Mucin-coated electrode and uses thereof

Abstract

Provided herein an electrode coated with mucin-incorporated hydrogel film, a process for preparing of same, an electrochemical sensor comprising same, and methods for characterizing the redox properties of mucin and / or assessing permeability of the mucus to a redox-active compound of interest, and methods of diagnosing a disease or condition using said electrode or said sensor.

Description

MUCIN-COATED ELECTRODE AND USES THEREOFBackground of the invention

[0001] It has been shown that the human cells and the microbiome in the gastrointestinal tract are constantly communicating and affecting each other. Yet, there is a difficulty to study this interaction. These two biological kingdoms are separated by the mucus layer that governs their communication, mainly by transfer of electrons in the layer, oxygen gradients and nutrition availability. In addition, the mucus controls the interaction within the microbiome, mediating homeostatic conditions in the gut. The mucosa is the innermost layer within the gastrointestinal (GI) tract. Therefore diet, drugs and other external substances interact with the mucosa layer and the epithelial cells wall. Among the different parts, the mucosa is viewed as an enormous surface of contact with the environment and is colonized by hundreds of trillions of gut microbes coming in direct touch with the epithelium cell. Finally, the mucus layer acts as a barrier between the human epithelial cells and the external bacteria and substances.

[0002] The major component of the mucus is the protein mucin, that is responsible for the action of the mucus as a physical barrier, source of nutrition for bacteria and as a regulator of gut redox homeostasis. The concentrations of reactive oxygen species (ROS; H2O2 and 02') and antioxidant biothiols (cysteine, glutathione and thioredoxin) in the human gut control the oxidized (disulfide, R-S-S-R) and reduced (free dithiol, R-SH, R- SH) states of the redox couples in the mucin, affecting the spatial configuration of the mucus. These redox couples provide a functionally reversible redox based switching mechanism controlling oxidation stress and cellular-bacterial signaling. The oxidative homeostasis state in the gut seems to be crucial for understanding the complex relationship in the host-mucus-microbiota interkingdom and is important stage for learning the gut physiology.

[0003] To date, a few research groups have provided systems for mimicking the mucus and the mucosa layer. The mimic systems have focused on the physical and chemical properties of mucus and mucin [M. Boegh and H. M. Nielsen, “Mucus as a barrier to drug delivery - understanding and mimicking the barrier properties,” Basic Clin. Pharmacol. Toxicol., vol. 116, no. 3, pp. 179-186, Mar. 2015, doi: 10.1111 / BCPT.12342; L. M.Ensign, R. Cone, and J. Hanes, “Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers,” Adv. Drug Deliv. Rev., vol. 64, no. 6, pp. 557-570, May 2012, doi: 10.1016 / J.ADDR.2011.12.009; and R. Bansil and B. S. Turner, “Mucin structure, aggregation, physiological functions and biomedical applications,” Curr. Opin. Colloid Interface Sci., vol. 11, no. 2-3, pp. 164-170, Jun. 2006, doi: 10.1016 / J. COCIS.2005.11.001], A successful structure of mucus has been shown based on a range of approaches:1) Mucin-based structures [for example, M. Boegh, S. G. Baldursdottir, A. Miillertz, and H. M. Nielsen, “Property profiling of biosimilar mucus in a novel mucus-containing in vitro model for assessment of intestinal drug absorption,” Eur. J. Pharm. Biopharm., vol. 87, no. 2, pp. 227-235, Jul. 2014, doi: 10.1016 / J.EJPB.2014.01.001],2) Biosimilar [for example, D. Birch, R. G. Diedrichsen, P. C. Christophersen, H. Mu, and H. M. Nielsen, “Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium,” Eur. J. Pharm. Sci., vol. 118, pp. 144-153, Jun. 2018, doi: 10.1016 / J.EJPS.2018.02.032],3) cell-culture secreted [for example, N. Navabi, M. A. McGuckin, and S. K. Linden, “Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation,” PLoS One, vol. 8, no. 7, p. e68761, Jul. 2013, doi: 10.1371 / JOURNAL.PONE.0068761],4) ex-vivo [for example, J. Sotres, S. Jankovskaja, K. Wannerberger, and T. Amebrant, “Ex- Vivo Force Spectroscopy of Intestinal Mucosa Reveals the Mechanical Properties of Mucus Blankets,” Sci. Reports 2017 71, vol. 7, no. 1, pp. 1-14, Aug. 2017, doi: 10.1038 / s41598-017-07552-7] .

[0004] The works reported in the publications mentioned above focus on mucus secretion, bacterial movement and substance diffusion, protection from inflammation and structural form of the mucus (see also L. Wright, P. Joyce, T. J. Barnes, and C. A. Prestidge, “Mimicking the Gastrointestinal Mucus Barrier: Laboratory-Based Approaches to Facilitate an Enhanced Understanding of Mucus Permeation,” doi: 10.1021 / acsbiomaterials. lc00814). However, it appears that little was reported on controlling the redox behavior of mucin.The summary of the invention

[0005] It has now been unexpectedly found that it may be possible to detect and control mucin redox state and changes thereof in a specialized electrode surface mucin coating, and to utilize these measurements to detection of various chemical or microbiological effectors. This, in turn, may be conductive to diagnostic methodology for determination of various analytes in biological samples, and / or to the diagnosis of a variety of pathological conditions, e.g., of inflammatory bowel diseases, as shown om the appended examples.

[0006] Thus, in a first aspect provided herein is an electrode coated with mucinincorporated hydrogel film. Optionally, said electrode is wherein the hydrogel comprises alginate. Further provided herein is a process for preparing a mucin-coated electrode, comprising fabricating hydrogel film from mucin-containing gelator solution, on a surface of an electrode. Optionally, the process is wherein the hydrogel is electrodeposited on the electrode from a deposition solution comprising from 1-1.8% water-soluble gelator and from 0.05 to 0.15 mg / ml mucin. Optionally, the process is wherein the deposition solution comprises, as the water-soluble gelator, a monovalent alginate salt, and further comprises a carbonate of an alkaline earth metal. Optionally, the process is further comprising postdeposition addition of alkaline earth metal ions to crosslink and stabilize the alginate. In a further aspect provided herein is a microfabricated electrochemical sensor, comprising: a base substrate; optionally an adhesion layer applied on the base substrate; and at least one working microelectrode deposited on the base substrate or on the adhesion layer, wherein the working microelectrode is coated with mucin-incorporated hydrogel film. Optionally, the microfabricated electrochemical sensor is wherein the base substrate is made of glass or silicon / silicon oxide, with a titanium adhesion layer applied on the base substrate, and at least one disc-shaped working microelectrode deposited on the adhesion layer, wherein said disc-shaped working microelectrode is coated with mucin-incorporated alginate film and is encircled by a cylindrical wall defining a chamber. Optionally, the microfabricated electrochemical sensor is wherein the cylindrical wall encircling the coated microelectrode is made of epoxy polymer. Optionally, the microfabricated electrochemical sensor is further comprising a counter electrode and Ag / AgCl reference electrodes deposited on the base substrate. Further provided herein an electrochemicalmethod of characterizing the redox properties of mucin with the aid of a redox mediator, comprising recording an electrochemical signal at a working electrode coated with mucinincorporated hydrogel film, and measuring the charge delivered to the electrode by the redox mediator to determine the reduced / oxidized state of the mucin. Further provided herein an electrochemical method of assessing permeability of the mucus to a redoxactive compound of interest, comprising recording in a solution of said redox-active compound an electrochemical signal at a working electrode coated with mucinincorporated hydrogel film, wherein passage of said redox active molecule across the mucus is indicated by one or more features of the electrochemical signal, such as intensity, position, and trend of recurrence. Further provided herein a method of diagnosing of an inflammatory process in the bowel in a subject, comprising obtaining a sample of bowel contents from said subject, incubating said sample on a working detection electrode coated with mucin-incorporated hydrogel film, incubating a blank solution to said sample on a reference working electrode identical to said working electrode, recording an electrochemical signal obtained at said detection electrode and at said identical reference electrode, comparing said recorded signals with standard signals obtained with a confirmed pathological sample or a confirmed healthy sample, and sorting said sample as normal, pathologic, or borderline based on said comparing. Optionally, the method is wherein said process is characterized by a microbiome shift or a dysbacteriosis. Optionally, the method is wherein said recording comprises cyclic voltammetry measurements. Optionally, the method is wherein said comparing comprising obtaining ratios between the signal obtained at said detection electrode and at said identical reference electrodes. Optionally, the method is wherein said sorting is based on comparing ratios obtained between the signal obtained at said detection electrode and at said identical reference electrodes, with ratios obtained from said standard signals, wherein said recording comprises cyclic voltammetry measurements, and said comparing further comprises evaluating the cyclic voltammetry signals at at least two cycles separated by at least 30% of the total number of cycles. Optionally, the method is wherein said recording comprises cyclic voltammetry measurements, wherein said sorting comprises a log-linear regression of the signals obtained at said detection electrode and at said identical reference electrodes, wherein said log-linear regression is in form of Q cyc = A * eA(-k*cyc) + Q_inf and wherein Q_cyc is anodic charge in each cycle, k isthe decay constant, eye is the cycle number, Q inf is the constraint of basal anodic charge at the tested conditions, and A is the coefficient representing the initial additional charge contributed by said mucin-incorporated hydrogel film, and wherein said sorting further comprises comparing A and k parameters obtained parameters obtained with said standard signals.Brief description of figures

[0007] Figure 1 schematically demonstrates an exemplary proposed mechanism of oxidation and reduction of mucin using redox mediators and antioxidants on an electrode according to an embodiment of the invention.

