Porous electrode

Abstract

A porous electrode used, for example, in the detection of harmful microorganisms. A porous electrode comprising a polymeric foam coated with a conductive ink, wherein the conductive ink comprises the mixture of: 92-95% (v / v) of a polymer mixture of two ionomers, 1-2% (v / v) of a bifunctional organosilane and 4-6% (v / v) of a organosulfur compound; wherein the conductive ink is uniformly distributed over the polymeric foam forming a conductive film; wherein said coated polymeric foam comprises a surface area from 1 cm2 to 400 cm2, a porosity of 35 to 85% and an average porous size inferior to 300 µm; wherein the electrode conductivity ranges from 10 to 20 S / m.

Description

POROUS ELECTRODETECHNICAL FIELD

[0001] The present disclosure relates to a porous electrode designed for use in the detection of harmful microorganisms. The electrode comprises a coated porous substrate, facilitating the efficient detection and quantification of microorganisms within a sample. The porous structure enhances surface area, promoting effective interaction between the electrode and microorganisms used, for example, in the detection of harmful microorganisms.BACKGROUND

[0002] The early detection of harmful microorganisms in marine and freshwater settings is becoming globally important. Per instance, Harmful Algal Blooms (HABs) are a severe environmental problem, with critical impacts on human health, aquatic ecosystems and services costing over €750 million / year to the European Union alone. HABs have detrimental consequences in a wide variety of industries, particularly in human health hazards in the drinking water industry. On the other hand, the US$260 billion aquaculture industry is hindered by debilitating attacks from pathogenic bacteria which affect food production and are expected to generate global economic losses of at least US$26 billion within 2 years.

[0003] The technology used in the field of porous electrodes for the detection of harmful microorganisms in marine and freshwater environments is centered around the development of advanced materials and sensor designs that enhance the sensitivity, selectivity, and durability of electrochemical biosensors.

[0004] In this technical field, the development and optimization of electrode materials with high surface area and tailored porosity is critical. These characteristics are critical for enhancing the sensitivity and selectivity of biosensors used in detecting pathogens, toxic algae, and other hazardous microorganisms in aquatic ecosystems.

[0005] Porous electrodes are designed to increase the interface between the electrode surface and the water sample, facilitating greater interaction with target microorganisms. The high surface area provided by the porous structure allows for a more efficient immobilization of biological recognition elements, such as antibodies, nucleic acids, or enzyme receptors, that specifically bind to the microorganisms of interest.

[0006] The development of these electrodes involves selecting the appropriate materials, such as carbon-based materials, metal oxides, or conductive polymers, that not only provide the necessary porosity and surface area but also exhibit good conductivity, chemical stability, and biocompatibility. Advanced fabrication techniques such as electrospinning, template synthesis, and additive manufacturing are often employed to achieve the desired nano- or micro-structured porous architecture.

[0007] These porous electrodes are integrated into electrochemical biosensors that operate based on various detection principles, including amperometric, voltammetric, impedimetric, and field-effect transistor (FET)-based sensors. The choice of detection principle depends on the type of microorganism, the required sensitivity and selectivity, and the specific environmental conditions of the water body being monitored.

[0008] The porous electrode materials usually used include carbon-based materials, such as graphene, carbon nanotubes, and activated carbon which are popular for their high conductivity, surface area, and chemical stability. It can also be used some metal oxides, such as zinc oxide (ZnO) and titanium dioxide (TiOz), which are known for their unique electrochemical properties and ability to form porous structures. Finally, some conductive polymers, such as polypyrrole, polyaniline, and polythiophene are conductive and can be synthesized with porous structures to facilitate biofunctionalization and electron transfer.

[0009] For the fabrication of this porous electrode, some different fabrication techniques can be used, such as electrospinning, which produces fibrous mats with high porosity and surface area, suitable for electrode materials, template synthesis, which uses a sacrificial template to create pores in materials, allowing precise controlover pore size and distribution and additive manufacturing, which enables the fabrication of electrodes with complex structures and tailored porosity.

[0010] To enhance the interaction between the electrode and target microorganisms, the surface is often modified with biological recognition elements, such as the immobilization of biomolecules, antibodies, DNA probes, or enzymes that specifically bind to target microorganisms are attached to the electrode surface, or the incorporation of nanoparticles or quantum dots that can improve sensitivity and facilitate multiplexed detection.

[0011] In practical applications, sensors must withstand environmental variations, prevent biofouling, and provide accurate readings despite the presence of complex biological matrices.

[0012] Existent monitoring solutions for water quality in water supply reservoirs, and offshore aquaculture facilities utilize measurements of chemical parameters such as nitrogen and phosphorus fractions, environmental parameters including temperature and dissolved oxygen or basic biological parameters such as phytoplankton biomass including chlorophyll as a proxy. Yet, these existing approaches fail to provide near-real time prediction capacity for HABs and bacterial pathogenesis affecting fish diseases.

[0013] Document W02004010102A2 discloses a device for monitoring the migration or invasion of a biological particle such as a cell. The device includes an upper chamber adapted to receive and retain a cell sample, a lower chamber having at least two electrodes, and a biocompatible porous membrane having a porosity sufficient to allow cells to migrate therethrough. The membrane is disposed in the device so as to separate the upper and lower chambers from one another. Migration of cells through the porous membrane permits contact between the migrating cells and one or more electrodes of the lower chamber. The contact provides a detectable change in impedance between or among the electrodes.

[0014] Document KR102567966B1 discloses a water purification device and a water purification method using a conductive membrane and energy harvesting, and more particularly to a water purification device using a conductive membrane and energy harvesting, characterized in that it comprises a conductive membrane coated with aconductive material on a porous membrane; a power supply supplying electricity to said conductive membrane; and an energy harvesting unit for filtering and sterilizing water through said conductive membrane, said power supply unit harvesting ambient mechanical energy and converting it into electrical energy.

[0015] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0016] The present disclosure relates to a porous electrode designed for use in the detection of harmful microorganisms. The electrode comprises a coated porous substrate, facilitating the efficient detection and quantification of microorganisms within a sample. The porous structure enhances surface area, promoting effective interaction between the electrode and microorganisms used, for example, in the detection of harmful microorganisms.

[0017] The present disclosure comprises a porous electrode comprising a polymeric foam coated with a conductive ink, wherein the conductive ink comprises the mixture of: 92-95% (v / v) of a polymer mixture of two ionomers, 1-2% (v / v) of a bifunctional organosilane and 4-6% (v / v) of a organosulfur compound; wherein the conductive ink is uniformly distributed over the polymeric foam forming a conductive film; wherein said coated polymeric foam comprises a surface area from 1 cm2to 400 cm2, a porosity of 35 to 85% and an average porous size inferior to 300 pm; wherein the electrode conductivity ranges from 10 to 20 S / m.

[0018] Surprisingly, it is now possible to use electrochemical impedance spectroscopy (EIS) to monitor and model algae grown, such as Lobochlamys segnis, based on the use of the mentioned porous electrode. Equivalent circuit and logistic growth modelling of the low frequency impedance and cell-density over time reveal that L. segnis cells proliferate within the porous electrodes in a similar rate as in traditional growth curves, exhibiting a growth rate of k = 0.51 cells. day1. Furthermore, the contribution of extracellular polysaccharides (EPS) formation during growth was quantified.

[0019] The porous electrode morphology and arrangement allows to electrically perform electrophysiology on large cyanobacteria cohorts, particularly those responsible for harmful algal blooms (HABs) and taste and odor (T&O) issues in freshwater reservoirs, and in aquaculture pathogens such as Pseudomonas species; Aeromonas species and Vibrio species. The obtained values for sensitivity scales with electrode area, which is proportional to the number of cells being measured simultaneously, were not obvious as cells were not known to communicate with each other in this way. Also, the electrical detection is made in the time and in the frequency domain using the noise spectra, which was found to be unconventional but very useful.

