent substrate (polyimide, PI) without XRD signal in the range of 10 to 45 degrees. To prepare the sample, we deposited a thin layer of PI in a SiO2 wafer. As conductive substrate, 15 nm of Ti and 150 nm of Au were deposited using an e-beam evaporator with an open window of 8 mm of diameter. GO (d=10mm) was transferred to the substrate by wet transfer and hydrothermally reduced. PI/Ti/Au/rGO electrode was peeled off to perform the measurement. Fig. 21b depicts the custom-made electrochemical cell consisting of: i) a methacrylate substrate to couple the cell to the XRD diffractometer, ii) a methacrylate reservoir to fill with the working solution (PBS 150 mM) and with space to add the counter electrode (Pt wire) and reference electrode (Ag/AgCI), iii) Al current collector, iv) rGO sample in Pl/Au flexible substrate passivated with PDMAto avoid electrochemical reactions on the gold film, and v) aluminium cover with an open window sufficiently large in diameter to allow for the necessary low 0 angle XRD measurement in a reflection geometry. In this way the penetration of the X-rays is limited to the back rGO electrode minimising the strong signal from the liquid solution. The electrochemical cell was connected to a Biologic SP-200 potentiostate to perform the electrochemical measurements.

Table 1. Fitting parameters extracted from the PEIS measurements before and after different EA timepoints (after 1^st, 10^th and 100^th CV cycles) using Zview software.

0 180 382800169550 34111 0.5210.009

1 290.7120.0 254951920 7.9911.35 0.4810.007

10 28817.6 26761220 0.8510.1 0.45110.007

100 205.811.5 686121 0.2410.01 0.43310.002

Table 2. Parameters extracted from the QCM-D experiment to ensure gravimetric regime of rGO

Table 3. Parameter extracted from the fitting of in-situ FTIR, Raman spectroscopy and XRD in OCV at different EA stages. Bibliography

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Claims

Claims

1. A method of activating a porous or nanoporous structure made of a carbon-based porous or nanoporous material, wherein the step of activating said porous or nanoporous structure is implemented in an aqueous solution characterized by a pH level that is substantially neutral.

2. The method of claim 1, characterized in that the step of activating the porous or nanoporous structure comprises electrochemically activating the structure by applying a plurality of cyclic voltammetry (CV) cycles with a scan rate below 100 mV/s, while maintaining said structure immersed in the aqueous solution.

3. The method of claim 2, characterized in that the step of activating the porous or nanoporous structure comprises electrochemically activating the structure by applying 100 CV cycles in a potential window between -0.9 V and 0.8 V, while maintaining said structure immersed in the aqueous solution.

4. The method of claim 1, characterized in that the step of activating the porous or nanoporous structure comprises electrochemically activating the structure by applying a sequence of biphasic current pulses, while maintaining said structure immersed in the aqueous solution.

5. The method of claim 4, characterized in that the step of activating the porous or nanoporous structure comprises electrochemically activating the structure by applying a sequence of 1000 biphasic current pulses at equal time intervals with increasing values of current amplitude in a potential window between -0.9 V and 0.8 V, while maintaining said structure immersed in the aqueous solution.

6. The method of claim 5, characterized in that the increasing values of current amplitude are defined based on charge injection of the porous or nanoporous structure.

7. The method of claim 1, characterized in that the step of activating the porous or nanoporous structure comprises thermally activating said structure in the aqueous solution.

8. The method of claim 7, characterized in that the step of thermally activating the porous or nanoporous structure comprises maintaining said structure immersed in the aqueous solution at a temperature ranging between 50 °C and 80 °C , preferably of 60°C, for 5 minutes.

9. The method of any of claims 4 to 8, characterized in that the method further comprises: applying one or more final cyclic voltammetry (CV) cycles after activation of the porous or nanoporous structure, while maintaining said structure immersed in the aqueous solution, preferably wherein said one or more final CV cycles comprise one to three CV cycles.

10. The method of any of the preceding claims, characterized in that said porous or nanoporous carbon-based material is reduced graphene oxide (rGO), preferably hydrothermally-reduced graphene oxide (HT-rGO).

11 The method of any of the preceding claims, characterized in that said aqueous solution is a saline solution, preferably a Phosphate-buffered saline (PBS) solution, more preferably a PBS 500 to 150 mM solution, even more preferably a PBS 150 mM solution, or said aqueous solution is one or more among: a LiCI solution, a NaCI solution, a KCI solution, and a CsCI solution.

12 The method of any of the preceding claims, characterized in that said porous or nanoporous structure is an electrode made of a nanoporous carbonbased material, preferably rGO, more preferably HT-rGO.

13. A carbon-based porous or nanoporous structure, said structure being activated through the method according to any of claims 1 to 11.

14. An electrode made of a carbon-based porous or nanoporous material, said electrode being activated through the method according to any of claims 1 to 11 , preferably wherein said carbon-based porous or nanoporous material is rGO, more preferably wherein said carbon-based porous or nanoporous material is HT-rGO.

15. A medical device comprising one or more electrodes according to claim 14.

16. A method of storing and re-activating an electrode according to claim 14, the method comprising: storing the electrode in a storage medium comprising an aqueous solution characterized by a pH level that is substantially neutral, preferably a PBS 150 mM solution, or under dry conditions, for a period of time up to one year, and re-activating the electrode, wherein the step of re-activating the electrode comprises: thermally re-activating the electrode in an aqueous solution characterized by a pH level that is substantially neutral, or electrochemically re-activating the electrode by applying a plurality of cyclic voltammetry (CV) cycles, preferably one to three CV cycles, while maintaining the electrode immersed in an aqueous solution characterized by a pH level that is substantially neutral.

17. The method of claim 16, characterized in that the electrode is thermally re-activated by immersion in a PBS solution, preferably a PBS 500 to 150 mM solution, more preferably a PBS 150 mM solution, for 30 minutes at a temperature of 60°C.