[0008] Figure 2 schematically demonstrates a process of preparing of an electrode according to an embodiment of the invention.

[0009] Figure 3 A shows schematically a process described in the Example 1. Figure 3B demonstrates a micrograph of an assembly of 24 microelectrodes prepared according to an embodiment of the invention demonstrated in the Example 1. Figure 3C demonstrates a chronopotentiometry plot of an electrodeposition according to an embodiment of the invention demonstrated in the Example 1.

[0010] Figure 4 A demonstrates a representative light micrograph of dried calcium alginate coating on Au electrode according to an embodiment of the invention demonstrated in the Example 1. Figure 4B demonstrates a representative scanning electron micrograph of a bare electrode according to an embodiment of the invention demonstrated in the Example 1. Figure 4C demonstrates a representative scanning electron micrograph of a alginate-coated electrode according to an embodiment of the invention demonstrated in the Example 1. Figure 4D demonstrates a representative scanning electron micrograph of a alginate-mucin-coated electrode according to an embodiment of the invention demonstrated in the Example 1.

[0011] Figure 5A demonstrates voltammograms corresponding to the first voltametric cycle obtained with low-mucin electrode according to an embodiment of the inventiondemonstrated in the Example 2. Figure 5B demonstrates voltammograms corresponding to the first, fifth, and tenth voltametric cycles obtained with intermediate-mucin electrode according to an embodiment of the invention demonstrated in the Example 2. Figure 5C demonstrates an exemplary voltammogram used for calculation of oxidation and reduction charges. Figure 5D demonstrates a chart of calculated oxidation charge versus voltametric cycle number for electrodes according to embodiments of the invention demonstrated in the Example 2. Figure 5E demonstrates a chart of calculated reductive charge versus voltametric cycle number for electrodes according to embodiments of the invention demonstrated in the Example 2.

[0012] Figure 6 A demonstrates a summary of measurements performed on mucin-coated electrode according to an embodiment of the invention demonstrated in the Example 3. Figure 6B demonstrates a summary of measurements performed on alginate-only electrode according to an embodiment of the invention demonstrated in the Example 3. Figure 6C demonstrates box and whiskers plot of fluorescence intensity obtained on electrodes according to an embodiment of the invention and a control electrode demonstrated in the Example 3.

[0013] Figure 7A demonstrates a summary of measurements performed on alginate- coated electrode with or without model bacteria, according to an embodiment of the invention demonstrated in the Example 4. Figure 7B demonstrates a summary of measurements performed on oxidized mucin-coated electrode with or without model bacteria, according to an embodiment of the invention demonstrated in the Example 4. Figure 7C demonstrates a summary of measurements performed on reduced mucin-coated electrode with or without model bacteria, according to an embodiment of the invention demonstrated in the Example 4. Figure 7D demonstrates a summary of TOC and TAS measurements performed on mucin-coated electrodes with or without model bacteria, according to an embodiment of the invention demonstrated in the Example 4.

[0014] Figure 8A demonstrates a summary of measurements performed on oxidized mucin-coated electrode with model bacteria in absence or in presence of oxidative stress, according to an embodiment of the invention demonstrated in the Example 4. Figure 8Bdemonstrates a summary of measurements performed on reduced mucin-coated electrode with model bacteria in absence or in presence of oxidative stress, according to an embodiment of the invention demonstrated in the Example 4.

[0015] Figure 9 A demonstrates a summary of measurements performed on reduced mucin-coated electrode in absence or in presence of humanized murine microbiome, according to an embodiment of the invention demonstrated in the Example 5. Figure 9B demonstrates a summary of measurements performed on oxidized mucin-coated electrode in absence or in presence of humanized murine microbiome, according to an embodiment of the invention demonstrated in the Example 5. Figure 9C demonstrates a chat of the ratio of oxidative charge for bacteria on mucin and on alginate electrodes bearing humanized murine microbiome, according to an embodiment of the invention demonstrated in the Example 5.The detailed description

[0016] Experimental work conducted in support of this invention shows that mucin can be applied on an electrode surface via encapsulation in an electrochemically fabricated hydrogel (i.e., hydrogel film deposited on the surface of an electrode), and that an electrode coated with mucin-incorporated hydrogel film can be used to control and manipulate electrochemically the mucin redox behavior, i.e., interconversion of dithiol / disulfide in the presence of redox mediators, as shown by cyclic voltammetry experiments reported below (the interconversion between the dithiol and disulfide groups in mucin cysteine residues is a redox reaction; the dithiol form is the reduced state, and the disulfide form is the oxidized state).

[0017] Cyclic voltammetry was conducted using a three-electrode setup by scanning the potential applied on the mucin-coated working electrode across the range of 0.1 to 0.8V (vs. Ag / AgCl), in a solution of the hexachloroiridate(IV) / hexachloroiridate(III) redox couple (also referred to herein interchangeably as “Ir”, “iri”, and like). Hexachloroiridate(IV), whose structure is shown below, is a strong oxidant (available as the potassium salt):

[0018] Hexachloroiridate(IV) (sometimes abbreviated herein iridate or iri) oxidizes dithiol to disulfide, and transforms into the reduced form hexachloroiridate(III). The voltammogram recorded with the hexachloroiridate(IV) / hexachloroiridate(III) redox pair indicates the effect of the mucin incorporated into the coating of the working electrode, showing enhancement of the anodic (oxidation) current with increasing loading of mucin in the coating; the enhancement was observed only when the mucin concentration in the coating was sufficiently high. However, the amount of charge delivered to the anode upon oxidation of hexachloroiridate(III) (oxidation charge) gradually decreased with increasing number of voltametric cycles in the cyclic voltammetry, suggesting that the number of reduced thiols groups is progressively lessened with each consecutive cycle, leading to a decrease in the ability of the mucin to reduce the oxidized form (the tetravalent iridium), with eventually lower quantity of charge transferred to the working electrode with each successive cycle. That is, oxidized mucin, in the disulfide state, is steadily becoming the predominant form of mucin in the coating during repeated potential voltametric cycling.

[0019] But the coated electrode can restore its reductive state, as the oxidized mucin is reducible to the full open state (free dithiol) with the aid of a strong reductant, such as Tris(2-carboxyethyl)phosphine (TCEP), whose structure is shown below (available as the hydrochloride salt):

[0020] The newly restored reduced mucin (the dithiol form) has shown a strong enhancement in iridate oxidation peak, comparable in magnitude to the enhancement observed in the first cycle of a previously conducted cyclic voltammetry.

[0021] The proposed mechanism of oxidation and reduction of mucin using redox mediators and antioxidants that was explained above is also shown in the scheme depicted in Figure 1, i.e., the electron transfer through redox mediators to the electrode. The phase 1 process represents oxidation and discharging of mucin, and enhanced oxidation charge delivered to the anode, with the (hexachloroiridate(IV) / hexachloroiridate(III)) redox cycle. That is, the strong oxidant hexachloroiridate(IV) (denoted as “Iri(OX)”) accepts the electron donated by the reduced dithiol, with conversion into hexachloroiridate(III) (denoted as “Iri(RED)”) and the oxidized disulfide, respectively. As the potential is swept forward to positive, oxidative potential, hexachloroiridate(III) is oxidized and the electron lost is transferred to the electrode (anode). The strong oxidant hexachloroiridate(IV) is regenerated, and can again oxidize the dithiol to disulfide, and so on. This shows the ability of the mucin-coated electrode to sense a redox solution.

[0022] The Phase II process represents reduction and charging of mucin using TCEP treatment. The electron donated by the reductant TCEP is gained by the oxidized mucin (disulfide state) whereby the free dithiol is regenerated. Through operation in this cyclic manner, i.e., discharge (phase I) and recharge (phase II) of the mucin, a stable and repeatable electrochemical signal is achieved.

[0023] Thus, without wishing to be bound by theory, it appears that the strong oxidant hexachloroiridate(IV) closes the sulfur gates, i.e., creates the disulfide bonds, and the strong reductant TCEP opens the gates back, i.e., restores the free dithiol groups. In this manner, the mucin's gates are controlled electrochemically using redox mediators, determining the spatial structure of the artificial mucosa layer deposited on the electrode.

[0024] Accordingly, the invention is primarily directed to an electrode coated with mucin-incorporated hydrogel film. Another aspect of the invention is a process for preparing a mucin-coated electrode, comprising fabricating (e.g., electrodepositing) hydrogel film from mucin-containing gelator solution, on a surface of an electrode. Another aspect of the invention is a kit for extemporaneous fabricating of a mucin-coated electrode, an electrode assembly, a hydrogel film precursor solution and mucin solution, or comprising ready mucin-containing gelator solution, for electrodeposition onto asurface of an electrode. A further aspect is a method of diagnosis of a pathological condition by measuring a biological sample of a patient in need thereof electrochemically using a mucin-coated electrode.

[0025] Mucin-containing gelator solution is prepared by dissolving in water a gelator selected from the group consisting of alginic acid and salts thereof, chitosan, collagen, carboxymethyl cellulose, hyaluronic acid, and salts thereof, and poly acrylic acid and salts thereof, followed by addition of mucin. The concentration of the gelator in the solution is usually from 1.0 to 3.0% w / v, e.g., 1.0 to 1.8% w / v. To load an effective amount of mucin onto an electrodeposited hydrogel film, mucin should be present in the gelator solution at a concentration of not less than 0.03 mg / ml, e.g., from 0.05 to 0.15 mg / ml, for example, from 0.05 to 0.1 mg / ml mucin.