[0020] The measured impedance over time was quantified using optical analysis for determination of the contributed area of cell-bound polysaccharides. The ability to detect and model microalgae growth in real-time opens new avenues for more effective environmental diagnostics and predictive management in water reservoirs, in real time and in situ monitoring of algal productivity and biomass as an early warning system, which is not anticipated by any piece of the prior art.

[0021] Effective methods with rapid response indicators of microalgal growth are useful to predict catastrophic scenarios or even to simulate over population of harmful microorganisms or microalgal growth assessment for water resource management in large scale compared to others (Maruccio et al. 2019; Wungmool et al. 2019; Rashidi et al. 2021; Han et al. 2019). Since EIS performed with high area electrodes are an important tool for investigating collective cell adhesion, the fabrication of these three- dimensional porous electrodes is central to their application. EIS technique was performed combined with a homemade transducer setup with a porous electrode to monitoring microalgal growth. Until now, this approach has never been anticipated by any piece of prior art in microalgal assessments. The distinct growth curve phases which are composed by lag, exponential, stationery and decline were analysed by EIS values extracted at 0.1 mHz frequency over 14 days. In parallel, this analysis was supported by counting cells over time and observing biological events occurring in the cells and their surroundings.

[0022] Using standard microscopy analysis, it is possible with the porous electrode now disclose that within the first 6h, progenitor cells sediment to the porous electrode surface, attaching to this surface and after 40-96h, midbrain neurons start to emerge and after 120h, the maturation is likely to start developing.

[0023] The present disclosure comprises a porous electrode comprising a polymeric foam coated with a conductive ink, wherein the conductive ink comprises the mixture of: 92-95% (v / v) of a polymer mixture of two ionomers, 1-2% (v / v) of a bifunctional organosilane and 4-6% (v / v) of a organosulfur compound; wherein the conductive ink is uniformly distributed over the polymeric foam forming a conductive film; wherein said coated polymeric foam comprises a surface area from 1 cm2to 400 cm2, a porosity of 35 to 85% and an average porous size inferior to 300 pm and wherein the electrode conductivity ranges from 10 to 20 S / m.

[0024] The surface area of the porous electrode was determined by Brunauer- Emmett-Teller (BET) analysis with nitrogen, using a Micromeritics ASAP 2000 (n=10). The I- V curve was determined using a two-point probe setup in a Semiconductor Device Analyzer, such as a Keysight B15000A. The porosity and pore size distribution was determined by mercury porosimetry, such as a Micromeritics AutoPore IV 9500, Mercury Porosimeter. The thickness of a conductive thin film on a planar substrate was determined by a Dektak® Stylus Profiler. The thickness of PEDOT:PSS adhered to polyurethane was determined with SEM.

[0025] In an embodiment, the amount of conductive ink per foam area of the porous electrode ranges from 5.0e-6 to 1.0e-5 g / cm2; preferably from 6.0e-6 to 1.0e-5 g / cm2.

[0026] In an embodiment, the coated polymeric foam used in the porous electrode comprises a surface area from 30 cm2to 300 cm2, preferably from 50 cm2to 250 cm2, more preferably from 80 cm2to 220 cm2.

[0027] In an embodiment, the porosity of the polymeric foam of the porous electrode ranges from 37 to 82%; preferably from 40 to 79%; more preferably from 42 to 77%.

[0028] In an embodiment, the average porous size of the polymeric foam of the porous electrode ranges from 160 to 295 pm; preferably from 170 to 275 pm; more preferably from 180 to 255 pm.

[0029] In an embodiment, the polymeric foam of the porous electrode is selected from a list consisting of: polyurethane, thermoplastic polyurethane, cellulose, or their combinations.

[0030] In an embodiment, the polymer mixture of two ionomers of the porous electrode is selected from a list consisting of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate, graphene ink, printable conductive nanocomposites of poly lactic acid and multi-walled carbon nanotubes ink, or their combinations.

[0031] In an embodiment, the bifunctional organosilane of the porous electrode is selected from a list consisting of (3-glycidyloxypropyl) trimethoxysilane, polyethylene glycol)diglycidyl ether (PEGDE), or their combinations.

[0032] In an embodiment, the organosulfur compound of the porous electrode is dimethyl sulfoxide.

[0033] In an embodiment, the conductive ink of the porous electrode comprises the mixture of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate and (3- glycidyloxypropyl) trimethoxysilane, in a proportion of 0.4:100 (v / v), and dimethyl sulfoxide.

[0034] In an embodiment, the porous electrode further comprising at least one capture reagent immobilized on the surface, preferably wherein the said capture reagent is capable of binding target cells and / or particles.

[0035] In an embodiment, the conductive film of the porous electrode comprises a thickness from 0.1 to 1.4 pm, preferably from 0.15 to 1.3 pm, more preferably from 0.2 to 1.25 pm.

[0036] The present disclosure also comprises the use of a porous electrode in the detection of harmful microorganisms in water and electrically detect bacteria and diatoms, preferably wherein the harmful microorganisms in water include bacteria, diatoms and aquaculture pathogens.

[0037] The present disclosure also comprises a device for monitoring and control biological particle, comprising the porous electrode hereby described in this disclosure.

[0038] In an embodiment, the device further comprises means for detecting changes in the electrical properties of the porous electrode upon interaction with microorganisms.

[0039] The present disclosure also comprises a method for obtention of the porous electrode comprises the following steps: cleaning and adapting the dimension of the polymeric foam; a plurality of coating steps of the polymeric foam with a conductive ink; an annealing step.

[0040] In an embodiment, the double coating step of the method comprises a first dipcoating by immersing the polymeric foam into the conductive ink solution; drying the coated polymeric foam; a second dip-coating by immersing the polymeric foam into the conductive ink solution.

[0041] In an embodiment, the annealing step of the method is made with conducting cylinders placed on top of rounded shape electrodes.

[0042] In an embodiment, the method further comprises the step of application of at least one capture reagent immobilized on the porous electrode surface after the annealing step.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0044] Figure 1: Schematic representation of an embodiment of the method of fabrication of a porous electrode and his characterization.

[0045] Figure 2: Schematic representation of an embodiment of the method of fabrication of a porous electrode and his characterization.

[0046] Figure 3: Graphic representation of the electrochemical impedance spectroscopy test for an embodiment of the porous electrode in BG11 medium for 14 days (n=3).

[0047] Figure 4: Graphic representation of the impedance as a function of molarity.

[0048] Figure 5: Photographic and graphic representation of the morphology characterization of the polymeric foam.

[0049] Figure 6: Photographic and graphic representations of an embodiment of the porous electrode.

[0050] Figure 7: Graphic representations of the monitoring of Lobochlamys segnis as a function of time.

[0051] Figure 8: Photographic and graphic representations of the extracellular polymeric substances formation over time in Lobochlamys segnis.

[0052] Figure 9: Graphic representation of a sensing network with real-time telemetry data for predicting HABs and bacteria pathogenesis.

[0053] Figure 10: Graphic representation of the volumetric capacitance and electromechanical characterization of the polymeric foam.

[0054] Figure 11: Graphic and photographic representation of the extracellular electrical recordings of Oscillatoria sp. on an embodiment of a porous electrodes, in the dark.DETAILED DESCRIPTION

[0055] The present disclosure relates to a porous electrode used, for example, in the detection of harmful microorganisms.

[0056] The porous electrode of the present disclosure addresses the aforementioned need by providing an efficient and scalable platform for the detection of harmful microorganisms, namely in the monitorization and control units for water and wastewater treatment. The electrode of the present disclosure maximizes surface area, facilitating control of microorganism growth. Additionally, the electrode structure allows for easy integration into microbial detection systems, offering a versatile solution for various applications.

