Solid state reference electrodes and methods of producing thereof

Abstract

The invention provides a membrane for a solid-state reference electrode, the membrane formed from a cross-linked zwitterionic polymeric matrix loaded with an inorganic salt, the inorganic salt being dispersed in the cross-linked zwitterionic polymer matrix. The invention also provides a solid-state reference electrode comprising a reference electrode with the membrane disposed on a surface of the reference electrode and a method of producing thereof. In addition, the invention provides a potentiometric sensor comprising the solid-state reference electrode and an ion-selective electrode.

Description

[0001] SOLID STATE REFERENCE ELECTRODES AND METHODS OF PRODUCING THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention provides solid-state reference electrodes suitable for potentiometric applications, more particularly, solid-state reference electrodes comprising a reference electrode and a membrane disposed on a surface of the reference electrode, the membrane formed from a cross-linked zwitterionic polymeric matrix loaded with an inorganic salt.

[0004] BACKGROUND

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Electrochemical sensors are essential tools used in a wide span of applications such as environmental monitoring, healthcare, and industrial process control. Particularly, potentiometry is a commonly used electrochemical technique for ion-sensing applications. In potentiometry, the potential difference is measured across the reference electrode and an ion- selective electrode (ISE). (FIG. 1) For certain ISEs, the ISE’s selective response to a specific ion is generally attributed to the ionophores present in the ion-selective membrane of the ISE. When the target ion binds to the ionophore in the membrane, it induces a potential difference proportional to its concentration, enabling ion quantification. On the other hand, the reference electrode serves a crucial role by maintaining a stable electrochemical potential and remains relatively unaffected to external ion concentration changes during sampling. This stability is essential to ensure that the potential generated by the ISE accurately reflects the concentration of the ion of interest. The absolute potential generated in the reference electrode and the ISE is governed by the Nernstian equation. (FIG. 2). For example, a monoanionic ISE with ideal behaviour exhibits a Nernstian slope of -59 mV / decade. In contrast, an ideal reference electrode should remain unaffected by changes in sampling concentration, exhibiting a slope close to 0 mV / decade.

[0007] The double junction silver-silver chloride (Ag / AgCI) electrode is one of the most frequently used reference electrodes for electrochemical measurements. The double junction Ag / AgCI reference electrode is typically constructed with a silver wire coated with silver chloride paste, and is immersed in a saturated 3M potassium chloride (KCI) solution (FIG. 4). The operating principle of the reference electrode is based on two simultaneous, reversible reactions on the surface of the electrode which is in contact with the solution (FIG. 3).The saturated KCI serves as a reference electrolyte, helping to maintaining a constant potential. The saturated KCI solution is prevented from contamination by the testing solution through the use of an outer salt bridge electrolyte such as 1 M lithium acetate solution (LiOAc). KCI and LiOAc are allowed to move across the boundaries through a porous frit. While the double junction Ag / AgCI reference electrode is commonly used, there are some problems associated with this type of reference electrode: (1 ) leaching of reference electrolyte and salt bridge electrolyte (2) contamination of salt bridge during sampling (3) frequent maintenance of reference electrode and electrolyte replacement (4) large form factor which make integration into small devices difficult. Hence, this prompted the development of solid-state reference electrodes as a solution to eliminate the use of liquid reference electrolyte and allowing the miniaturization of reference electrodes.

[0008] In recent years, various strategies have been explored to overcome the problems associated with the use of traditional liquid junction reference electrodes. Various methods of encapsulation have been explored, such as the use of thermal and photocurable polyacrylate membranes used for the encapsulation of the KCI particles (Rius-Ruiz, F. X.; Kisiel, A.; Michalska, A.; Maksymiuk, K.; Riu, J.; Rius, F. X. Solid-State Reference Electrodes Based on Carbon Nanotubes and Polyacrylate Membranes. Anal. Bioanal. Chem. 2011 , 399 (10), 3613- 3622). In another strategy, an equitransferent salt, tetrabutylammonium tetrabutylborate (TBA- TBB), was used in place of KCI to allow more effective reference electrolyte retention since these salts are usually bulkier and more lipophilic (Anastasova- Ivanova, S.; Mattinen, U.; Radu, A.; Bobacka, J.; Lewenstam, A.; Migdalski, J.; Danielewski, M.; Diamond, D. Development of Miniature All-Solid-State Potentiometric Sensing System. Sensors Actuators B Chem. 2010, 146 (1 ), 199-205). While these strategies showed promise in improving drift and lifetimes of the solid-state reference electrodes, these solid-state reference electrodes suffered from long conditioning times and sensitivity to light and redox reactions due to use of transduction layer for effective electron transport. Similarly, ionic liquids, instead of KCI, were used in conjunction with PVC-based reference membrane to suppress drift due to leaching of reference electrolyte (Lindner, E.; Guzinski, M.; Khan, T. A.; Pendley, B. D. Reference Electrodes with Ionic Liquid Salt Bridge: When Will These Innovative Novel Reference Electrodes Gain Broad Acceptance? ACS Sensors 2019, 4 (3), 549-561 ; Chopade, S. A.; Anderson, E. L.; Schmidt, P. W.; Lodge, T. P.; Hillmyer, M. A.; Buhlmann, P. Self-Supporting, Hydrophobic, Ionic Liquid- Based Reference Electrodes Prepared by Polymerization-Induced Microphase Separation. ACS Sensors 2017, 2 (10), 1498-1504). However, its stability was affected by the presence of surfactants and more hydrophobic ions. Guinovart et al. (Guinovart, T.; Crespo, G. A.; Rius, F. X.; Andrade, F. J. A Reference Electrode Based on Polyvinyl Butyral (PVB) Polymer for Decentralized Chemical Measurements. Anal. Chim. Acta 2014, 821, 72-80), developed a polybutyral-based reference membrane in attempt to develop a solid-state reference membrane with a shorter conditioning time, reducing the conditioning time from days to only 12 hours. One major drawback of the above-mentioned strategies is the construction on reference membranes on rod-shaped glassy carbon or gold electrodes which makes them difficult to be integrated into devices.