[0026] The electrodeposition of a hydrogel film from mucin-containing gelator solution on a surface of an electrode involves the creation of a solubility gradient across the deposition solution, such that the solubility of the gelator increases with increasing distance from the electrode's surface. The solubility of gelators is usually pH dependent, and therefore creation of a pH gradient across the solution achieves the desired effect and drives the deposition of the hydrogel film onto the electrode surface. The approach is shown in Figure 2, where alginate is the hydrogel of choice, owing to its good filmforming ability. The gelator in the deposition solution is an alginate salt that dissolves well in water at nearly neutral or alkaline pH but is insoluble in an acidic environment. Suitable alginate salts include monovalent alginates such as sodium alginate, potassium alginate and ammonium alginate; the sodium salt is preferred. The pH gradient is triggered by oxidation of H2 molecules, to generate an acidic pH (around 4-5) near the electrode surface. It is seen in Figure 2 that the pH varies with distance (denoted as “pH Gradient”), with slightly alkaline values in the bulk solution owing to the presence of an alkaline earth metal carbonate compound in the bulk solution, which creates an alkaline pH (9-10). The alginate film is progressively grown on the electrode surface in the acidic environment. Postdeposition addition of polyvalent metal cations, such as Ca2+(shown in Figure 2) or Mg2+, which can crosslink alginate chains, stabilizes the electrodeposited hydrogel films, with the mucin encapsulated inside.

[0027] A preferred deposition solution for use in the invention contains from 0.5 to 1.5% w / v, e.g., -1.0% w / v sodium alginate and from 0.03 to 0.1 mg / ml mucin, e.g., 0.05 to 0.1 mg / ml mucin. On industrial scale, the deposition solution is prepared by gradual addition of the sodium alginate powder to water (e.g., DDW) under stirring using a high-speed propeller type stirrer to uniformly disperse and dissolve the alginate in a fairly short time. On a lab scale, sodium alginate powder can be dispersed and dissolved in water using magnetic stirrer over a couple of hours followed by a short sonication. After the formation of an alginate viscous solution, the alkaline earth metal carbonate is added, for example, at concentration of 0.1 to 0.5% w / v, e.g., 0.25-0.45%. Lastly, mucin powder is dispersed in the solution by constant stirring for 20-30 minutes.

[0028] Sodium alginate grades suitable for use in the invention show viscosity of 20 to 1000 mPa- s as measured on a 1% aqueous solution thereof at a temperature of 20°C, e.g., with a Brookfield viscometer, e.g., viscosity >100 mPa s. Alginate extracted from brown algae such as Laminaria Hyperborea can be used, for example, medium viscosity sodium alginate such A2203 from Sigma Aldrich. Suitable sodium alginates are available on the marketplace from various manufacturers such as FMC corporation (Protanal®) and Dupont (Manucol®) and Biosynth, usually with viscosity ranging from 100 to 150 mPa- s, 200 to 350 mPa s or 300 to 700 mPa s.

[0029] Mucin for use in the invention is obtained from natural sources (e.g., mammalian gastrointestinal and buccal mucins such as porcine gastric mucin, bovine gastric mucin, or bovine submaxillary glands). Commercial products are available as 'mucin type If and 'mucin type IIP from a range of suppliers such as Sigma Aldrich (M2378 M1778, M3895 and M4503) and A / S Orthana Kemisk Fabrik ('Orthana mucin'). Methods to extract mucin are known (Glenister and Salmon, K. Micro bial Ecol. in Health & Disease 1, 31, (1988) (from pork stomach); or Deshmukh et al. Am. J. Pathol. 38, 446-54, (2008) (from bovine submaxillary gland). Methods of purifying mucin are also known (see for example Marczynski et al. in Separation and Purification Technology 294 (2022) 121209). It is not uncommon for commercial mucins to be only partially purified and prior to use in the invention a further purification may be useful; a detailed purification procedure is shownin the experimental section below. The mucin that is used in the invention is preferably lyophilized mucin.

[0030] Alternatively, mucin glycoproteins may be produced in recombinant or nonrecombinant cells lines (see Backstrom et al., Biochem. J.376:677-86, 2003: Batra et al., J. Cell. Sci. 100:841-9, 1991; Dabbagh et al., J. Immunol. 162:6233-7, 1999: Kim et al., Mol. Pharmacol. 62:1112-8, 2002; and Link et al., J. Biotechnology 110:51-62, 2004). Mucin glycoproteins may be extracted and isolated from recombinant and nonrecombinant cell lines, as described in, e.g., Davies and Carlstedt, Methods Mol. Biol, in Glycoprotein Methods and Protocols, 125:3-13, 2000; Car raway, Methods Mol. Biol, in Glycoprotein Methods and Protocols, 125:15-26, 2000; and Bhavanandan et al., Glyco conjugate !., 15:37-49, 1998.

[0031] The mucin-incorporated hydrogels can be deposited on electrodes made of noble metals, e.g., gold, platinum, rhodium, and iridium. Other electrodes, such as glassy carbon and modified tin oxide electrodes (e.g. indium doped tin oxide), can also be coated with the mucin-incorporated hydrogel film. An electrochemical set-up for fabricating the hydrogel is usually a three-electrode configuration consisting of a working electrode as outlined above, a counter electrode and a reference electrode.

[0032] Turning now to the dimensions and geometry of the mucin-coated electrode, some applications, e.g., a mucosa-on-chip microsystem for microbiome-human body interaction studies, call for miniaturization of the electrode, i.e., the use of mucin-coated microelectrodes. Thus, another aspect of the invention relates to a microfabricated electrochemical sensor, comprising: a base substrate (made, for example, of glass or silicon / silicon oxide); optionally an adhesion layer (made, for example, of titanium) applied on the base substrate; and at least one working microelectrode deposited on the base substrate or on the adhesion layer, wherein the working microelectrode is coated with mucin-incorporated hydrogel film.

[0033] The counter and reference electrodes may be either external (e.g., commercial Pt wire and Ag / AgCl electrodes, respectively), or may be also patterned on the substrate as described below.

[0034] Microfabrication, i.e., the creation of microstructures (microelectrodes, wall defining microchambers encompassing the microelectrodes), can be created on the substrate by techniques such as etching and photolithography. The latter technique is preferred and is illustrated in detail in the experimental work reported below, in reference to Figure 3 A.

[0035] Briefly, as can be seen from Figure 3 A, microelectrodes (1) are patterned on a substrate (2), e.g., a wafer made of oxidized silicon, e.g., a glass substrate or silicon / silicon oxide. The wafer is usually 0.5 to 1 mm thick and its diameter may vary from 10 to 15 cm. The fabrication consists of the following steps. The substrate is cleaned, e.g., with 'piranha’ solution dried. A first photoresist (3) is applied (either negative, positive or image reversal resist), e.g., by spin coating, spray coating or dip coating, to produce a thin uniform layer on the substrate, followed by soft baking. A first mask (4) is aligned (different from denoted as the legend entry “Ti metal”), to transfer the pattern corresponding to electrodes' sites onto the surface of the substrate. In Figure 3A, photoresist (3) is a positive photoresist (denoted as the legend entry “AZ-nLOF”). The photoresist (3) is exposed through the pattern on the mask (4) with UV light (5), followed by a development step (6), to wash away the exposed areas. Next, bare microelectrodes are deposited in the intended sites, e.g., first titanium which serves as an adhesion layer (7) and then electrode active material (1). Deposition of an adhesion layer (7) e.g., a titanium layer or chromium layer (which adheres strongly to the glass substrate) onto the cleaned substrate is by electron beam evaporation or magnetron sputtering, to create 10 to 30 nm thick adhesion layer, followed by deposition of the electrode active metal, e.g., 100 to 300 nm thick gold layer, onto the adhesion layer; also by the same techniques. Figure 3B shows the deposited electrode material, e.g., gold, consisting of a disc-shaped area (1) (to function as the electrode), joined by a thin wire (21) to a square ped (22), which in turn is connected to a potentiostat. This is followed by lift off procedure (8) thatresults in creation of individual, spaced apart gold microelectrodes protruding from the surface of the glass substrate.

[0036] Each of the titanium / gold microstructures need to be encircled by a wall, to define the active surface area of the electrode. This is accomplished again by photolithography, as shown in Figure 3 A and explained in detail in the experimental part below. After spin coating of a negative photoresist (9) (e.g., SU8 3005, as denoted as the legend entry “SU- 8 3005”) and soft baking step, a suitably designed mask is applied, creating the pattern around the electrode and defining its geometrical shape.

[0037] Figure 3A shows just one approach to the structuring of microelectrodes on a wafer. The application of positive, negative or image reversal photoresists is often a matter of choice and convenience. Also, structuring via etching (i.e., wet etching / dry etching) could be used in place of lift-off procedures.

[0038] Having patterned the microstructures on the substrate, the desired coatings are applied on the microelectrodes, for example, by electrodeposition. In general, formation of a coating onto the surface of a microelectrode can be accomplished from the previously described gelator / mucin deposition solutions by the following electrodeposition techniques:(i) galvanostatic electrodeposition (chronopoten-tiometry), in which a constant current is passed through the microelectrode(s) to be coated;(ii) potentiostatic electrodeposition (chronoamper-ometry), in which a constant potential is applied on the working microelectrode(s) to be coated; or(iii) cyclic voltammetry electrodeposition.