[0057] The porous electrode disclosed in the present disclosure is surprisingly able to detect harmful microorganisms in water and electrically detect antibiotic resistant bacteria and diatoms from a very early stage. The large electrode area allows detectionof microalgae growth dynamics with EIS. The relative change in impedance, using a low impedance, is found to be a good method for early detection of growth and EPS production, meaning a lower background noise, which means a better Signal-to Noise Ratio (SNR), when recording from these electrodes

[0058] The present disclosure comprises a porous electrode comprising a polymeric foam coated with a conductive ink, wherein the conductive ink comprises the mixture of: 92-95% (v / v) of a polymer mixture of two ionomers, 1-2% (v / v) of a bifunctional organosilane and 4-6% (v / v) of a organosulfur compound; wherein the conductive ink is uniformly distributed over the polymeric foam forming a conductive film; wherein said coated polymeric foam comprises a surface area from 1 cm2to 400 cm2, a porosity of 35 to 85% and an average porous size inferior to 300 pm; wherein the electrode conductivity ranges from 10 to 20 S / m.

[0059] The surface area of the porous electrode was determined by Brunauer- Emmett-Teller (BET) analysis with nitrogen, using a Micromeritics ASAP 2000 (n=10). The I- V curve was determined using a two-point probe setup in a Semiconductor Device Analyzer, such as a Keysight B15000A. The porosity and pore size distribution was determined using by mercury porosimetry, such as a Micromeritics AutoPore IV 9500, Mercury Porosimeter. The thickness of a conductive thin film on a planar substrate was determined by a Dektak® Stylus Profiler. The thickness of PEDOT:PSS adhered to polyurethane was determined with SEM.

[0060] In an embodiment, the amount of conductive ink per foam area of the porous electrode ranges from 5.0e-6 to 1.0e-5 g / cm2; preferably from 6.0e-6 to 1.0e-5 g / cm2, for better results.

[0061] In an embodiment, the coated polymeric foam used in the porous electrode comprises a surface area from 30 cm2to 300 cm2, preferably from 50 cm2to 250 cm2, more preferably from 80 cm2to 220 cm2, for better results.

[0062] In an embodiment, the porosity of the polymeric foam of the porous electrode ranges from 37 to 82%; preferably from 40 to 79%; more preferably from 42 to 77%, for better results.

[0063] In an embodiment, the average porous size of the polymeric foam of the porous electrode ranges from 160 to 295 pm; preferably from 170 to 275 pm; more preferably from 180 to 255 pm, for better results, allowing cells to adhere and light and medium to enter - hence maintaining cells viable for a longer time inside the internal pores.

[0064] In an embodiment, the polymeric foam of the porous electrode is selected from a list consisting of: polyurethane, thermoplastic polyurethane, cellulose, or their combinations, for better results.

[0065] In an embodiment, the polymer mixture of two ionomers of the porous electrode is selected from a list consisting of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate, graphene ink, printable conductive nanocomposites of poly lactic acid and multi-walled carbon nanotubes ink, or their combinations, for better results.

[0066] In an embodiment, the bifunctional organosilane of the porous electrode is selected from a list consisting of (3-glycidyloxypropyl) trimethoxysilane, polyethylene glycol)diglycidyl ether (PEGDE), or their combinations, for better results.

[0067] In an embodiment, the organosulfur compound of the porous electrode is dimethyl sulfoxide, for better results in what concerns the conductivity.

[0068] In an embodiment, the conductive ink of the porous electrode comprises the mixture of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate and (3- glycidyloxypropyl) trimethoxysilane, in a proportion of 0.4:100 (v / v), and dimethyl sulfoxide, for better results.

[0069] In an embodiment, the porous electrode further comprising at least one capture reagent immobilized on the surface, preferably wherein the said capture reagent is capable of binding target cells and / or particles, for better results.

[0070] In an embodiment, the conductive film of the porous electrode comprises a thickness from 0.1 to 1.4 pm, preferably from 0.15 to 1.3 pm, more preferably from 0.2 to 1.25 pm, for better results.

[0071] The present disclosure also comprises the use of a porous electrode in the detection of harmful microorganisms in water and electrically detect antibioticresistant bacteria and diatoms, preferably wherein the harmful microorganisms in water include bacteria, diatoms and aquaculture pathogens.

[0072] The present disclosure also comprises a device for monitoring and control biological particle, comprising the porous electrode hereby described in this disclosure.

[0073] In an embodiment, the device further comprises means for detecting changes in the electrical properties of the porous electrode upon interaction with microorganisms, for better results.

[0074] The present disclosure also comprises a method for obtention of the porous electrode comprises the following steps: cleaning and adapting the dimension of the polymeric foam; a double coating step of the polymeric foam with a conductive ink; an annealing step.

[0075] In an embodiment, the double coating step of the method comprises a first dipcoating by immersing the polymeric foam into the conductive ink solution; drying the coated polymeric foam; a second dip-coating by immersing the polymeric foam into the conductive ink solution, for better results.

[0076] In an embodiment, the annealing step of the method is made with conducting cylinders placed on top of rounded shape electrodes, for better results.

[0077] In an embodiment, the method further comprises the step of application of at least one capture reagent immobilized on the porous electrode surface after the annealing step, for better results.Fabrication and electrochemical characterization of porous electrodes

[0078] Commercially available polyurethane (PU) foams, such as Eurospuma, 3049PR, where cut using a Gunville Foam Cutter, into a cylinder shape having 5.5 mm diameter and 1 cm heigh. PU cylinders were first cleaned with soap and distilled water, followed by sonication with acetone and distilled water for 10 minutes, on 1:1:1 by volume, then dried with compressed air. PU cylinders were further cleaned and hydrophilized with oxygen plasma, using Diener Atto, 40kHz 200 W, for 20 minutes, as illustrated in Figure 1 A), i). After cleaning, the PU cylinders were dip-coated in a conductive ink, prepared by mixing 19 mL of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT: PSS) mixture, such as Clevios PH 1000, 94.25 vol%, with 80 pL (3-glycidyloxypropyl) trimethoxysilane (GOPS, SigmaAldrich, USA) in a proportion of 0.4:100 (v / v) for enhancing adherence and mechanical stability of the coating. The solution was mixed with 1 mL of 5% (v / v) of dimethyl sulfoxide (DMSO, SigmaAldrich, USA) for minimising the conductivity loss due to adding GOPS. The mixture was continuously stirred for 6h at room temperature.