[0009] The development of wearable electronics has sparked tremendous interest in screen-printed electrodes due to their potential as low-cost, flexible, and robust options for in-situ sensing applications. In the works of Shitanda and co-workers, an aqueous KCI-PDMS reference electrolyte layer was used in conjunction with a small liquid junction layer (1 mm in width) (Shitanda, I.; Kiryu, H.; Itagaki, M. Improvement in the Long-Term Stability of Screen-Printed Planar Type Solid-State Ag / AgCI Reference Electrode by Introducing Poly(Dimethylsiloxane) Liquid Junction. Electrochim. Acta 2011 , 58, 528-531 ). The narrow liquid junction allows for ion exchange with external test solution while effectively reducing the exposure and leaching of the electrolyte into the test solution. While effective, these screen-printed electrodes were tailored to specific application and lack broader usability. More recently, Bananezhad and coworkers developed a large-scale fabrication of flexible solid-state reference electrodes, by using UV curable acrylate ink, KCI and tetradodecylammonium tetrakis(4-chlorophenyl)borate lipophilic salt in the reference membranes (Bananezhad, A.; Jovic, M.; Villalobos, L. F.; Agrawal, K. V.; Ganjali, M. R.; Girault, H. H. Large-Scale Fabrication of Flexible Solid-State Reference Electrodes. J. Electroanal. Chem. 2019, 847, 113241 ). While the performance of these flexible solid-state reference electrodes seems promising, the electrodes have a short shelf life of 4 weeks when stored dry, and their lifespans remain a challenge to be resolved.

[0010] Thus, there is a need for alternative and / or improved solid-state reference electrodes suitable for potentiometric applications and methods of preparing such solid-state reference electrodes

[0011] SUMMARY

[0012] Aspects and embodiments of the current invention will now be described by reference to the following numbered clauses.

[0013] 1 . A membrane for a solid-state reference electrode, the membrane formed from a crosslinked zwitterionic polymeric matrix loaded with an inorganic salt, the inorganic salt being dispersed in the cross-linked zwitterionic polymer matrix.

[0014] 2. The membrane of clause 1 , wherein the cross-linked zwitterionic polymeric matrix is formed from a zwitterionic acrylate copolymer and a crosslinking agent. 3. The membrane of clause 2, wherein the zwitterionic acrylate copolymer comprises constitutional units containing a quaternary ammonium group and a sulfonate group, and / or comprises constitutional units containing a phosphorylcholine group.

[0015] 4. The membrane of clause 2 or 3, wherein the zwitterionic acrylate copolymer is a copolymer of:

[0016] (a) one or more zwitterionic monomers according to formula (I): wherein,

[0017] R1is H or methyl,

[0018] R2and R3are each independently Ci^alkylene; and

[0019] A is NH, O, or S, for example, NH or O;

[0020] X is -N+(Ra)2- and Y is -SO3-, or X is -O-P(CT)(O)-O- and Y is -N+(Ra)3; and each Rais independently Ci-i2alkyl; and

[0021] (b) one or more of additional monomers selected from the group consisting of C1-20alkyl acrylate, C1-20alkyl methacrylate, and methacrylate, optionally wherein the C1-20alkyl acrylate and Ci20alkyl methacrylate are substituted with one or more substituents selected from halo, C1-20 alkoxy and Het, wherein halo represents F, Cl, Br or I and Het represents a 4- to 10- membered heterocyclic groups containing one or more heteroatoms selected from O, S and N.

[0022] 5. The membrane of clause 4, wherein each Rais methyl.

[0023] 6. The membrane of clause 4 or 5, wherein R2is C^alkylene and R3is Ci ^alkylene.

[0024] 7. The membrane of clause 4 or 5, wherein R2is C^alkylene and R3is Ci^alkylene. 8. The membrane of clause 7, wherein the zwitterionic monomer according to formula (I) is selected from the group consisting of:

[0025] [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1 -sulfonate, [2-(acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [3-(methacryloylamino)propyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1 -sulfonate, 3-[(3-acrylamidopropyl)dimethylammonio]propane-1 -sulfonate, 2-methacryloyloxyethyl phosphorylcholine, and a combination of two or more thereof.