[0039] For example, electrodeposition of a hydrogel coating from alginate / mucin deposition solution (1-1.8% alginate and 0.05-0.15 mg / ml mucin in water) onto a discshaped electrode with diameter of 2 mm can be accomplished by applying chronopotentiometry with current density of 4 A / m2for 180 sec, using a microfabricated electrode described above as a working electrode, and commercial Pt wire as short circuited RE and CE, immersed in the deposition solution. Upon completion of theelectrodeposition, the coated electrode is immersed in an aqueous solution of a polyvalent metal ion (e.g., Ca2+) as previously explained.

[0040] Thus, a specific aspect of the invention relates to a microfabricated electrochemical sensor, comprising abase substrate made of glass or silicon / silicon oxide, with a titanium adhesion layer applied on the base substrate, and at least one disc-shaped working microelectrode deposited on the adhesion layer, wherein said working microelectrode is coated with mucin-incorporated hydrogel film and is encircled by a cylindrical wall defining a chamber (the ring-shaped microelectrode and the cylindrical wall are contiguously, coaxially and concentrically aligned).

[0041] For example, the disc-shaped electrode coated with mucin-incorporated hydrogel film, that is placed on the glass substrate, is encircled by a wall made of epoxy polymer, e.g., bisphenol A Novolac epoxy such as SU-8 photoresist, which is 50 pm thick.

[0042] Conversely, the electrode may be supplied as a prefabricated kit of parts. The kit may be intended for extemporaneous fabricating of a mucin-coated electrode as described herein, by the end user, using conventional methodology. The kit may therefore comprise an electrode assembly, ready for electrodeposition of mucin-comprising hydrogel. The hydrogel and mucin may be supplied as dry powders, or as ready separate or mutual solutions. Preferably, the kit may comprise a hydrogel film precursor solution or a lyophilized powder for reconstitution of same, and mucin solution or a lyophilized powder for reconstitution of same, or the ready mucin-containing gelator solution or a lyophilized powder for reconstitution of same. The kit preferably may be accompanied by instructions on performing electrodeposition using conventional methods as described herein. The instructions may be in form of printed instructions, e.g., on the packaging of the kit, or as a package insert. The instructions may also be provided by referring the user to an online resource, e.g., via an Internet address, a QR code, or like methods.

[0043] As pointed out above, in operation, the microfabricated electrochemical sensor of the invention may be combined with commercial counter and reference electrodes to be connected to a potentiostat or galvanostat to run the electroanalytical method, e.g.,voltammetry. However, a disc-shaped counter microelectrode, e.g., of diameter in the range from 2 to 10 mm, and Ag / AgCl electroplated reference microelectrode may be applied on the base substrate adjacent to the working electrode. Silver / silver chloride miniature electrodes are prepared as described in WO 2022 / 137238, Example 6.

[0044] The mucin-incorporated, hydrogel -coated electrode, that mimics and controls mucin’s redox behavior, can be used in various applications, such as gut-on-chip biomicrosystems, energy storage devices, and sensors to recognize oxidative or reductive environments. Specifically, mucus like models can be used by tracking the sulfur gates in the mucin proteins with electrode and redox mediators. When the cyclic voltammogram oxidative signal is low, then the sulfur biosystem is in disulfide state, i.e., the system is oxidized; when the oxidative signal is high, then the sulfur biosystem is in thiol state, i.e., the system is reduced.

[0045] Thus, an additional aspect of the invention is an electrochemical method of characterizing the redox properties of mucin with the aid of a redox mediator, comprising recording an electrochemical signal at a working electrode coated with mucinincorporated hydrogel film, and measuring the charge delivered to the electrode by the redox mediator to determine the reduced / oxidized state of the mucin.

[0046] The electrode coated with a mucin-incorporated hydrogel film can also be used for studying the passage of substances through mucus layers. A range of techniques were tested for this purpose, as shown in a review paper by Lock JY, Carlson TL, Carrier RL. Mucus models to evaluate the diffusion of drugs and particles. Adv Drug Deliv Rev. 2018 Jan 15; 124:34-49. doi: 10.1016 / j .addr.2017.11.001. However, it appears that the electrochemical responsiveness of the mucus to redox molecules acting as redox mediators to manipulate the interconversion of the dithiol / disulfide system, was not exploited for the assessment of, for example, drug delivery across the mucosal barrier.

[0047] Therapeutically active compounds with redox properties include, for example, organometallic complexes used in anticancer therapy, based on redox couples of transition metals MP+ / Mn+(p>n), such as Co3+ / Co2+; Pt4+ / Pt2+, Fe3+ / Fe2+, Ru3+ / Ru2+,Ir4+ / Ir3+and Osp+ / Os2+, and organic compounds with redox-active sites, such as quinone- containing compounds (quinone derivatives were developed by the pharmaceutical industry as antibacterial, antifungal and antitumoral agents). Drug design may benefit from the screening of redox-active candidate compounds with the aid of an electrode coated with a mucin-incorporated hydrogel film, to determine the penetrability of the mucus barrier to such candidate compounds, i.e., passage of the tested candidate compound is indicated by an enhancement, recurrence or a trend observed in an electrochemical signal generated by the electrode (e.g., an increased oxidation current collected by the anode during cyclic voltammetry due to the cyclic action of Mp+ / Mn+redox couple (p>n), namely, oxidation of the mucin by the high oxidation state Mp+to give the reduced state Mn+and the anodic oxidation of the latter to reproduce the MP+which may again act on remaining dithiol groups, and so forth).

[0048] Accordingly, another aspect of the invention is an electrochemical method of assessing permeability of the mucus to a redox-active compound of interest (e.g., a redoxactive drug candidate), comprising recording in a solution of said redox-active compound an electrochemical signal at a working electrode coated with mucin-incorporated hydrogel film (for example, by voltametric technique, e.g., measuring the charge delivered to the electrode by the reduced state of said redox active-compound upon sweeping the potential to the positive direction in a cyclic voltammetry setup), wherein passage of said redox active molecule across the mucus is indicated by one or more features of the electrochemical signal, such as intensity, position and trend of recurrence.

[0049] In an additional aspect of the invention provided herein is an electrochemical method of diagnosis of a pathological condition by measuring a biological sample of a patient in need thereof electrochemically using a mucin-coated electrode. The method comprises recording an electrochemical signal at a working electrode coated with mucinincorporated hydrogel film exposed to the biological sample, measuring the charge delivered to the electrode, and comparing the results obtained with charge delivered to a different unit of the electrode by a known sample, such as a pathogen.

[0050] Specifically, the biological sample may be a microbiome sample. In these instances, microbiome sample may be incubated on the surface of the electrode coated with mucin-incorporated hydrogel film, for a time interval of between 2 and 4 hours, e.g., between 135 and 165 minutes, followed by characterizing the redox properties of mucin with the aid of a redox mediator. Cyclic voltametric signal may be recorded on the working electrode, by measuring the charge delivered to the electrode by the redox mediator. The resulting signal is compared with the control signal obtained without the biological sample but in presence of the blank solution used for the biological sample, and the difference may be compared to the difference obtained from signals recorded in presence of a pathogen, e.g., E. coli. The coated electrode may be primed by the redox mediators prior to the measurements, to transform the deposited mucin into fully oxidized state, or into fully reduced state.

[0051] Thus, provided herein a method of diagnosing of an inflammatory process in the bowel in a subject. The process is usually characterized by microbiome shifts and dysbacteriosis, including overgrowth of pathological bacteria. It has been unexpectedly found that normal microbiome reacts differently to reduced mucin electrodes than the opportunistic pathogenic bacteria E. coli. For example, normal microbiome reduced the anodic discharge relative to the blank over the tested cyclic voltametric measurements, as demonstrated in the appended examples. Conversely, pathogenic microbiota signals converged as cycling progressed under otherwise identical experimental conditions. Therefore, comparing the cyclic voltammogram of the microbiome sample versus the blank may enable determination the presence or absence of pathologic overgrowth therein, or other redox-shifting inflammatory response, being an important diagnostic factor to inflammatory diseases of the bowel in the subject. Thus, a method of diagnosing of an inflammatory process in the bowel in a subject may comprising obtaining a sample of bowel contents from the subject, and incubating the sample on a working detection electrode coated with mucin-incorporated hydrogel film. Preferably, in parallel, a blank solution to the sample may be incubated on a reference working electrode identical to the working electrode. Following this incubation, an electrochemical signal obtained at the detection electrode and at the identical reference electrode may be recorded. As demonstrated in the appended examples below, comparing the recorded signals withstandard signals obtained with a confirmed pathological sample or a confirmed healthy sample, may enable sorting the microbiome sample as normal, pathologic, or borderline based on said comparing.

[0052] The diagnostic method may be advantageously employed when the pathogenic process is characterized by a microbiome shift or a dysbacteriosis, although other conditions affecting the redox behavior of mucin are readily envisaged, as discussed above. Preferably, the pathogenic process in an inflammatory bowel disease as generally known in the medicine, e.g., as classified in a subclass 13 of the 11th revision of World Health Organization International Classification of Diseases (herein: “ICD-l l”), particularly under the codes DD70-DD7Z, or elsewhere, provided the pathogenic process involves an inflammatory response within gastrointestinal system.