[0079] Along this description, it is considered that room temperature is a comfortable temperature range indoors, usually considered to be in the range of 20 to 25°C.In the first dip-coating cycle, PU cylinders in the pristine state (PU only) where immersed into the PEDOT: PSS mixture which from now on we refer to as PEDOT:PSS only and left overnight on an orbital shaker at 200 rpm (Figure 2, B). The dip-coated cylinders were then hot-baked at 120°C in a muffle for lh (Figure 2, C), with the first 20 minutes inside a revolving grid at 0.4 rpm for an even distribution of the conductive ink. The cylinders went through 4 coating cycles towards achieving a uniform conductive coverage of the insulating polyurethane walls while maintaining the porosity and material integrity. In the 4th dip-coating cycle, the cylinder is placed on top of a thermally evaporated circular Au pad, with 10.7 mm2area. The placement of the foams on top of the planar Au electrodes, in an embodiment through the use of PEDOT:PSS is a non-obvious way of robustly connecting the foams to the external instrumentation. The metals are evaporated though a shadow mask using a Edwards High Vacuum 4P thermal evaporator and comprise a thin 10 nm layer of Ti and 50 nm of Au on a 2 mm thick borosilicate glass slide. Figure 2, D illustrates the PU / PEDOT:PSS sponge adhered on a circular Au electrode. The same electrodes were used to electrically record cells on planar Au electrodes only. It was then developed that the placement of the foam on the metal electrodes is a non-obvious way to electrically connect these foams.Fabrication of the PEDOT:PSS thin films

[0080] Borosilicate glass slides were sonicated in a mixed solution of pure ethanol, isopropanol and MilliQ water (1:1:1 by volume) and were placed under oxygen plasma (200W) for 10 min. Then, a thin PEDOT:PSS film was spin coated for 10s at 300 rpm and 60s at 1500 rpm followed by hot baking at 120°C for 1 hour. This was repeated 4times. The electrical conductivity was obtained according to the van der Pauw four- point-probe method and by measuring the film thickness with a Dektak® Stylus Profiler. PEDOT:PSS films exhibited an averaged conductivity of 520 S / cm, as indicated in Table 1, in close agreement with others.Table 1 - Sample weight according to the heightSurface area determination

[0081] The surface area of the PU / PEDOT: PSS electrodes was determined by Brunauer-Emmett-Teller (BET) analysis with nitrogen, using a Micromeritics ASAP 2000 (n=10). All samples were outgassed using heat and / or vacuum prior to analysis.I-V Curves

[0082] The l-V curve was determined using a two-point probe setup in a Semiconductor Device Analyzer, such as Keysight B15000A. Cylinders of 12 mm height and 20 mm diameter were fabricated with 1 to 4 dip-coating cycles. Two square standard Printed Circuit Boards (PCBs), measuring 22 x 22 mm and comprising a thin 1.6 mm thick FR-4 and a copper layer of 35 pm, were cut and positioned with the copper layer facing inward to the PU / PEDOT:PSS cylinder face. To ensure electrical continuity between the porous electrode and the copper layer of the supporting PCBs, 200 pL of liquid silver or SCP, such as ElectroLube SCP50G, was deposited onto the copper layer. After 2 minutes, the cylinder was placed over the deposited SCP, at the PCB, for lh. The same procedure was performed to the opposite face of the porous cylinder. After this process, the cylinder was pressed to achieve a 6.8 mm distance between the two parallel PCB squares, for 24h.Electrochemical Impedance Spectroscopy (EIS)

[0083] The volumetric capacitance was measured with cylindrical PU / PEDOT:PSS sponges with a diameter of 5.5 mm and a height of 1, 2.5, 5, 7.5, and 10 mm. The volumetric capacitance was extracted from recordings in 54 distinct samples, with up to 4 dip-coating cycles. The sample volume was achieved by the difference in the final weight of the sample compared with the initial one and assuming a volume density of PEDOT:PSS of 1 g / cm3.

[0084] Electrochemical Impedance Spectroscopy (EIS) measurements were carried out with a potentiostat, a PGSTAT 302N-Basic from Autolab, equipped with a MULTI4 multiplexer with 3 individual channels. EIS was measured in the frequency range of 0.1 mHz to 1 MHz, with an AC voltage of 20 mV. The equivalent circuit model was extracted using ZVIEW software.

[0085] A homemade transducer was created to evaluate the electrochemical behaviour of the PU / PEDOT:PSS porous electrodes. Two similar PU / PEDOT:PSS electrodes separated by an inter-electrode distance of 7 mm were used in EIS recordings. A drilled Polyether ether ketone (PEEK) well was screw fixed on top of the substrate with the prefabricated electrodes. This served as a container for the electrolyte solution or for the cell suspension. The PU / PEDOT: PSS electrodes were located inside the well and connected with a small strip-line to the contact pad outside the well. Hence, the impedance measurements were performed by connecting the contact pads outside the electrolyte solution. Due to large surface area of the AU / PU / PEDOT: PSS electrode, the area of the strip-line was disregarded.

[0086] The sensitivity of the system was characterized by recording EIS of aqueous KCI electrolyte solutions of growing concentration, 10 pM, 100 pM, 1 mM, lOmM, 100 mM and 1 M, as a function of frequency. Three independent wells were loaded with 3 ml of L. segnis culture with a cell density of lxlO5cell / mL. The cells were contained in the cultivation well fixed on top of the transducer and the outside pads were connected to the potentiostat, for acquisition with the same settings used for BG11. The measurements were continuous for a period of 14 days with no nutrient refreshment. Each transducer was kept in standard growth conditions. The frequency range of the measurements was the same as used with BG11, 0.1 mHz to 1 MHz.Growth curve of Lobochlamys segnis and extracellular polymeric substances (EPS) formation

[0087] A microalgal strain identified as Chlamydomonas sp. A2O 2268 was purchased from the company A2O and cultured in BG11 culture medium (Sigma). Cells were collected by centrifugation of 0.5 mL culture and disrupted using a mixer mill, such as MM200 Retsch, Haan, Germany, for 5 min. Genomic DNA was extracted using NucleoSpin Plant II from Macherey-Nagel. The PCR and sequencing service was outsourced to StabVida. Primers used for obtaining sequences of the 18S rRNA gene included the universal amplification primers used for eukaryotic algae 18S-F and 18S-R (Katana et al., 2001) and originated a sequence of 1255 bp. Primers used for amplification of rbcL were lAB_rbcLF and lAB_rbcLR (Ghosh and Love, 2011) and originated a sequence of 580 bp. Sequencing reads were assembled with SeqAssem and manually edited by visual inspection of sequencing chromatograms. The sequences were loaded into GenBank blastn search engine from NCBL The combined results from the two gene sequences indicate that the organism is highly similar to Lobochlamys segnis, a species of microalgae previously known as Chlamydomonas segnis (Proschold et al. 2001).

[0088] The culture was started by diluting an inoculum, which was pre-cultivated in batch for 3 weeks in standard conditions, with medium to a final cell density of 4xl05cell / mL. The growth curve was set in a multiwell plate and cell density was estimated by counting a different well every other day with a Brand-Blaubrand Sigma-Aldrich counting chamber (n=3) therefore enabling undisturbed estimations of density over time. Cells were cultivated for 14 days with standard conditions in a growth chamber, such as aralab FITOCLIMA 600 PDH, with temperature of 18°C, photoperiod of 12h:12h and a light intensity of 30 pmol / m2 / s provided by cool white daylight fluorescent lamps.

[0089] The cell density (cell / mL) over time (days) originated a growth curve. Growth of Lobochlamys segnis was modelled with MATLAB R2020b, using a logistic function, which describes the relationship between growth and density of a microorganism in limiting environmental conditions originally devised as a model of bacterial population growth, by Verhulst 1847; 1845, also applied to microalgal growth.

[0090] The logistic function is described by equation [1],where x is cell density at a given time (cells / mL); t is the time (days), tmid is the value of the logistic function midpoint (day); k is the logistic growth rate of the curve (day-1). The upper part of the equation x(to) + x(tmax) is the carrying capacity viz., the maximum density achieved during growth, a is the logistic model constant indicating the relative position to the origin.