[0026] 9. The membrane of any one of clauses 4 to 8, wherein the one or more of additional monomers are selected from the group consisting of n-butyl acrylate, s-butyl acrylate, n-hexyl acrylate, n-octyl acrylate, n-dodecyl acrylate, 2-ethylhexyl acrylate, 2,2,2-trifluoroethyl acrylate, 2-methoxyethyl acrylate, tetrahydrofurfuryl acrylate, methyl methacrylate, n-hexyl methacrylate, n-octyl methacrylate, n-dodecyl methacrylate, octadecyl methacrylate and methyl methacrylate, for example the one or more additional monomers are n-butyl acrylate and methyl methacrylate.

[0027] 10. The membrane of any one of clauses 4 to 9, wherein the monomer according to formula (I) is 2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, and the one or more of additional monomers are n-butyl acrylate and methyl methacrylate.

[0028] 11 . The membrane of any one of clauses 2 to 10, wherein the crosslinking agent comprises two or more groups selected from the groups consisting of aryl azide, azidoformate, a- diazoester, a-diazoamide, and diazirine, for example the crosslinking agent may comprise two or more aryl azide groups.

[0029] 12. The membrane of clause 11 , wherein the crosslinking agent comprises two or more perfluorophenyl azide groups.

[0030] 13. The membrane of clause 12, wherein the crosslinking agent is selected from the group consisting of: and a combination of two or more thereof.

[0031] 14. The membrane of any one of clauses 2 to 13, wherein the cross-linked zwitterionic polymeric matrix is formed by reacting the zwitterionic acrylate copolymer with from about 1 wt% to about 10 wt% of the crosslinking agent based on the combined mass of the crosslinking agent and zwitterionic acrylate copolymer, for example the cross-linked zwitterionic polymeric matrix may be formed by reacting the zwitterionic acrylate copolymer with from about 1 wt% to about 5 wt% (e.g. about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt% or about 5 wt%) of the crosslinking agent based on the combined mass of the crosslinking agent and zwitterionic acrylate copolymer.

[0032] 15. The membrane of any one of clauses 2 to 14, wherein the zwitterionic acrylate copolymer is formed by the polymerisation of the zwitterionic monomer and the one or more of additional monomers at a molar ratio of the zwitterionic monomer to the one or more of additional monomers of from about 1 :20 to about 1 :250, for example about 1 :90.

[0033] 16. The membrane of clause 15, wherein the one or more of additional monomers are n- butyl acrylate and methyl acrylate at a molar ratio of n-butyl acrylate to methyl methacrylate of from about 3:1 to 20:1 , for example about 7:1 .

[0034] 17. The membrane of any one of the preceding clauses, wherein the inorganic salt is provided in a particle size of less than or equal to about 45 pm, for example, less than or equal to about 30 pm or less than or equal to about 25 pm. 18. The membrane of any one of the preceding clauses, wherein the inorganic salt is a chloride salt.

[0035] 19. The membrane of any one of the preceding clauses, wherein the chloride salt is selected from the group consisting of potassium chloride, sodium chloride, lithium chloride, and a combination of two or more thereof, for example, the chloride salt is potassium chloride.

[0036] 20. The membrane of any one of the preceding clauses, wherein the membrane comprises from about 10 wt% to about 60 wt% of the inorganic salt based on the total mass of the membrane, for example the membrane may comprise from about 10 wt% to about 55 wt% (e.g. about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt%) of the inorganic salt based on the total mass of the membrane.

[0037] 21 . A solid-state reference electrode comprising a reference electrode with the membrane of any one of clauses 1 to 20 disposed on a surface of the reference electrode.

[0038] 22. The solid-state reference electrode of clause 21 , wherein the reference electrode is selected from the group consisting of a silver-silver chloride electrode, a calomel electrode, silver-silver sulfate electrode, mercury-mercury sulfate electrode, and copper-copper(ll) sulfate electrode.

[0039] 23. The solid-state reference electrode of clause 22, wherein the reference electrode is a silver-silver chloride electrode.

[0040] 24. The solid-state reference electrode of clause 23, wherein the silver-silver chloride electrode comprises a solid electron-conducting material coated with a layer of silversilver chloride.

[0041] 25. The solid-state reference electrode of clause 24, wherein the solid electron-conducting material is silver, gold, platinum, copper, steel, carbon (or carbon nanomaterials), metal oxide (e.g. indium-tin-oxide, titanium dioxide, fluorine-doped tin oxide) or aluminium.

[0042] 26. The solid-state reference electrode of clause 24 or 25, wherein the solid electronconducting material is disposed on a substrate, optionally wherein the substrate is a ceramic substrate or a polyimide substrate. 27. The solid-state reference electrode of any one of clauses 22 to 26, wherein the reference electrode is a screen-printed electrode, for example a screen-printed silver electrode.

[0043] 28. A method of producing the solid-state reference electrode of any one of clauses 21 to 27, the method comprising: i) preparing a mixture of the zwitterionic acrylate copolymer, the crosslinking agent, and the inorganic salt in an organic solvent by dissolving the zwitterionic acrylate copolymer and the crosslinking agent in the organic solvent, followed by adding the inorganic salt to the organic solvent; ii) applying the mixture from step i) to a surface of the reference electrode followed by evaporating the organic solvent; iii) subjecting the reference electrode to a temperature of at least 100 °C for a time period sufficient to form a cross-linked zwitterionic polymeric matrix of the surface from the zwitterionic acrylate copolymer and the crosslinking agent; iv) submersing the reference electrode following step iii) in an aqueous solution of the inorganic salt, thereby producing the solid-state reference electrode of any one of clauses 21 to 27.