[0053] Specifically, the diagnostic method recording the electrodes’ signals may comprise a variety of measurements, however preferably the measurements are cyclic voltammetry measurements. As demonstrated in the appended examples, recording the electrochemical response of the electrode bearing a sample may be used to sort the sample to either pathogenic or normal, or borderline, depending on the pattern of the electrical signals obtained from the electrode. Thus, for example, obtaining ratios between the signal obtained at the detection electrode and at the reference electrodes may be compared. The appended results demonstrated distinct patterns when the sample contains pathological microbiota, and when the sample contains healthy humanized microbiome from a laboratory animal. Specifically, the sorting may be performed by comparing the ratios obtained between the signal obtained at the detection electrode and at the identical reference electrodes, with the ratios obtained from the standard signals, using cyclic voltammetry measurements and evaluating the cyclic voltammetry signals at several cycles along the timeline of the measurement, but preferably at least two cycles separated by at least 30% of the total number of cycles, e.g., the first and the last cycle of three- cycle measurement, the second and fifth cycle of five-cycle measurements, and so on. Preferably, more than just two cycles may be used, and the whole voltammogram of the test electrode and the reference electrode may be used to sort the sample as pathogenic,borderline, or healthy, depending on the similarity between the overall patterns, e.g., as expressed in the ratios between the signals, as explained above.

[0054] However, in some preferred embodiments, the diagnostic method may employ said a log-linear regression of the signals obtained with cyclic voltammetry as described above. The log-linear regression may be preferably in the form of Q cyc = A * eA(-k*cyc) + Q inf, such that Q cyc is anodic charge in each cycle, k is the decay constant, eye is the cycle number, Q inf is the constraint of basal anodic charge at the tested conditions, and A is the coefficient representing the initial additional charge contributed by the mucin-incorporated hydrogel film. Thus, fitting the data to the regression may furnish the A and k parameters, which may be then used for sorting the specimen as to healthy or pathogenic, or borderline, based on the A and / or k parameters obtained with the standard pre-sorted signals.

[0055] Generally, various other features according to the invention as described herein for one aspect are applicable mutatis mutandis to other disclosed aspects according to the teachings herein; such, various features according to the invention as described herein for the aspect of electrodes and / or electrochemical sensor comprising same, are applicable mutatis mutandis to the methods of manufacturing of said electrodes, said electrochemical sensors, and to methods of characterizing the redox properties of mucin and / or assessing permeability of the mucus to a redox-active compound of interest, and methods of diagnosing a disease or condition using said electrode or said sensor, according to the teachings herein, and vice versa. The herein described preferred embodiments as provided herein demonstrating some of the embodiments of the present disclosure are provided to better understand the present disclosure, which however does not limit the invention in any respect. Some variants and equivalents may be readily envisaged by the skilled artisan; the invention therefore encompasses all these variations and equivalents. It must also be noted that, as used in this specification and the appended claims: all scientific and technical terms have meanings commonly used in the art unless otherwise specified; the definitions inasmuch as provided herein are given with the purpose to facilitate understanding of certain terms used frequently herein and are not necessarily meant to limit the scope of the present disclosure; as used herein the term "about", “c.a ”,and like, as used interchangeably herein, refers to the value and the range of ± 10 %; the terms"comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to", with this terms also encompassing the terms "consisting of and "consisting essentially of, which have their narrower meaning as known in the art, thus an embodiment described as comprising something also discloses embodiments consisting essentially of same and consisting exclusively of same; the singular forms “a”, “an”, and “the”, include plural referents unless the context clearly dictates otherwise; as used herein, a phrase in the form “A and / or B” means a selection from the group consisting of (A), (B), and (A and B); as used herein, a phrase in the form “at least one of A, B, and C” means a selection from the group consisting of (A), (B), (C), (A and B), (A and C), (B and C), and (A, and B, and C), and further combinations are envisaged for the lists comprising larger number of terms. It is appreciated that, certain features of the invention, which are, for brevity, described in the context of separate embodiments, may also be provided in combination with other features in a single embodiment, unless technically infeasible. Conversely, features described in specific combinations of various features, which are, for clarity and demonstration, are described in the context of a single embodiment, may also be provided as separate embodiments individually or in any suitable sub-combination with other features and / or embodiments, as reasonable to the skilled artisan, suitable, and operative. Certain features described in the context of various embodiments, including preferred features, are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.ExamplesMaterials

[0056] Methanol (001368052100, Bio-Lab, Ltd.), acetone (376, Bio-Lab, Ltd.), potassium chloride (11595, Alfa Aesar), 2-propanol (1301221, Bio-Lab, Ltd.), Sulfuric acid (258105, Bio-Lab, Ltd.), hydrogen peroxide (1.07210.1000, Merck), calcium chloride (10195054, Alfa Aesar), sodium acetate anhydrous 99% (A13184, Alfa Aeser), acetic acid glacial (100063.2500 Merck), potassium hexachloroiridate (III) hydrate (011887.1, Holand Moran), potassium hexacyanoferrate(II) trihydrate (‘Ferrocyanide’, 1.04984.0100, Merck), potassium hexacyanoferrate (III) (‘Ferricyanide’, 1.04973.0100, Merck), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) bond-breaker (TS-77720, ThermoFisher) were used without further purification. Deionized water (‘DI’; resistivity > 18 MQ) was obtained from a Super Q water system (Millipore). Mucin type II from porcine (M2378, Merck) was cleaned before use. PBS tablets (BP2944- 100, ThermoFisher) were used for 0.01M PBS solution preparation. Alginic acid sodium salt from brown algae (Medium viscosity, A2203, Sigma Aldrich) was stirred (1% w / v) in DDW over-night, following 15 min sonication and 15 min stirring at the day of the experiment.Electrodeposition & electrochemical measurements

[0057] Measurements were performed using a PalmSens4 and MUX16 multiplexer plugin (Palmsens, Ltd.) with a three-electrode cell configuration consisting of a microfabricated electrode (working electrode; ‘WE’), an externally applied commercial Pt wire (CHI115, CH Instruments; counter electrode; ‘CE’), and an externally applied commercial Ag / AgCl electrode (CHI11 IP, CH Instruments, reference electrode; ‘RE’). Electrochemical potentials are vs Ag / AgCl half-cell potential.Data analysis

[0058] Data was analyzed by Pyt hon 3.8 using Spyder 5.2 interface.Example 1 - Electrodes coated with mucin-incorporated alginate filmA) Fabrication of microelectrodes on a glass substrate

[0059] Photolithography and thin film deposition-based techniques were used to manufacture gold electrode onto a glass substrate (prime grade, wafer diameter 100 mm, wafer thickness 500 pm, and double side polished, University Wafer, Ltd.). The sequence of steps is shown in Figure 3 A.

[0060] The glass substrate (2) was cleaned with a ‘piranha’ solution (a 1 :3 ratio mixture of hydrogen peroxide and sulfuric acid, HMxSquare SUSS MicroTec system) followed by dehydration on a contact hot plate at 120 °C for 10 minutes. Then, the cleaned substrate was left at room temperature for 10 minutes to cool down. An AZ-nLOF photoresist (product no. 97, Micro chemicals) (3) was spin-coated (80RCDelta, Universal Spin- Coating system, SUSS MicroTec) onto the glass wafer (2200 RPM for 12 s at an acceleration rate of 800 RPM / s). The coated substrate was left at room temperature on the spinner chamber for 5 minutes to settle down, followed by a ‘soft bake’ step on the contact hot plate (110 °C for 2.5 minutes). The substrate was cooled down to room temperature for 10 minutes, followed by exposure to a transparency mask (4) (a light flux of 7.6 mW / cm2for 65 s; Karl Suss Mask aligner MA6 system, SUSS MicroTec). A ‘postexposure bake’ was performed on the contact hot plate (120 °C for 2.5 minutes) and the substrate was left to cool down to room temperature for 10 minutes. A flood exposure step (5) was performed (a light flux of 7.6 mW / cm2) and the exposed substrate was developed (6) (AZ 726 MIF developer, Micro Chemicals) for 6 minutes, rinsed with distilled water for 5 minutes, and dried with nitrogen gas, followed by oxygen plasma cleaning for 0.5 minutes. Next, 20 nm -thick titanium (7) and 200 nm-thick gold layers were evaporated onto the developed substrate using an E-gun deposition system (VST Service, Ltd.). The substrate was then dipped in an acetone solution (8) for 60 minutes, followed by rinsing with distilled water and drying with nitrogen gas. SU8-3005 was used to define the microelectrode chamber (9); this allows cleaning the microelectrode with an AMI (acetone, methanol, and isopropanol) solution without destroying the chamber before using it. First, SU8-3005 was spin coated at 3000 RPM for 30 s, followed by a soft bake on a hot plate at 95 °C for 15 min. Second, the photoresist was exposed to light using a hard contact of 7.6 mW / cm2for 50s at a Mask Aligner (MA6, SUSS MicroTec). Then,Post Exposer Bake (PEB) was used for 5 min at 95 °C. The exposed wafer was then developed in PGMA ERB developer solution for 8 min and washed in isopropanol for 10s. The hard bake on a contact hot plate at 150 °C for 5 min was carried out to remove any hydration on the substrate, and oxygen plasma cleaning (30W, 500 mTorr, 2 min, 3 seem) was used after the hard bake to remove any residues or impurities on the substrate. Finally, the fabricated substrate was diced into single microchamber chips (Dicer ADT- 7100, ADT) (Figure 3B). A total of twenty-four electrodes is seen in Figure 3B. An individual disc-shaped electrode (1) is shown in the insert on the right side of the Figure, with diameter of 2 mm, encircled by SU8-3005 wall which is 50 pm thick. Prior to electrochemical testing, the microfabricated chips were sequentially cleaned by rinsing with acetone, methanol, isopropanol, and then cleaned with “piranha” solution and distilled water solutions to remove the organic residues, and finally dried using a nitrogen gun.B) Mucin purification