[0091] Chlamydomonas and many related species such as Lobochlamys segnis have a bound layer of extracellular polymeric substances (EPS) and excessive production leads to dispersion of EPS to the surrounding medium. Here, a study was conducted for determining the EPS production of the cultivated cells over time. A culture was started by diluting an inoculum, which was precultivated in 250 mL Erlenmeyer flask batch for 3 weeks, with medium to a final cell density of 4 xlO5cell / mL. A volume of 2 mL was distributed by different tubes and the EPS production was recorded by sampling one tube and imaging the cells every other day. Microscopic slides were prepared by adding 3 pL of contrasting ink, such as drawing ink, Pelikan, to 25 pL of cells collected from the bottom of the tube. The ink colours the surrounding medium and highlights the EPS. Micrographs were obtained with a Zeiss axiocam 305 color microcamera coupled to an Axio Zoom microscope, such as Zeiss Axio Zoom V16, software ZEN 3.3 blue edition. Areas occupied by cells and by EPS were determined through Fiji 1.54f.Morphological characterization with Scanning Electron Microscopy (SEM)

[0092] Cylinders of 1 mm height and 5.5 mm diameter were analysed, from pristine to 4x dip-coating cycles. The samples were prepared for scanning electronic microscopy (SEM) analysis by the deposition of a 10 nm thick metallization layer. For that purpose, the samples were placed inside a plasma generator Quorum SC7620 Mini Sputter Coater with a Glow Discharge System with a gold / palladium (Au / Pd) sputter target. The microstructural analysis was performed using a TESCAN VEGA 3 SBH Easy Probe SEM with a tungsten-heated cathode. The images were acquired with a working voltage of 5kV and using the secondary electrons detector.

[0093] Surface area of fabricated porous electrodes was determined by examining samples of 10 mm by the volumetric method with nitrogen with Brunauer-Emmett- Teller (BET) analysis, such as Micromeritics ASAP 2000 instrument, (n=10). All samples were outgassed using heat and vacuum prior to analysis. Median pore diameter and porosity were determined by mercury porosimetry, such as Micromeritics AutoPore IV 9500, Mercury Porosimeter. The instrument enables the intrusion of mercury into the porous structure of samples with a known dimension under controlled pressure. The samples were submitted to 0.50 to 33000 psi.Characteristics of porous electrodes

[0094] Long-term stability was assessed by continuous EIS recordings of one independent pair of AU / PU / PEDOT:PSS electrodes with BG11 culture medium. Figure 3 A) shows the small-signal impedance as a function of the frequency between 0.1 mHz to 100 kHz, with only BG11 which we consider our baseline. No significant impedance variations occurred over 14 days as shown by the neglectable standard deviation of the three independent recordings. The inset provides a representation of the equivalent circuit characterizing an electrode-electrolyte interface. This circuit comprises an ideal capacitor (Cp) in parallel with a charge transfer resistance (Ret) connected in series to the solution resistance (Rsoi). Additionally, there is a parallel capacitive effect, denoted as Csoiwhich is negligible.

[0095] The impedance can be calculated by the given equation [2],

[0096] The solution resistance is represented by the spreading of current from the local electrode to a distant counter electrode, within the electrolyte media. The spreading resistance is given by Equation [3],R,=P_ [3]4r where p is the specific resistivity of the solution.

[0097] The real and imaginary parts of the impedance, Z' and Z” are given by equation[4] and [5] respectively:Z' = Rsol +

[0098] The extracted values for the charge transfer resistance Rct, capacitance Cp, and spreading resistance Rsoiare presented in Table 2. Table 2 shows that by using equation [3], a specific resistivity, p, for the BG11 medium is calculated to be ~400 Qcm, in agreement with the value obtained with a standard pH / conductivity meter, such as 3320 SET 2 WTW, Germany GmbH.Table 2. Extracted impedance parameters of 3D porous electrode by fitting the experimentally measured impedance with BG11 culture medium, equivalent circuit seen in Figure 3 A), and with cells, see the full EIS spectra partly used in figure 6, to Equations [7,8]; fitting impedance parameters of 3D porous electrode / BGll interface with average and standard deviation (n=14); fitting impedance parameters of 3D porous electrode / cells interface values after cells seeding are shown over time. The porous electrode surface area was calculated using BET analysis, 240 cm2.

[0099] Figure 3 B) illustrates the extracted phase. Under the influence of Cpat low frequencies, the phase angle reveals an almost ideal capacitor behavior, e ~ 90°. Inset shows the extracted equivalent parallel capacitance, Cp(red) intercepted by loss curve. The black arrow corresponds to the relaxion frequency, fr, which indicates the dispersion in capacitance obtained from the maximum in the corresponding dielectric loss spectrum. At this frequency fr, the system transitions from being capacitive- dominated to being resistive-dominated. The loss is given by the equivalent parallel conductance, Gpover the angular frequency (oo). The maximum loss is obtained at the Maxwell-Wagner frequency, fr, that is given by Equation [6],

[0100] Recently, EIS recordings have been used to monitor biofilm formation in E. coli and Salmonella, in medically used material, and in Pseudomonas aeruginosa. Although bacteria adhesion to functionalized microelectrode surfaces and fractional biofilm formation have been monitored with EIS, long term proliferation of bacteria with doubling times of a few ten's of minutes remains impaired due to small, pm2-mm2electrode surfaces. Hence, it was developed a porous electrode with a surface area of 240 cm2. According to Figure 2 A), the measure EIS is from 0.1 mHz to 100 kHz with algal culture medium BG11 to account for the relaxation frequency shift to lower frequencies, given the large electrode size, as compared to gold planar electrodes of per instance, 10.5 mm2.

[0101] The larger area of the porous electrodes is reflected in a larger capacitance comparing to smaller and planar electrodes. Specifically, below the relaxation frequency, the capacitance reads 0.05 Farad (F), as can be seen in Figure 3 B) inset, as opposite to planar electrodes with a much lower capacitance of 6xl0-7F.

[0102] In 3D porous electrodes, the impedance | Z | after fr is one order of magnitude lower than in gold planar electrodes.

[0103] The conductivity of the electrodes is linked to the number of dip-coating rounds in PEDOT:PSS, as illustrated in Figure 1 B). For double rounds of cycles which is the final dip-coating cycle, the l-V curves allowed to extract a conductivity of 18 S / m. Although more dip-coating rounds would increase the conductivity and maintained porosity integrity, double dip-coating rounds allowed a uniform distribution of PEDOT:PSS solution, reasonable conductance and excellent electrochemically stability while maintaining sponge porosity at around 50%. In fact, the electrochemical stability of electrodes has been ascertained with long-term impedance recordings. The impedance was measured for 14 days in culture medium only, as illustrated in Figure 1 A) and B), and for calibration purposes was also measured upon varying the concentration of KCI between 10 pM to 1 M, as represented (in logarithmic scale) in Figure 4 A) and B).

[0104] The extracted values for the charge transfer resistance Ret, capacitance Cp, and spreading resistance Rsoiare given in Table 3.Table 3 - Extracted impedance parameters of Figure 11 D) and E) by fitting the mean experimentally measured impedance, of two replicates.

[0105] At low frequency the impedance of the electrode prevails whereas at high frequency the impedance of the series solution is notorious. The impedance of the electrodes decreases at low frequencies as a function of dip-coating cycles. Similarly, the capacitance increases from 0.02 F (lx) to 0.07 F (4x). At high frequencies, the capacitance is neglected owing to the low capacitance of 100 mM KCI solution. We repeated the same analysis with 3 replicates, with 4x PU / PEDOT:PSS electrodes on cell media BG11. We extracted a mean capacitance of 2.49e-2±0.21e-2 F, an Rct of 9.34e5±2.73e5 and an Rsoiof 471.4±71.83 Q as depicted in Table 4.Table 3 - Extracted impedance parameters by fitting the mean experimentally measured impedance using 4x PU / PEDOT:PSS porous electrode, to Eqs [6,7],

[0106] By using Eq. 6, the fitted Rsoiof 470 Q, and a measured radius of a 4x dip-coated cylinder of 0.26 cm we extract a BG11 solution resistivity, p of ~490 Q-cm in agreement with the reference conductivity of BG11 provided by a pH / conductivity meter.