[0044] 29. The method according to clause 28, wherein the zwitterionic acrylate copolymer used in step i) is prepared by a polymerisation reaction between (methacryloyloxy)ethyl]dimethyl- (3-sulfopropyl)ammonium hydroxide, n-butyl acrylate and methyl methacrylate.

[0045] 30. The method according to clause 28 or 29, wherein the inorganic salt is KCI.

[0046] 31 . The method according to any of clauses 28 to 30, wherein the reference electrode is a silver-silver chloride electrode.

[0047] 32. The method according to clause 31 , wherein the silver / silver chloride electrode comprises a screen-printed silver electrode coated with a layer of silver-silver chloride.

[0048] 33. The method according to any one of clauses 28 to 32, wherein the crosslinking agent is selected from the group consisting of: and a combination of two or more thereof.

[0049] 34. The method according to clause 33, wherein step iii) comprises subjecting the reference electrode to a temperature of about 140 °C for about 4 to 5 hours under vacuum, optionally wherein step iii) comprises subjecting the reference electrode to a temperature of about 140 °C for about 4 to 5 hours under vacuum at a pressure of from 100 Pa to 5,000 Pa, such as from 133 Pa to 5,000 Pa, such as from 2,000 Pa to 5,000 Pa, such as from 4,000 Pa to 5,000 Pa, such as from 100 Pa to about 4,000 Pa, such as from 100 Pa to 2,000 Pa, such as from 100 Pa to 133 Pa or such as from 133 Pa to 4,000 Pa.

[0050] 35. The method according to any one of clauses 28 to 34, wherein step iv) comprises submersing the reference electrode following step iii) in a 1 M KCI solution for about 48 hours.

[0051] 36. A solid-state electrode obtained by the method according to any one of clauses 28 to 35.

[0052] 37. A potentiometric sensor comprising the solid-state reference electrode of any one of clauses 21 to 27 or 36 and an ion-selective electrode.

[0053] 38. Use of the solid-state reference electrode of any one of clauses 21 to 27 or 36 in an electrochemical analysis. 39. The use according to clause 38 comprising determining a concentration of a solute in a solution using the solid-state reference electrode and an ion-selective electrode.

[0054] 40. The use according to clause 38 comprising determining a pH of a solution using the solid-state reference electrode and an ion-selective electrode.

[0055] BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1 is an illustration of a conventional potentiometric system.

[0057] FIG. 2 depicts the Nernst equation.

[0058] FIG. 3 depicts the Ag / AgCI equilibrium equation.

[0059] FIG. 4 is an illustration of a conventional double junction Ag / AgCI reference electrode.

[0060] FIG. 5 depicts the fabrication of a reference membrane material according to an embodiment of the present disclosure.

[0061] FIG. 6 depicts the chemical structures of the azide crosslinkers used according to an embodiment of the present disclosure.

[0062] FIG. 7 depicts the fabrication steps of solid-state reference electrode according to Example 1 of the present disclosure.

[0063] FIG. 8 depicts the electrode design of the discrete layers of the solid-state reference electrode, including the reference membrane material and the reference electrode, according to Example 1 of the present disclosure.

[0064] FIG. 9 depicts the comparison of NMR spectra of (a) zwitterionic copolymer, nBA monomer, MMA monomer and SBMA monomer; and (b) zwitterionic copolymer and nBA-MMA (10:1 ) polymer according to Example 1 of the present disclosure.

[0065] FIG. 10 depicts the results of the sensitivity and interference measurements of solid-state reference electrodes (a) R1 (b) R2 (c) R3 (d) R4 (e) R5 (f) R6 (g) R7 (h) S1 (I) A1 (j) P1 according to Example 2 of the present disclosure. FIG. 11 depicts the results of the pH study of solid-state reference electrodes (a) R2 and (b) R4 according to Example 3 of the present disclosure.

[0066] FIG. 12 depicts the results of the lifespan study of (a) R2 (b) R4 over 32 days at room temperature and (c) R2 (d) R4 over 18 days at 50 °C according to Example 4 of the present disclosure.

[0067] FIG. 13 includes pictures showing corrosion observed on the electrode connectors after heating for 18 days at 50 °C according to Example 4 of the present disclosure.

[0068] FIG. 14 depicts the comparison of calibration curves obtained using solid-state reference electrode and conventional liquid double junction electrode for (a) NOs' ISE and (b) K+ISE according to Example 5 of the present disclosure.

[0069] DESCRIPTION

[0070] The present inventors have surprisingly found that the use of zwitterionic polymers in a membrane of a solid-state reference electrode would enable the retention of the electrolyte (e.g., KCI) via ionic interactions (FIG. 5). Coupled with crosslinking with a suitable reagent (FIG. 6), the resultant mesh-like structure serves as a barrier to prevent the leaching of the electrolyte. The present solid-state reference electrodes possess advantages as compared to the conventional double junction Ag / AgCI reference electrode, such as (I) not requiring electrolyte replacement, (ii) enhanced durability and (iii) small form factor which allows integration into small devices, portability, and cost effectiveness. As demonstrated in the Examples section in the present disclosure, the present solid-state reference electrodes also display insensitivity / resistance towards interfering ions, making them useful for potentiometric applications such as measuring pH or a concentration of a solute in a solution.