[0061] Mucin type II from porcine stomach was dissolved in acetic buffer (pH 5.5) to saturation (30mg / ml) and stirred over-night. The dissolved solution was centrifuged (Eppendorf™ Centrifuge 5810, ThermoFisher) - 8000rpm for 20 min following lOOOOrpm for 20 min. The centrifuged solution was filtered (Millipore Express™Plus 0.22pm) using vacuum pump (MRC, VP- 17, 650mmHg max). The filtered solution was dialysed using 12kDa dialysis sack (Yair Technologies, Cellulose Tubing MWCO 12kDa) for 2 days (DI water was changed after 1 day). The dialyzed solution was feezed using liquid nitrogen and lyophilized (Labconco, Triad freeze dryer) for 3 days.C) Electrodeposition of films onto the electrodes

[0062] Alginate-mucin2 (alg-muc) films were electrodeposited onto the gold electrodes from solutions prepared by mixing 0.01 mg / ml, 0.05mg / ml or 0.1 mg / ml cleaned mucin in sodium alginate (1% w / v) and calcium carbonate (0.25% w / v) solutions. For comparison, a mucin-free alginate film was also produced, using sodium alginate and calcium carbonate deposition solution. Electrodeposition was carried out with a three- electrode electrochemical setup consisting of a microfabricated Au electrode as WE, commercial Pt wire as short circuited RE and CE, positioned in the deposition solutions, by chronopotentiometry (j = +4A / m2, 180 sec, as shown in the potential versus time plotsof Figure 3C, with the red, blue, black, and green curves corresponding from top downwards to 0.1 mg / mL mucin, 0.05 mg / mL, 0.01 mg / mL, and alginate as the lowest curve), followed by 1 min of cooling in the solution and additional 20 min dipping in 1% w / v calcium chloride for stabilization of the hydrogel coating.

[0063] Figure 4A is a microscope image of dried calcium alginate coating on Au electrode (i.e., a mucin-free coating). Figures 4B-4D are SEM images of (B) bare, (C) alginate and (D) alginate-mucin coatings. All SEM images (SEM -Quanta 200, magnification) are magnified by 10000. Alginate-mucin coatings showed different surface topography / morphology compared to regular alginate films (Fig 4C-4D). The red size bar denoted as “4pm” represents a length of 4 pm.

[0064] To examine the electrochemical performance of the electrodes coated with alginate hydrogel, cyclic voltammetry was performed, using 100 pM of the positively charged redox couple hexaaminoruthenium (II / III). The potential scanned was between - 0.4[V] and 0.1 [V] vs. Ag / AgCl, with can rate of 0.1 V / s and three 3 cycles, and the total charge in a single voltametric cycle (during the anodic and cathodic sweep) was calculated. The total charge is related to the area under the curve and is given by Q =where Q represents charge[pC], I represents current[pA], V represents potential[V] vs. Ag / AgCl). The charge collected by an alginate-coated electrode was significantly higher compared to the bare electrode (820±210pC vs 397±91pC, three electrodes for each group, pVaiue = 0.069). The result is in line with the fact that the alginate hydrogel film assumes negative charge in solution and is therefore able to attract electrostatically the positively charged redox couple, and additionally, the deposition of an alginate film on the Au electrode leads to increased active surface area of the electrode.Example 2 - Mucin coating increases the anodic signal of a strong oxidant: testing an electrode coated with mucin-incorporated alginate film by cyclic voltammetry in the presence of a strong oxidant

[0065] Cyclic voltammograms were recorded using the three-electrode described above in 200 pM solution of KjIrCls. Potassium hexachloroiridate(IV) is a strong oxidant, withE°=0.6V vs. Ag / AgCl. The potential between 0.1V and 0.8V vs. Ag / AgCl was cycled at a scan rate of 0.1 V / s, over ten cycles.

[0066] The surface modified electrodes tested were the alginate-coated gold electrodes and different alg-muc coated electrodes, which were electrodeposited from solutions with varied mucin concentration as described in Example 1 [0.01, 0.05 and 0.1mg / mL]. Each electrode (labeled alg, alg-muco.oi, alg-muco.os and alg-muco.i) was tested in triplicate (3 electrodes in each group).

[0067] The voltammograms corresponding to the first voltametric cycle are shown in Figure 5 A (alg-coated electrode: solid triangles (A) yellow voltammogram; alg-muco.oi- coated electrode: solid rectangles (■) green voltammogram; alg-muco.os-coated electrode: solid circles (•) red voltammogram; and alg-muco.i -coated electrode: solid rhombi (♦) magenta voltammogram). Alg-muco.os-coated electrode and alg-muco.i-coated electrode showed an increase in cyclic voltammograms with similar voltammogram shapes and anodic and cathodic peak currents [pVaiue > 0.4, non-significant: (Alg-muco.os-coated electrode anodic peak: 0.58±0.33pA, cathodic peak: -0.280±0.1 lOpA; alg-muco.i- coated electrode anodic peak: 0.58±0.17pA, cathodic peak: -0.088±0.009pA).

[0068] The voltammograms recorded in the 1st, 5th, and 10thcycles for the Alg-muco.os- coated electrodes are shown in Figure 5B, with solid red line corresponding to 1stcycle, solid triangles (A) yellow line to 5thcycle, and solid rectangles (■) magenta line to 10thcycle. A decrease in the anodic and cathodic peak currents was observed as the experiment progressed, with the anodic peak dropping from 0.58±0.33pA in first cycle to 0.44±0.23pA in last cycle, and the cathodic peak changing from -0.280±0.110pA in first cycle to -0.260±0.121pA in last cycle. A similar trend was observed for the alg- muco.i-coated electrode, with the anodic peak decreasing from 0.58±0.17pA in first cycle to 0.40±0.12pA in last cycle, and the cathodic peak changing from -0.088±0.009pA in first cycle to -0.208±0.105pA in last cycle (results not shown). These results attest to an electrochemical non-reversibility of the process for highly loaded mucin coatings.

[0069] To assess the influence of mucin peptides on the oxidation and reduction of the Hexachloroiridate(III) / Hexachloroiridate(IV) redox mediator, the charges collected by the electrode during an anodic sweep and a cathodic sweep over a single voltametric cycle were calculated. That is, a voltammogram was divided into two I versus E curves: positive current (anodic) and negative current (cathodic) curves, as shown in Figure 5C, and the areas under the anodic curve (green, above zero) and cathodic curve (pink, below zero)were each determined by calculating the integral Q =scanrateJ IdV as explained above, to give the oxidation and reduction charges, respectively. The results are shown in Figures 5D and 5E, as calculated oxidation charge versus voltametric cycle number (Figure 5D) and calculated reductive charge versus voltametric cycle number (Figure 5E) for each of the four types of electrodes (alg, alg-muco.oi, alg-muco.os and alg-muco.i).

[0070] Starting with Figure 5D, the results show the effect of the concentration of mucin incorporated into the alginate film deposited on the electrode, on the oxidation charge (Q oxidation) collected by the electrode during the anodic sweep. The electrodes coated with high mucin-loaded films, namely alg-muco.os and alg-muco.i, showed increased oxidation charge compared to the mucin free and the low loading mucin films. Data is tabulated in Table 1 below:Table 1Pvaiue [alg vs. alg-muco.O5]=O.O8-O. 15, pvaiUe [alg vs. alg-muco.i]=O.O4-O.O9)

[0071] Another trend seen in the data plotted in Figure 5D is that the anodic charge gradually decreases with increasing number of voltametric cycles, for the alg-muco.os and alg-muco.i -coated electrodes, namely, the concentrated mucin coatings. The trend can be explained by a decrease in the number of reduced dithiol groups in the protein cysteine gates, leading to a decrease in reduction of the oxidized form of [IrCE]2-(the tetravalent iridium) and lower quantity of charge transferred to the working electrode.

[0072] Turning now to Figure 5E, the electrodes coated with high mucin-loaded films, namely alg-muco.os and alg-muco.i, delivered higher amount of charge during the cathodic sweep as compared to the mucin free and the low loading mucin films (alg and alg- muco.oi). The data extracted from Figure 5E for the first and tenth voltametric cycles is tabulated in Table 2 below:Table 2[alg vs. muco.i] > 0.4Example 3 - Mucin redox behavior is controlled by redox mediators and electrochemistry

[0073] The behavior of two types of electrodes was studied, alg and alg-muco.os-coated gold electrode, using the three-electrode cyclic voltammetry set-up described above. Each type of electrode was tested in triplicate. Each experiment consisted of two stages, labeled Phase I and Phase II, respectively, which were performed in a cyclic manner:

[0074] Phase I: the potential of the working electrode was varied over the range between 0.1V and 0.8V vs. Ag / AgCl at a scan rate of 0.1 V / s, over ten cycles (1-10), in 200 pM solution of the strong oxidant K^IrCle (tetrapositive iridium). Phase II: the tested electrode was dipped (1 min) in 50pM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) bondbreaker solution for reduction of mucin’s disulfide bonds. Next, Phase I was repeated, recording additional set of ten voltammograms for cycles 11-20, followed by the treatment of the working electrode with the strong reductant according to Phase II. Lastly, Phase I was conducted once again, cycling the potential of the working electrode over ten cycles 21-30.