[0107] At high frequencies the system is dominated by the electrolyte solution, while at low frequencies the contribution of double layer capacitance dominates. The increase of KCI concentration turns the solution more conductive yielding a low impedance at high frequencies - because ions facilitate the flow of current through the solution accompanied by a gradual shift of the Maxwell-Wagner relaxation frequency. Figure 4 A) shows the influence of the KCI molarity on the relaxation frequencyextracted. The specific conductivity of the KCI solution is directly related to the solution resistance, cf. Eq. [3], The trendline with a slope of 0.95 represents the linearity of the molarity and the relaxation frequency. Figure 4 B) shows the KCI variation from 10 pM to 1 M as a function of frequency, at room temperature.Microalgal cells in 3D sensorsCell adhesion to 3D porous electrode and EIS measurements

[0108] A culture of Lobochlamys segnis cells was pre-grown for 3 weeks with standard growth conditions for microalgae. L. segnis cells have a very high EPS production in batch culture and are rich in sulfate groups. L. segnis cells were viable and mostly sessile, with some motile cells seldom seen. Upon cessation of the experiments, L. segnis cells were confirmed to be adherent to the surface of the porous electrodes, as illustrated Figure 6 A).

[0109] The electrodes surface, coated with PEDOT:PSS, was electrochemically stable over time. No degradation of the system was detected upon dismantling of the cellelectrode setup, at the end of the experiment. The biocompatibility of PEDOT:PSS porous electrodes allow L. segnis growth, as illustrated in Figure 6 A) and B), for over 14 days.

[0110] Each EIS recording cycle comprises a spectrum from 0.1 mHz to 100 kHz, with a relaxation frequency (fr) determined to be 0.01 Hz. In order to select a representative frequency that best captures microalgae growth, several frequencies were tested in the whole measured spectra. It was noted that frequencies bellow frdisplayed a higher experimental variability, particularly at 0.1 mHz. Hence, 0.1 mHz was selected for analysing cell-electrode interface events such as cell adhesion and biofilm formation, as illustrated in Figure 6 C) and D). A frequency above fr, at 100 Hz, is also shown to illustrate the smaller variations detected over time, as illustrated in Figure 6 C) and D).

[0111] At low frequency, such as 0.1 mHz, a substantial increase of impedance of over 3 kQ between day 0 and day 14 was recorded, as illustrated in Figure 6 C). This may be explained by cell proliferation and adhesion to the electrode surface since cell density increased as a function of time. This increase is also notorious, yet less evident beyond frat 100 Hz, with a 40 Q increment over 14 days, as illustrated in Figure 6 C).

[0112] The increment over time in impedance coincides with biological events in the measured cohort. On day 2 some flagellated cells, i.e. zoospores, were observed, which were more frequent on day 4 and day 6, after which they were very rarely seen. Zoospores swim freely all over the system, which may have contributed to the electrolyte-related impedance change at high frequencies on days 2 to 6. Biological events related to the extracellular polymeric substances (EPS) production and release by the cells may explain the impedance fluctuation recorded at day 10. As will be explained in the following paragraphs, the EPS production by cells achieved its maximum at day 8, and then after day 10 the EPS starts to detach the cells followed by its dispersion to the surrounding medium, likely causing electrochemical changes in the electrolyte. These alterations may be the cause of impedance fluctuation recorded at high frequencies.Growth curve of Chlamydomonas and EIS evolution

[0113] The biological events occurring on the electrodes, related to L. segnis, could be assessed by the growth determinations set in parallel to the impedance recordings. L. segnis growth followed a typical microalgal growth curve, with exponential phase from day 2 until day 6 followed by linear growth and then cell density starts to decline at day 10.

[0114] Microalgal growth kinetic models spin off from microbiological methods and did not yet reach a consensus due to the influence of simultaneous factors on growth. Attempts to calculate Monod's growth rate considering multiple limiting factors range from 0.041 day1for Tetraselmis suecica to 3.6 day1for Pseudochlorococcum sp. Many species follow a logistical tendency of cell proliferation until the point that growth stops at the plateau phase of culture maturing. It was determined the growth rate of k =0.85 cells day-1, as can be seen on Figure 7 A), by employing a logistical model fit to the data, implemented with Matlab R2023b. The growth rate is higher yet in accordance with other estimations in Chlamydomonas species in low light ~40 pmol / m2 / s, batch cultivation, with a k = 0.628 cells day-1.

[0115] We observed that the EIS recordings at 0.1 mHz followed a similar trend to the growth curve, with an impedance increase at the beginning of the growth curve coinciding to the exponential and linear phase of growth, followed by anelectrochemically stable stationary stage, with a logistic growth of kz=0.51, as illustrated in Figure 7 A).Extracellular polymeric substances (EPS) formation and their contribution to EIS

[0116] The initial part of the impedance curve, between day 0 and day 6, seen in Figure 7 A) is higher than explained by numeric increase of cells on the sensor, i.e. on a growth curve. The studies that were made indicate that high production of extracellular polymeric substances (EPS) by each cell in this initial growth phase may be contributing to higher impedance at the beginning of growth. The cells produce and excrete EPS which is bound to the cell surface at the beginning and eventually it detaches and dissolves to the surrounding medium around day 8-10. The average size of the cells per day remains stable over time, however their variety is notable, as illustrated in Figure 8 B i). The highest production of EPS formation in L. segnis cells occurred on day 8, as illustrated in figure 8 A), when impedance is very high. By intercepting impedance measurements with biological events, we could determine that the cells and EPS are both occupying sensor surface area and causing the impedance change. Figure 8 B) ii) shows the occupied area (pm2), by a single cell and with EPS over time in growth curve cultivated in multiwell. Therefore, we could estimate the occupied area by the total number of cells adhered in the 3D porous electrode in table 4 and compare with the impedance both normalized, as illustrated in Figure 8 B) iii).

[0117] Table 4. Estimative number of cells over time in 3D porous electrode.

[0118] The bound portion of the EPS in L. segnis is constituted by unique capsular polysaccharides (CPS) with a highly branched random coil structure made of galactose, glucuronic acid and glucose sugars, contrary to the more common hyaluronan-based linear polysaccharides (HP). EPS are produced by many mucilaginous species for creating a water retention layer buffering the cell from the surrounding osmotic pressure. Rheological properties of L. segnis CPS include a higher water retention capacity as well as thickening comparing with other microalgal EPS. While these characteristics are quite interesting as food enhancers in biotechnology applications, the presence of a thick, water-rich layer adjacent to the cell contributes to the impedance area occupied by the cells, therefore causing an overestimation of density at the beginning of the EIS "growth curve". This overestimation must be considered when using EIS for cell growth estimations in microalgae.Morphological characteristics of PU / PEDOT: PSS electrodes

[0119] Conducting PU / PEDOT:PSS electrodes were fabricated based on 4 iterations of dip-coating cycles. As depicted in (Figure 5, A-E), SEM images were taken from the pristine to 4x dip-coating cycles. We observed a residual pore-clogging effect of PEDOT:PSS, yet generally, after the 1st round, the PEDOT:PSS was uniformly distributed through the internal sponge structure. We noticed that after 4 dip-coating cycles, at 5x, the original oval pore shape degrades and porosity decreases to 45% (data not shown), possibly due to the degradation of solid polyurethane walls caused by DMSO used in preparing the PEDOT:PSS solution.