[0071] Thus, in a first aspect of the invention, there is provided a membrane for a solid-state reference electrode, the membrane formed from a cross-linked zwitterionic polymeric matrix loaded with an inorganic salt, the inorganic salt being dispersed in the cross-linked zwitterionic polymer matrix.

[0072] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0073] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0074] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of two or more such compounds, reference to “a composition” includes mixtures of two or more such compositions, and the like.

[0075] As disclosed herein, the term “reference electrode” refers to any device in direct contact with the test solution that maintains a constant or predictable interfacial potential difference despite changing type and concentration of chemical species in the test electrolyte. As will be appreciated, the term “solid state” in respect of a reference electrode refers to a reference electrode that does not contains any liquid (e.g. any liquid filling solution).

[0076] As disclosed herein, the term “zwitterionic polymeric matrix” refers to one that is formed from at least one zwitterionic monomer, which contains both positive and negative charges in the same molecule. As will be appreciated, the zwitterionic polymeric matrix be formed as a copolymer, where an additional component that may be a neutral monomer may be used.

[0077] The present cross-linked zwitterionic polymeric matrix is loaded with an inorganic salt which serves as a solid electrolyte for the solid-state reference electrode. In other words, the inorganic salt (in the particulate form) is dispersed in the cross-linked zwitterionic polymer matrix. This is achieved by, for example, mixing the zwitterionic polymer, the crosslinking agent and the inorganic salt (in the particulate form) in a suitable organic solvent to form a dispersion and evaporating the organic solvent thereafter. As mentioned above, the inorganic salt is held by ionic interactions with the zwitterionic polymer and retained in the polymeric matrix further by crosslinking of the zwitterionic polymeric matrix. In certain embodiments, the cross-linked zwitterionic polymeric matrix may be formed from a zwitterionic acrylate copolymer and a crosslinking agent. In certain embodiments, the zwitterionic acrylate copolymer may comprise constitutional units containing a quaternary ammonium group and a sulfonate group. In other embodiments, the zwitterionic acrylate copolymer may comprise constitutional units containing a phosphorylcholine group. For the avoidance of doubt, the constitutional units referred to herein may be derived from a monomeric material having the same functional groups.

[0078] In certain embodiments, the zwitterionic acrylate copolymer may be a co-polymer of

[0079] (a) one or more zwitterionic monomers according to formula (I): wherein,

[0080] R1is H or methyl,

[0081] R2and R3are each independently C1-12alkylene; and

[0082] A is NH, O, or S, for example, NH or O;

[0083] X is -N+(Ra)2- and Y is -SO3- or X is -O-P(O-)(O)-O- and Y is -N+(Ra)3; and each Rais independently Ci i2alkyl; and

[0084] (b) one or more of additional monomers selected from the group consisting of C1-20 alkyl acrylate, C1.20 alkyl methacrylate, and methacrylate, optionally wherein the C1.20 alkyl acrylate and C1-20 alkyl methacrylate are substituted with one or more substituents selected from halo, C1.20 alkyoxy and Het, wherein halo represents F, Cl, Br or I and Het represents a 4- to 10-membered heterocyclic groups containing one or more heteroatoms selected from O, S and N (e.g. one or more of additional monomers selected from the group consisting of C1-12alkyl acrylate, C1-12 alkyl methacrylate, and methacrylate, optionally wherein the C1-12 alkyl acrylate and C1-12 alkyl methacrylate are substituted with one or more substituents selected from halo, C1.4 alkyoxy and Het, wherein halo represents F, Cl, Br or I and Het represents a 4- to 10-membered heterocyclic groups containing one or more heteroatoms selected from O, S and N, such as e.g. one or more of additional monomers selected from the group consisting of C1-6 alkyl acrylate, C1-6 alkyl methacrylate, and methacrylate, optionally wherein the C1-6 alkyl acrylate and Ci s alkyl methacrylate are substituted with one or more substituents selected from halo, C1-4 alkyoxy and Het, wherein halo represents F, Cl, Br or I and Het represents a 4- to 10-membered heterocyclic groups containing one or more heteroatoms selected from O, S and N).

[0085] As used herein, “alkyl” refers to an unbranched or branched, cyclic, saturated hydrocarbyl radical, which may be substituted or unsubstituted.

[0086] In certain embodiments, each Ramay be methyl. In certain embodiments, R2may be Ci- 4alkylene and R3may be Ci ^alkylene. In other embodiments, R2may be Ci 4alkylene and R3may be Ci-4alkylene. In further embodiments, the zwitterionic monomer according to formula (I) may be selected from the group consisting of:

[0087] [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1 -sulfonate, [2-(acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [3-(methacryloylamino)propyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1 -sulfonate, 3-[(3-acrylamidopropyl)dimethylammonio]propane-1 -sulfonate, 2-methacryloyloxyethyl phosphorylcholine, and a combination of two or more thereof.