[0075] For each voltammogram recorded, the charge received by the anode (oxidationcharge) was determined by calculating the integral Q =scanrateJ IdV over the anodic curve. The results calculated for the alg-muco.os -coated gold electrode and for the alg- coated gold electrode are shown in Figures 6 A and 6B. Figure 6 A summarizes the measurements performed on mucin-coated electrode, and Figure 6B on alginate-only electrode. In the appended graphs, the abscissa indicates the number of cycles, and the ordinate is the calculated oxidation charge. The vertical lines positioned after the tenth and twentieth cycles mark the intervention with the strong reductant.

[0076] The maj or results extracted from the voltammograms recorded for the alg-muco.os- coated gold working electrode are tabulated in Table 3.Table 3

[0077] Akin to the previous set of experiments reported in Example 2, a steady decrease in the charge collected by the alg-muco.os-coated gold electrode was observed over the ten successive voltammetry cycles 1 to 10 (1.738±0.283pC — 1.217±0.080pC). But the treatment of the working electrode in the TCEP reductant solution leads to a sharp increase in the oxidation charge collected during the anodic sweep in the subsequent (11thcycle) voltammetry cycle (1.217±0.080pC — 3.975±0.551pC). The reason is that the immersion of the electrode in the TCEP reductant solution, which takes place after the 10thcycle, converts the mucin, "saturated" with the oxidized disulfide form (R-S-S-R), to the free thiol reduced form (RSH, RSH), which is amenable to oxidation by tetrapositive iridium. In the next set of voltammetry cycles, the oxidation charge drops gradually, down to 1.614±0.372pC in the 20thvoltammetry cycle.

[0078] The immersion of the electrode in the TCEP reductant solution for the second time, which takes place after the 20thcycle, has the same effect discussed above: conversion of the mucin, "saturated" with the oxidized disulfide form (R-S-S-R), to thefree thiol reduced form (RSH, RSH), as shown by high oxidation charge determined in the 21stvoltammetry cycle.

[0079] Turning now to Figure 6B, a different pattern of the cyclic voltammogram and oxidative charge is observed, suggesting that TCEP is reacting differently on alg coating, underscoring its effect on mucin-containing coating applied on electrodes.

[0080] In addition thereto, fluorescent thiol detection was performed on a hydrogel with a protective coating to assess the oxidation and reduction of mucin sulfur switches. Thiolspecific fluorescent test was performed on the coated hydrogels. Four testing groups — alginate, alginate-mucin, oxidized alginate-mucin, and reduced alginate-mucin — were evaluated, with each group undergoing eight repetitions. The hydrogels were removed from the electrode and transferred into a buffer solution using a scalpel knife, followed by stirring for two minutes. A thiol detection assay kit (Item 700340, Cayman Chemicals) was utilized to measure the fluorescence signal from the test solutions. Fluorescence readings were obtained using a 96-well plate and a plate reader (Infinite M Plex 200Pro, Tecan; excitation - 380 nm, emission - 510 nm). Additionally, two thiol standard solutions, Glutathione and Cysteine, were analyzed alongside the test solutions, producing clear linear model fitting curves. Briefly, gels containing mucin showed a higher number of thiol groups than with the alginate gels. Oxidation of alg-muc hydrogels (10 voltametric cycles with Ir) resulted in a lower number of thiol groups, whereas reduction of the coatings (Imin TCEP treatment) resulted in a higher number of thiol groups compared with the default alg-muc coating.

[0081] The results are shown in Figure 6C, wherein box and whiskers plot of fluorescence intensity is demonstrated for pure alginate electrode, alg-muc electrode as obtained, oxidized alg-muc electrode (designated as “Ox Mucin”), and reduced alg-muc electrode (designated as “Red Mucin”). Statistical significance designated as “***” is derived by p value being smaller then 0.001, after one-way ANOVA and Tukey HSD post-hoc analysis.

[0082] The results demonstrated a significantly elevated fluorescent signal in gels containing mucin protein compared to those with an alginate coating. This outcome indicates the presence of thiol groups in mucin-containing gels. Upon testing the fluorescence signal in alg-muc gel after oxidation (10 voltametric cycles with iri mediator), a notably lower signal was observed compared to the regularly deposited alg- muc coating, suggesting a reduced number of thiol functional groups due to their oxidation to disulfides. Conversely, when testing the fluorescence signal in alg-muc gel after reduction (1 -minute TCEP treatment), a significantly higher signal was obtained compared to the regularly deposited alg-muc coating, indicating an increased number of thiol functional groups due to mucin reduction and the opening of sulfur gates.Example 4 - Effects of bacteria on mucin redox behavior and electrochemistry

[0083] E. coll was used as a model bacterium to evaluate if microbiota affect the oxidative state of the mucin. The alg-muc hydrogel was electrodeposited on the electrodes as in Examples above and the mucin state was change to oxidized and reduced using redox mediators.

[0084] To allow following the procedure, fluorescent E. coli ((AlacU169, ASoxRS901) were used. Prior to the experiment, the culture was maintained at glycerol with 1% v / v ampicillin. At the day of the experiment, the cells were grown in 1.5% LB Miller solution overnight, following by growing in 1% LB Miller solution up until OD600 = 0.2 . At the day of experiment bacteria seeded at OD600 = 0.2 in LB Miller medium on the hydrogels. After 10 hours of incubation of the bacteria on the coating, 10 voltammogram cycles using Ir as the redox mediator were performed and the anodic charge was calculated as in Example 3.

[0085] The results are shown in Figures 7A-7C, wherein charge in micro-Coulombs is plotted along the ordinate axis, versus the cycles plotted along the abscissa axis (designated as “Cycle[#]”). Similar anodic charge was observed for the groups with and without E. coli in alginate (designated as “alg cells” and “alg LB”, Figure 7A), suggesting that the cells do not interact with the Ir analyte and do not change the redoxstate of the alginate hydrogel, whereas in the oxidative mucin group, an increase in anodic charge in presence of E. coli was observed for all the cycles with a similar decaying factor (designated as “mucOx cells” and “mucOx LB”, Figure 7B), suggesting a reductive effect of the E. coli bacteria on the oxidized mucin, leading to higher availability of thiol groups in the hydrogel. In the reductive mucin group, a decrease in anodic charge in presence of E. coli was observed for the first 5 cycles, and convergence to a similar anodic charge afterwards (designated as “mucRed cells” and “mucRed LB”, Figure 7C), suggesting a mild oxidative effect of the E. coli bacteria on the reduced mucin, leading to lower availability of thiol groups in the hydrogel.

[0086] The major results extracted from the voltammograms recorded for the alg-muco.os- coated gold working electrode are tabulated in Table 6 below.

[0087] A total antioxidant capacity (TAC) and total oxidative stress (TOS) measurements were performed as follows Electrochemical treatment (oxidation / reduction) of alg-muc hydrogel and 10 h seeding ofE.coli was performed prior to the test. Then, the hydrogels were removed from the electrode and transferred into a buffer solution using a scalpel knife, followed by stirring for two minutes. TAC and TOS tests were performed using T- AOC and TOS assay kits and using kits manual (Assay Genie, CAS: MAES0147 and MAES0199, respectively). The results are shown in Figure 7D. In the Figure, TAC values are charted at left ordinate axis and TOS responses are charted along the right ordinate axis, with the groups of bars referring to the tested conditions, with leftwards bars indicate TAC, and rightwards bars indicate TOS, and the groups represent reduced or oxidized native or bacteria-coated electrodes, designated as “Red LB”, “Red Bac”, “Ox LB”, and “Ox bac”. The presence of bacteria in the oxidative mucin group significantly increased (one-way Anova, p < 0.05) the antioxidant species presence in the hydrogel, strengthening the electrochemical results and showing antioxidative behavior of the E. coli culture. In addition, the results show a decrease of oxidative species when E. coli are present, for both oxidative and reductive mucin coatings.

[0088] The major results are tabulated in Table 4, wherein TAC / TOS measurements of hydrogel w / wo mucin after E. coli seeding are displayed.Table 4.

[0089] The distribution of the E. coli on the coating was followed using confocal microscopy both in xy and in z axes. From a plan view a uniform distribution of bacteria was observed on alginate hydrogel, a clustered distribution on the oxidized mucin, and a uniform yet thinner distribution with empty areas in the reduced mucin group. To test the distribution uniformity, the fluorescent intensity from cropped images 20x20 pixels was averaged and displayed the intensity of each cropped image, as well as their distribution in form of a histogram, and qq-plot test to test for normality of intensity distribution. In line with the microscopic observations, the intensity was distributed uniformly for the alginate group, clustered to lone areas of high intensity for the oxidized mucin group, and uniformly distributed at a lower-intensity range with a significant number of frames presented with zero intensity, for the reduced mucin group. The z-axis distribution revealed a high penetration of cells through the hydrogel for alginate with bimodal distribution (highest density 160 pm deep), almost no penetration through oxidized mucin (highest density 30 pm deep), and uniform penetration distribution (highest density 80- 100 pm) through reduced mucin. The results are in good concordance with the present disclosure. It is believed without being bound by any particular theory that alginate gel, being porous, allows the bacteria to distribute uniformly in the gel and penetrate the coating. On the other hand, oxidized mucin does not allow bacteria to penetrate, while the reduced mucin with less crosslinks is less dense and allows bacteria to distribute and penetrate to higher extent.