[0120] An important aim for fabricating porous electrodes is to achieve a large surface area, which is only possible if internal clogging doesn't occur. Indeed, the surface area of our porous electrodes is not significantly altered as a function of dipcoating cycles. On average the surface area changes around 25%, from a pristine (PU only) mean surface area of 3.75 m2 / g to 2.83 m2 / g in PU / PEDOT:PSS electrodes with 4x dip-coating cycles (see Figure 5 F). The median pore diameter also changes due to PEDOT:PSS adherence to the pore walls. On average, we observed a 35% change, from 219 pm to 143 pm in pore diameter as shown in Table 5, influenced by the increase of PEDOT:PSS which we estimate to be around 0.25 pm per dip-coating cycle (see Figure 5H), extracted from SEM images (Figure 51) of peeled PEDOT:PSS films induced bysubmerging the PU / PEDOT:PSS sponges with 90% ethanol under mechanical deformation. The porosity of PU / PEDOT:PSS electrodes as a function of dip-coating cycles was barely affected. On average we measure a decrease around 1-2% per dipcoating cycle, varying from ~56% in pristine samples, to ~52% in 4x dip-coated electrodes.Table 5 - Morphological changes of porous PU / PEDOT:PSS electrodes as a function of dip-coating cyclesElectrical and Mechanical characterization of PU / PEDOT: PSS electrodes

[0121] On porous materials, the conductivity is strongly dependent on dip-coating cycles. Hence, we performed l-V measurements as a function of dip-coating cycles to extract the conductivity. The first dip-coating cycle (lx) increases the sample conductivity to 0.06 ±0.01 S / cm (Figure 10 B). The second (2x) dip-coating cycle reaches 0.18±0.03 S / cm and the following coating cycles increases the conductivity by 0.16 S / cm per cycle. The conductivity reaches 0.55± 0.11 S / cm after 4 dip-coating cycles, or 4x.

[0122] Porous sponges are often desirable for their piezoresistive properties. Yet, for extracellular electrophysiology, mechanical deformation is generally not preferred due to producing electric interference. To evaluate the mechanical performance of our porous electrodes and ascertain that they remain stable over time, even when subject to a strong (9 N) mechanical stress, we probe the l-V characteristics (from -lOOmV to lOOmV) under different forces exerted, from 0 to 9 N. In all l-V recordings, deviations from applied force are residual which means the PU / PEDOT:PSS electrodes have minimum deformation over mechanical strength. With 4 dip-coating cycles, the l-V characteristics under 0 N are similar to when subject to 9 N and similar to all coatingsunder maximum 9 N applied force. This suggests that 4x dip-coating cycles establishes a uniform coating across the internal porous structure, given that uncoated microregions are likely electrically connected under maximum pressure / compressibility. We also performed tensile testing, with up to 500 rounds and observed a residual change.

[0123] In figure 1, it is possible to check a schematic representation of an embodiment of the method of fabrication of a porous electrode and his characterization, where it is illustrated at the A) part, (i) the hydrophilization of three- dimensional electrodes by oxygen plasma treatment; (ii) double dip coating rounds; (iii) annealing at 120°C; and the B) l-V curves obtained with single and double dip coating rounds in a PEDOT:PSS solution.

[0124] In figure 2, it is possible to check a schematic representation of an embodiment of the method of fabrication of a porous electrode and his characterization, where it is illustrated in point A) cleaning and hydrophilization of the polyurethane pristine cylinders with oxygen plasma prior to the dip-coating cycles; B) impregnation of PU sponges with PEDOT: PSS solution by stirring at 200 rpm overnight at RT; C) annealing at 120°C for lh, D) representation from 1-4 dip-coating cycles and attachment to the Au electrode pad.

[0125] In figure 3, it is possible to see a graphic representation of the electrochemical impedance spectroscopy test for an embodiment of the porous electrode in BG11 medium for 14 days (n=3). A) Impedance as a function of frequency between 0.1 mHz and 100 kHz. The inset shows the equivalent circuit of an electrode-electrolyte interface, consisting of an ideal capacitor (Cp) representing the capacitance in parallel with a charge transfer resistance (Ret) connected in series to the solution resistance (Rsoi), the parallel capacitive effect, denoted as (Csoi) is typically negligible; B) Extracted Phase (e) as a function of frequency between 0.1 mHz and 100 kHz, measured (circles) and fitted (solid line) (n=3). The inset shows the extracted equivalent parallel capacitance, Cp and Loss, Gp / oj, fr with arrow indicates the Maxwell-Wagner relation frequency.

[0126] In figure 4, it is possible to see a graphic representation of the impedance as a function of molarity. A) Maxwell-Wagner relation frequency of molarity of an aqueous KCI solution. The frequency is extracted for the 3D porous electrodes, which have areaof 240 cm2. The dotted line represents a slope of 0.95. B) Impedance as a function of molarity of the KCI-varying electrolyte solution.

[0127] In figure 5, it is possible to see photographic and graphic representation of the morphology characterization of the polymeric foam. In the embodiment, the morphology characterization of PU / PEDOT: PSS sponges, (a-e) SEM images from pristine to 4x dip-coating rounds a) pristine, b) after lx, c) after 2x, d) after 3x and e) after 4x, red dotted line shows a guide to the eye for each case; (f-i) Physical properties of PU / PEDOT: PSS sponges from pristine to 4x dip coating rounds; (f) surface area determined by BET technique, n=3, g) median pore diameter, h) porosity was determined by mercury porosimetry technique, n = 3, i) PEDOT: PSS thickness formed until 4x dip coating rounds, j) SEM image containing the thickness measured of 0.24pm after lx dip coating round.

[0128] In figure 6, it is possible to see photographic and graphic representations of an embodiment of the porous electrode. Biocompatibility and growth of Lobochlamys segnis cells on 3D porous PU / PEDOT:PSS electrodes. A) Scanning electron microscopy visualization of L. segnis cells showing adherent cells on the 3D porous electrodes at the end of the experiment, yellow dashed line shows the zoom in of the region in (B); C) Impedance | Z| (kQ) as a function of time recorded at low frequency 0.1 mHz and at high frequency 100 Hz, inset illustrates the transducer comprised by 3D porous electrode, well and glass with cells; D) Evolution of capacitance; F) recorded at low frequency 0.1 mHz and at high frequency 100 Hz, during a time period of 14 days.

[0129] In figure 7, it is possible to see graphic representations of the monitoring of Lobochlamys segnis as a function of time. A) values of |Z| of 3D porous electrodes with cells at 0.1 mHz (n=3) and growth curve (n=3) normalized to percentage (min=0, max=100), as a function of time. The dashed lines represent the logistical fit, the growth rate of cells was determined, kcells=0.85. A k parameter was retrieved from the normalized EIS curve, kZ = 0.51. B) Delta variations of impedance and C) Cells density as a function of interval of days (interval 1 = 0-2 days variation). Red and black dashed lines represent the power law fit for impedance and cells density respectively. Black arrow indicates the exponential phase for each graph. The large electrode area allows detection of microalgae growth dynamics with EIS. The relative change inimpedance is found to be a good method for early detection of growth and EPS production, as illustrated in Fig. 7. The equivalent circuit parameter Rsoiis found to translate the electrolyte resistivity and concomitant EPS release from the electrode to the solution.

[0130] In figure 8, it is possible to see photographic and graphic representations of the extracellular polymeric substances formation over time in Lobochlamys segnis. A) Formation of bound EPS around the cells over time, from day 0 to day 10, evidenced by contrast with culture medium stained with drawing ink (scale bar 20 pm); B) L. segnis cell area determinations, i) cell area distribution (pm2) for 10 days (n=1000), the area occupied by cells was determined by image analysis and single cell area was determined based on the number of visible cells in the analysed area, ii) total area (pm2) occupied by a single cell, determined by image analysis every other day. Black circles refer to total area average occupied by a single cell (n=100), purple triangles refers to area occupied only by EPS and blue diamonds refer to the sum of cells and EPS; (iii) values of the occupied area by cells and EPS (blue diamonds) (n=100) and values of |Z|, normalized to percentage (min=0, max=10 days) (n=3) as a function of days.