[0088] In certain embodiments, the one or more of additional monomers may be selected from the group consisting of n-butyl acrylate, s-butyl acrylate, n-hexyl acrylate, n-octyl acrylate, n- dodecyl acrylate, 2-ethylhexyl acrylate, 2,2,2-trifluoroethyl acrylate, 2-methoxyethyl acrylate, tetrahydrofurfuryl acrylate, methyl methacrylate, n-hexyl methacrylate, n-octyl methacrylate, n-dodecyl methacrylate, octadecyl methacrylate and methyl methacrylate, for example the one or more additional monomers may be n-butyl acrylate and methyl methacrylate.

[0089] In certain exemplary embodiments, the monomer according to formula (I) may be [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide and the one or more of additional monomers may be n-butyl acrylate and methyl methacrylate.

[0090] According to the present invention, the zwitterionic polymeric matrix may be crosslinked by any suitable means. In certain embodiments, the zwitterionic polymeric matrix may be crosslinked via a suitable crosslinking agent. In certain embodiments, the crosslinking agent may comprise two or more groups consisting of aryl azide, azidoformate, a-diazoester, a- diazoamide, and diazirine. In certain embodiments, the crosslinking agent may comprise two or more perfluorophenyl azide groups. In more particular embodiments, the crosslinking agent may be selected from the group consisting of: and a combination of two or more thereof.

[0091] In certain embodiments, the cross-linked zwitterionic polymeric matrix may be formed by reacting the zwitterionic acrylate copolymer with from about 1 wt% to about 10 wt% of the crosslinking agent based on the combined mass of the crosslinking agent and zwitterionic acrylate copolymer, for example the cross-linked zwitterionic polymeric matrix may be formed by reacting the zwitterionic acrylate copolymer with from about 1 wt% to about 5 wt% (e.g. about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt% or about 5 wt%) of the crosslinking agent based on the combined mass of the crosslinking agent and zwitterionic acrylate copolymer.

[0092] When used herein, the term "about", in the context of particle size and molar ratios, may mean a variation of ± 5% of the stated value, more typically + / - 4% of the stated value, more typically ± 3% of the stated value, more typically, + / - 2% of the stated value, even more typically ± 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0093] In certain embodiments, the zwitterionic acrylate copolymer may be formed by the polymerisation of the zwitterionic monomer and the one or more of additional monomers at a molar ratio of the zwitterionic monomer to the one or more of additional monomers of from about 1 :20 to about 1 :250, for example about 1 :90. In certain embodiments, the one or more of additional monomers may be n-butyl acrylate and methyl acrylate at a molar ratio of n-butyl acrylate to methyl methacrylate of from about 3:1 to 20:1 , for example about 7:1 .

[0094] As mentioned above, the cross-linked zwitterionic polymeric matrix is loaded with a particulate inorganic salt, the particulate inorganic salt being dispersed in the cross-linked zwitterionic polymer matrix. In certain embodiments, the particulate inorganic salt may be provided in a particle size of less than or equal to about 45 pm, for example, less than or equal to about 30 pm or less than or equal to about 25 pm, as measured by a suitable method or device, such a particle size analyser.

[0095] As the skilled person will appreciate, the inorganic salt selected will be dependent on its application i.e., the reference electrode that the membrane is used for. For an Ag|AgCI reference electrode, the inorganic salt may be a chloride salt, for example, potassium chloride, sodium chloride or lithium chloride. In certain exemplary embodiments, the inorganic salt may be potassium chloride.

[0096] In certain embodiments, the membrane may comprise from about 10 wt% to about 60 wt% of the inorganic salt based on the total mass of the membrane, for example the membrane may comprise from about 10 wt% to about 55 wt% (e.g. about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt%) of the inorganic salt based on the total mass of the membrane.

[0097] In a second aspect of the invention, there is also provided a solid-state reference electrode comprising a reference electrode with the membrane as disclosed hereinbefore disposed on a surface of the reference electrode. As details of the membrane have already been described above, they are omitted here for brevity.

[0098] In certain embodiments, the reference electrode may be selected from the group consisting of a silver-silver chloride electrode, a calomel electrode, silver-silver sulfate electrode, mercurymercury sulfate electrode, and copper-copper(ll) sulfate electrode. In certain exemplary embodiments, the reference electrode may be a silver-silver chloride electrode.

[0099] In certain embodiments, the silver-silver chloride electrode may comprise a solid electronconducting material coated with a layer of silver-silver chloride. In certain embodiments, the solid electron-conducting material may be silver, gold, platinum, copper, steel, carbon (or carbon nanomaterials), metal oxide (e.g. indium-tin-oxide, titanium dioxide, fluorine-doped tin oxide) or aluminum.

[0100] In certain embodiments, the solid electron-conducting material may be disposed on a substrate. In further embodiments, the substrate may be a ceramic substrate or a polyimide substrate. In certain embodiments, the reference electrode may be a screen-printed electrode, for example a screen-printed silver electrode.

[0101] In a third aspect of the invention, there is provided a method of producing the solid-state reference electrode as disclosed hereinbefore, the method comprising: i) preparing a mixture of the zwitterionic acrylate copolymer, the crosslinking agent, and the inorganic salt in an organic solvent by dissolving the zwitterionic acrylate copolymer and the crosslinking agent in the organic solvent, followed by adding the inorganic salt to the organic solvent; ii) applying the mixture from step i) to a surface of the reference electrode followed by evaporating the organic solvent; iii) subjecting the reference electrode to a temperature of at least 100 °C for a time period sufficient to form a cross-linked zwitterionic polymeric matrix of the surface from the zwitterionic acrylate copolymer and the crosslinking agent; iv) submersing the reference electrode following step iii) in an aqueous solution of the inorganic salt, thereby producing the solid-state reference electrode as disclosed hereinbefore.