[0090] The effect of bacteria on the mucin redox state was tested state under simulated oxidative stress condition (hydrogen peroxide 100 pM). Here, electrodes were coated andprocessed as described above, i.e., oxidized mucin, reduced mucin, and the effect of E. coll culture on the redox state of the coating with and without presence of hydrogen peroxide was tested. Specifically, additional group was tested in presence of E.coli under oxidative stress conditions (physiological hydrogen peroxide conditions. All other aspects of the experiment were executed as mentioned above.

[0091] The results are presented in Figures 8A-8B, wherein charge in micro-Coulombs is plotted along the ordinate axis, versus the cycles plotted along the abscissa axis (designated as “Cycle[#]”). Increased anodic charge was observed for oxidized mucin group exposed to hydrogen peroxide in comparison to oxidized mucin group where bacteria were not in oxidative stress conditions (designated as “mucOx cells” and “mucOxHP cells”, Figure 8A). No significant difference was measured in oxidative charge in the reduced mucin group for bacteria with or without hydrogen peroxide (designated as “mucRed cells” and “mucRedHP cells”, Figure 8B). Without being bound by a particular theory it is currently believed that this may be due the bacteria releasing reductive enzymes to maintain oxidative homeostasis, thus reducing the system in a more efficient way when exposed to oxidative stress in a form of hydrogen peroxide elevated concentrations. The major results extracted from the voltammograms recorded for the three working electrode from each group are tabulated in Table 5.Table 5Example 5 - Effects of bacteria in humanized murine microbiome on mucin oxidative state

[0092] To evaluate the effect of humanized microbiome on the system, alg-muc hydrogels were electrodeposited on the electrodes as described in Example 1 above. Humanized murine microbiome was obtained from healthy humanized mice’s gut.Briefly, humanized mice faeces were stirred in PBS 0.15X solution, following by filtration (200 nm) for 2h. Then, the sediment was soaked in PBS 0.15X solution and stirred again, following by centrifugation. The sediment from the centrifuged tube was stirred in a fresh PBS solution.

[0093] At the day of experiment the extracted microbiome was seeded at OD600 = 0.2 in PBS medium on the hydrogels. After 2.5 hours of incubation of the microbiota on the coating, 10 voltammogram cycles using Ir as the redox mediator were performed and the anodic charge was calculated as in Example 3.

[0094] The results are presented in Figures 9A and 9B, wherein output charge in microCoulombs (designated “[pC]”) represents the oxidative charge measured by mucin coated electrodes, from IrCk, cyclic voltamogram [0.4 - 0.85V vs Ag / AgCl] after 2.5 h of incubation, plotted along the ordinate axis, versus the cycles plotted along the abscissa axis (designated as “Cycle[#]”). The major results extracted from the voltammograms recorded for the 3 working electrode in each group are tabulated in Table 6.

[0095] The presence of the microbiome on a reduced mucin decreased the oxidative charge of the electrodes in comparison to the group w / o microbiome (designated as “Muc / Red / Bac” and “Muc / Red / PBS”, Figure 9A). On the other hand, the presence of the microbiome on the oxidized mucin did not significantly affect the oxidative charge of the electrodes in comparison to the group w / o microbiome (designated as “Muc / Ox / Bac” and “Muc / Ox / PBS”, Figure 9B). These results demonstrate that physiological microbiome is oxidizing the reduced mucin, which is in contrary to the effect of E. coli (vide supra, a reducer of oxidized mucin), which may be employed to detect pathological bacterial infection or overgrowth in a variety of inflammatory bowel diseases. The ratio of oxidative charge for bacteria on mucin and on electrochemically non-active alginate is presented in Figure 9C, wherein emphasized the significant oxidation of reduced mucin in the first 5 voltammetric cycles, while the oxidized mucin shows no significant redox effect. The markers represent the mean oxidative charge and caps represent the standard deviation of 3 working electrodes.Example 6 - modelling of mucin oxidation charge decay

[0096] The data obtained above from the bioelectronic system was fitted to a first-order rate function to model mucin redox mediation. The anodic charge dependency was described by the equation:(1) dQ / dt = -kQ

[0097] By solving this equation with the constraints of the Ir basal anodic charge (*Q_inf*) and a coefficient (*A*) representing the initial additional charge contributed by mucin’s cysteine (Cys) groups, the following relationship for anodic charge was derived:(2) Q_cyc = A * eA(-k*cyc) + Q_infHere, *Q_cyc* denotes the anodic charge in each cycle, while *k* represents the decay constant. Table 6 provides details on the parameters and experimental conditions, including mucin presence, its redox state, and the influence of the physiological microbiome and / or E. coli. The results are present in Table 6 below, wherein first-order Rate Model Parameters of Anodic Charge Transfer in Mucin-Bacteria Biostructure are presented.Table 6.

Claims

CLAIMS1) An electrode coated with mucin-incorporated hydrogel film.2) An electrode according to claim 1, wherein the hydrogel comprises alginate.3) A process for preparing a mucin-coated electrode, comprising fabricating hydrogel film from mucin-containing gelator solution, on a surface of an electrode.4) A process according to claim 3, wherein the hydrogel is electrodeposited on the electrode from a deposition solution comprising from 1-1.8% water-soluble gelator and from 0.05 to 0.15 mg / ml mucin.5) A process according to 4, wherein the deposition solution comprises, as the water- soluble gelator, a monovalent alginate salt, and further comprises a carbonate of an alkaline earth metal.6) A process according to claim 5, further comprising postdeposition addition of alkaline earth metal ions to crosslink and stabilize the alginate.7) A microfabricated electrochemical sensor, comprising: a base substrate; optionally an adhesion layer applied on the base substrate; and at least one working microelectrode deposited on the base substrate or on the adhesion layer, wherein the working microelectrode is coated with mucin-incorporated hydrogel film.8) A microfabricated electrochemical sensor according to claim 7, wherein the base substrate is made of glass or silicon / silicon oxide, with a titanium adhesion layer applied on the base substrate, and at least one disc-shaped working microelectrode deposited on the adhesion layer, wherein said disc-shaped working microelectrode is coated with mucin-incorporated alginate film and is encircled by a cylindrical wall defining a chamber.9) A microfabricated electrochemical sensor according to claim 8, wherein the cylindrical wall encircling the coated microelectrode is made of epoxy polymer.10) A microfabricated electrochemical sensor according to any one of claims 7 to 9, further comprising a counter electrode and Ag / AgCl reference electrodes deposited on the base substrate.11) An electrochemical method of characterizing the redox properties of mucin with the aid of a redox mediator, comprising recording an electrochemical signal at a working electrode coated with mucin-incorporated hydrogel film, and measuring the charge delivered to the electrode by the redox mediator to determine the reduced / oxidized state of the mucin.12) An electrochemical method of assessing permeability of the mucus to a redox-active compound of interest, comprising recording in a solution of said redox-active compound an electrochemical signal at a working electrode coated with mucin-incorporated hydrogel film, wherein passage of said redox active molecule across the mucus is indicated by one or more features of the electrochemical signal, such as intensity, position, and trend of recurrence.13) A method of diagnosing of an inflammatory process in the bowel in a subject, comprising obtaining a sample of bowel contents from said subject, incubating said sample on a working detection electrode coated with mucin-incorporated hydrogel film, incubating a blank solution to said sample on a reference working electrode identical to said working electrode, recording an electrochemical signal obtained at said detection electrode and at said identical reference electrode, comparing said recorded signals with standard signals obtained with a confirmed pathological sample or a confirmed healthy sample, and sorting said sample as normal, pathologic, or borderline based on said comparing.14) The method according to claim 13, wherein said process is characterized by a microbiome shift or a dysbacteriosis.15) The method according to any one of the claims 13 or 14, wherein said recording comprises cyclic voltammetry measurements.16) The method according to any one of the claims 13 to 15, wherein said comparing comprising obtaining ratios between the signal obtained at said detection electrode and at said identical reference electrodes.17) The method according to any one of the claims 13 to 16, wherein said sorting comprises comparing the ratios obtained between the signal obtained at said detection electrode and at said identical reference electrodes, with the ratios obtained from said standard signals, wherein said recording comprises cyclic voltammetry measurements, and said comparing further comprises evaluating the cyclic voltammetry signals at at least two cycles separated by at least 30% of the total number of cycles.18) The method according to any one of the claims 13 to 17, wherein said recording comprises cyclic voltammetry measurements, wherein said sorting comprises a log-linear regression of the signals obtained at said detection electrode and at said identical reference electrodes, wherein said log-linear regression is in form of Q_cyc = A * eA(-k*cyc) + Q inf and wherein Q cyc is anodic charge in each cycle, k is the decay constant, eye is the cycle number, Q inf is the constraint of basal anodic charge at the tested conditions, and A is the coefficient representing the initial additional charge contributed by said mucin-incorporated hydrogel film, and wherein said sorting further comprises comparing A and k parameters obtained parameters obtained with said standard signals.

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