[0131] In figure 9, it is possible to see a graphic representation of a sensing network with real-time telemetry data for predicting HABs and bacteria pathogenesis. In point b), it is possible to see the equivalent circuit for the measured signal is(t) comprising the electrode cell-electrolyte system; point (c) is a SEM imaging of a PU / PEDOT:PSS pore with adherent cyanobacteria cells and point (d) without cells; point (e) is the electrical signalling over time of Oscillatoria sp. on PEDOT:PSS electrodes indicating the presence of cells; point (f) is electrochemically stable background noise with only cell media; point (g) symbols represent the average of five consecutive measurements of Si(w) with and without Oscillatoria sp. In the dashed line is illustrated the background noise calculated from the measured admittance.

[0132] In figure 10, it is shown the graphic representation of the volumetric capacitance and electromechanical characterization of the polymeric foam, wherein in the graphic A) shows the volumetric capacitance measured values, red dashed line shows the slope obtained by linear function fit, inset illustrates the recording setup; inthe graph B) shows the capacitance as a function of surface area, red dashed line is derived from linear function, the inset depicts the areal capacitance per volume, red dashed line represents the capacitance of 182.6 pF / cm2measured; in the graphic C) shows the equivalent circuit equivalent circuit of an electrode-electrolyte interface, consisting of an ideal capacitor (Cp) representing the capacitance in parallel with a charge transfer resistance (Ret) connected in series to the solution resistance (Rsoi), the parallel capacitive effect, denoted as (Csoi) is typically negligible; in graphic D) shows the electrochemical impedance spectroscopy of PU / PEDOT:PSS polymeric foams with 0.25 cm height, 5.55 mm diameter, from lx to 4x coating rounds of PEDOT: PSS solution in 100 mM KCI medium. The modulus of impedance as a function of frequency between 0.1 mHz and 100 kHz with four coating rounds, inset shows the phase as a function of frequency; in graphic E) shows the capacitance as a function of frequency between 0.1 mHz and 100 kHz with different dip coating rounds, inset depicts the extracted loss the peak in the dielectric loss corresponds to the Maxwell-Wagner relation frequency. The solid lines are fits derived by fitting the impedance using. As can be seen in figure 10, the volumetric capacitance and a highest / largest capacitance recorded in a sensing device - impacts on transconductance, meaning sensitivity for future electrochemical sensing devices or even capacitors. Unlike, the knowledge known in the prior art, it is not apparent the existence of a plateau in capacitance. The record low impedance data, as shown on Fig. 10, and record high capacitance are an important feature of the porous electrodes. Low impedance means a lower background noise which means a better Signal-to Noise Ratio (SNR) when recording from these electrodes.

[0133] In figure 11, it is shown the extracellular electrical recordings of Oscillatoria sp. on an embodiment of a porous electrodes, in the dark. The electrical activity is recorded in extended dark conditions, over 3 weeks, but also in normal light / dark cycles, e.g. 12h light and 12 hours dark. Graphic (A) illustrates the current response over time (i) of cells before treatment with gadolinium chloride (GaCH); (ii) Absence of electrical spikes and significative noise reduction, with addition of the ion blocker GaCH up to 800 pM, magnified view of noise (inset); (iii) current restored, after washing off the ion blocker from the cells. Graphic (B) shows the current noise spectraof Oscillatoria sp. The spectral power density Si(w) is presented as a function of frequency. The traces are reproduced from the recordings before, during and after treatment of gadolinium chloride (GaCh). The noise spectra power follows a 1 / fy behavior; graphic (C) shows a spike rate in response to GaCh concentration; Graphic (D) shows Oscillatoria sp. filaments immediately after addition of GaCh 800 pM. Live / dead stained filaments showing dead cells in red and alive in green.

[0134] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0135] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[0136] The following dependent claims further set out particular embodiments of the disclosure.

Claims

C L A I M S1. A porous electrode comprising a polymeric foam coated with a conductive ink, wherein the conductive ink comprises the mixture of: 92-95% (v / v) of a polymer mixture of two ionomers, 1-2% (v / v) of a bifunctional organosilane and 4-6% (v / v) of a organosulfur compound; wherein the conductive ink is uniformly distributed over the polymeric foam forming a conductive film; wherein said coated polymeric foam comprises a surface area from 1 cm2to 400 cm2, a porosity of 35 to 85% and an average porous size inferior to 300 pm; wherein the electrode conductivity ranges from 10 to 20 S / m.

2. The porous electrode according to the previous claim, wherein the amount of conductive ink per foam area ranges from 5.0e-6 to 1.0e-5 g / cm2; preferably from 6.0e-6 to 1.0e-5 g / cm2.

3. The porous electrode according to any of the previous claims, wherein the coated polymeric foam comprises a surface area from 30 cm2to 300 cm2, preferably from 50 cm2to 250 cm2, more preferably from 80 cm2to 220 cm2.

4. The porous electrode according to any of the previous claims, wherein the porosity of the polymeric foam ranges from 37 to 82%; preferably from 40 to 79%; more preferably from 42 to 77%.

5. The porous electrode according to any of the previous claims, wherein the average porous size of the polymeric foam ranges from 160 to 295 pm; preferably from 170 to 275 pm; more preferably from 180 to 255 pm.

6. The porous electrode according to any of the previous claims, wherein the polymeric foam is selected from a list consisting of: polyurethane, thermoplastic polyurethane, cellulose, or their combinations.

7. The porous electrode according to any of the previous claims, wherein the polymer mixture of two ionomers is selected from a list consisting of poly(3,4- ethylenedioxythiophene) polystyrene sulfonate, graphene ink, printable conductive nanocomposites of poly lactic acid and multi-walled carbon nanotubes ink, or their combinations.

8. The porous electrode according to any of the previous claims, wherein the bifunctional organosilane is selected from a list consisting of (3-glycidyloxypropyl) trimethoxysilane, polyethylene glycol)diglycidyl ether, or their combinations.

9. The porous electrode according to any of the previous claims, wherein the organosulfur compound is dimethyl sulfoxide.

10. The porous electrode according to any of the previous claims, wherein the conductive ink comprises the mixture of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate and (3-glycidyloxypropyl) trimethoxysilane, in a proportion of 0.4:100 (v / v), and dimethyl sulfoxide.

11. The porous electrode according to any of the previous claims, further comprising at least one capture reagent immobilized on the surface, preferably wherein the said capture reagent is capable of binding target cells and / or particles.

12. The porous electrode according to any of the previous claims, wherein the conductive film comprises a thickness from 0.1 to 1.4 pm, preferably from 0.15 to 1.3 pm, more preferably from 0.2 to 1.25 pm.

13. The use of a porous electrode as the one described in any of the claims 1 to 12, in the detection of harmful microorganisms in water, and electrically detect bacteria and diatoms, preferably wherein the harmful microorganisms in water include bacteria, diatoms and aquaculture pathogens.

14. A device for monitoring and control biological particle, comprising the porous electrode described in any of the claims 1 to 12.

15. The device according to the previous claim, further comprising means for detecting changes in the electrical properties of the porous electrode upon interaction with microorganisms.

16. A method for obtention of the porous electrode described in any of the claims 1 to12, comprising the following steps: cleaning and adapting the dimension of the polymeric foam; a plurality of coating steps of the polymeric foam with a conductive ink; an annealing step.

17. The method according to the previous claim, wherein the double coating step comprises a first dip-coating by immersing the polymeric foam into the conductive ink solution; drying the coated polymeric foam; a second dip-coating by immersing the polymeric foam into the conductive ink solution.

18. The method according to any of the previous claims 16 and 17, wherein the annealing step is made with conducting cylinders placed on top of rounded shape electrodes.

19. The method according to any of the previous claims 16 to 18, further comprising the step of application of at least one capture reagent immobilized on the porous electrode surface after the annealing step.

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