[0102] As details of the solid-state reference electrode have already been described above, they are omitted here for brevity. In certain embodiments, the zwitterionic acrylate copolymer used in step i) may be prepared by a polymerisation reaction between (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, n-butyl acrylate and methyl methacrylate, the inorganic salt may be KCI, the reference electrode may be a silversilver chloride electrode and the crosslinking agent may be selected from the group consisting of: and a combination of two or more thereof.

[0103] In certain embodiments, step iii) may comprise subjecting the reference electrode to a temperature of about 140 °C for about 4 to 5 hours under vacuum. In certain embodiments, step iii) may comprise subjecting the reference electrode to a temperature of about 140 °C for about 4 to 5 hours under vacuum at a pressure of from 100 Pa to 5,000 Pa, such as from 133 Pa to 5,000 Pa, such as from 2,000 Pa to 5,000 Pa, such as from 4,000 Pa to 5,000 Pa, such as from 100 Pa to about 4,000 Pa, such as from 100 Pa to 2,000 Pa, such as from 100 Pa to 133 Pa or such as from 133 Pa to 4,000 Pa.

[0104] In certain embodiments, step iv) may comprise submersing the reference electrode following step iii) in a 1 M KCI solution for about 48 hours.

[0105] In another aspect of the invention, there is provided a potentiometric sensor comprising the solid-state reference electrode as disclosed hereinbefore and an ion-selective electrode. The reference electrode according to the present disclosure finds particular utility in potentiometry. As the skilled person will appreciate, potentiometry is challenging since it is only a 2-electrode cell and all potentials are measured against the reference, so reference stability is extremely important for accuracy. In 3-electrode systems, especially in voltammetry, the potential applied is measured against the reference electrode, while the output voltage is measured against the counter electrode; in this case, slight variations in reference potential is not so detrimental to accuracy.

[0106] In another aspect of the invention, there is provided a use of the solid-state reference electrode as disclosed hereinbefore in an electrochemical analysis. In certain embodiments, the use may comprise determining a concentration of a solute in a solution using the solid-state reference electrode and an ion-selective electrode. In other embodiments, the use may comprise determining a pH of a solution using the solid-state reference electrode and an ion- selective electrode.

[0107] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0108] EXAMPLES

[0109] Example 1 : Fabrication steps of solid-state reference electrode

[0110] Step 1 : Synthesis of Zwitterionic Polymer

[0111] 25mg of 2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide was dissolved in 5ml DMSO solvent, and 877.5mg of n-butyl acrylate (nBA), 97.5mg methyl methacrylate (MMA), and 1 mg of azobisisobutyronitrile (AIBN) was added. The mixture was degassed under N2 for 20mins, followed by heating at 70°C for 24 hours. 50mg of 4-methoxy phenol was added to terminate the reaction, stirred at ambient temperature for 15 minutes. DMSO solvent was decanted and the polymer was washed once with 10ml of methanol. The resulting polymer was dissolved in chloroform and extracted with DI water. The mixture was extracted twice with chloroform. It was then extracted with brine and dried with magnesium sulfate. The polymer solution is filtered and left to evaporate overnight in a Teflon dish, followed by placing under vacuum for 3 hours. Polymer removed from Teflon dish and stored in glass vial.

[0112] Step 2: Preparation of Ag / AgCI solid contact

[0113] Commercial Silver SPE (Dropsens DRP-010-U75) was purchased from Metrohm Singapore Pte Ltd, and was used for the preparation of Ag / AgCI solid contact. 4mm puncher tool was used to create 4mm hole on the transfer tape to create a stencil. The stencil was pasted on the SPE, exposing the silver working electrode fully. Adequate amount of Ag / AgCI paste was applied to the left side of the stencil and it was spread from left to right of the stencil using a glass slide until the working electrode is fully covered. The Ag / AgCI paste was cured by heating at 80°C for 10 minutes, followed by removal of the stencil, and further heating at 120°C for 20 minutes.

[0114] Step 3: Preparation of reference membrane formulation and dropcasting onto Ag / AgCI surface 30mg of polymer was weighed, followed by adding adequate amount of crosslinker. 600ul of THF (50mg polymer per 1 ml) was added, followed by shaking for 2 hours to fully dissolve the polymer. KCI (< 25 pm particle size) was added, followed by shaking for 10 minutes on a vortex. 60ul of membrane solution was dropcasted on to the Ag / AgCI surface. The membrane was left to dry overnight, followed by placing under vacuum for 2 hours.

[0115] See Table 2 for the amounts of crosslinker and KCI added.

[0116] Step 4: Thermal curing of reference membrane (if crosslinker is added)

[0117] The membrane was crosslinked by thermal curing at 140°C for 4.5 hours under vacuum.

[0118] Step 5: Conditioning of all solid-state reference electrode

[0119] The crosslinked membrane was conditioned in 1 M KCI solution for 48 hours before it was used for further testing.

[0120] Results and discussion:

[0121] NMR analysis was performed to confirm the successful formation of the zwitterionic copolymer (FIG. 9a). It was observed that the peaks corresponding to the acrylate C=C bonds (5.5pm to 6.5 ppm) were not present in the product spectrum. In addition, peaks corresponding to the butyl groups can be observed in the region 0.7 to 1.7ppm, while peaks corresponding to the methyl groups can be observed at around 1.85 ppm. This suggests successful incorporation of both the nBA and MMA monomers.

[0122] Due to the small percentage of SBMA present within the polymer, the intensity of the peaks corresponding to SBMA function group was very low. Hence, a comparison with the nBA-MMA was performed to confirm the presence of SBMA within the zwitterionic copolymer. (FIG. 9b). It was observed that a new peak arose at about 3.0ppm, which can be attributed to the methyl groups present on the quaternary ammonium functional group on the SBMA, hence confirming the formation of the zwitterionic copolymer.

[0123] A gel fraction test was conducted to ascertain the degree of crosslinking of the polymers, and the results are summarized in Table 1. On Teflon dish, 200ul of zwitterionic polymer and crosslinker mixture was added. The mixture was dried overnight, followed by vacuum for 3h. The dried polymer and crosslinker mixture was thermally cured at 140°C for 4.5 hours. The resulting membrane was weighed, followed by addition of 2ml of THF. The mixture was heated at 70°C for 3 hours, followed by decanting the THF using a glass pipette. The remaining membrane material was washed twice by adding 2ml THF and removed using glass pipette. The membrane material was air dried and followed by placing under vacuum for 2 hours. The membrane was weighed to obtain the final weight. The gel fraction of the polymer was calculated based on Equation 1.

[0124] Equation 1. Formula for calculating gel fraction.

[0125] Table 1, Gel fraction for various crosslinker composition agel fraction was too small to determine as polymer was broken into small fragments.

[0126] The ester functionalized crosslinker was used in the initial stages of optimization, and the optimal composition was used to perform gel fraction for disulfide, acetal and phosphate functional groups. From the results, it indicated that 3wt% of ester crosslinker was sufficient to provide effective crosslinking. At 5wt% crosslinker, the gel fraction was very high (about 75%) and we expect that the membrane might be too rigid to allow ions to move across. Hence, 3wt% and 4wt% crosslinker was used for optimization of the membrane formulation. The disulfide, acetal and phosphate functionalized crosslinker also obtained good gel fractions at 3wt%.

[0127] Example 2: Testing and evaluation of reference electrode’s performance

[0128] The performance of the membrane was measured by open circuit potentiometry using a Dropsens Potentiostat (DRP-STAT8000, Metrohm Dropsens) in a 2-electrode setup with a Ag|AgCI|3M KCI|1 M LiOAc reference electrode.

[0129] Sensitivity measurements: The measurements were conducted in aqueous KCI solution with concentrations ranging from O.ljuM to 0.1 M, and the reference electrode was measured for at least 300 seconds for each solution. Three replicates of each solid-state reference electrode were tested.

[0130] Sensitivity towards other interfering ions: The performance of the membrane against interfering ions was tested using 1 mM solutions of KNOs, NaNOs, Na2SC>4, MgCh, CaCh, LiCI, NaCI, NaBr, Nal. These interference ions have been selected because they are commonly found in the real-world samples.

[0131] Results and discussion:

[0132] To optimize the reference membrane formulation, the amount of ester crosslinker and KCI was varied (Table 2, R1 to R7). The performance of the solid-state reference electrode was then determined by open circuit potentiometry. After optimizing the reference membrane composition with the ester crosslinker, other crosslinkers with disulfide, acetal and phosphate functional groups were also evaluated (Table 2, S1 , A1 and P1). Each measurement was done in triplicates, and the results are summarized in Table 2 and the potentiometric responses are depicted in FIG. 10.

[0133] Table 2. Comparison of performance of solid-state reference electrodes with varying amount of crosslinker and KCI (n = 3) aZwitterionic co-polymer was used except for R6.bSensitivity slope is measured from 1 mM to 0.1 pM.cpolymer used does not contain zwitterion monomerdmeasured EMF exhibited significant drift and instabilityeonly duplicate measurement was performed From the results, increasing amount of crosslinker from 3 wt% to 4 wt% showed similar sensitivity and potentiometric response (Table 2, R2 and R4). Hence, it was determined that 3wt% was sufficient to provide effective crosslinking and KCI retention. The amount of KCI was also optimized by preparing membranes with 15wt%, 30 wt% and 50 wt% KCI (Table 2, R1 to R3), and the sensitivity obtained was -0.98 ± 0.72 mV / decade, -0.09 ± 0.08 mV / decade and -0.90 ± 0.59 mV / decade respectively. From the potentiometry results, it could be observed that the R1 exhibited significant drift and noisy signals, which could possibly be attributed to inadequate amount of KCI within the membrane which is required to maintain its equilibrium with the external testing solution. The performance of R3 seem to exhibit poorer stability, possibly due to the high proportion of solid in the membrane suspension and hence good homogeneity was difficult to achieve. Similar trends were observed for membranes with 4wt% crosslinker with 30wt% and 50wt% KCI. (FIG. 10, R4 and R5). Control membrane R6 and R7 was prepared to determine the role of zwitterionic component as well as the crosslinker. The role of zwitterionic component was studied by preparing R6, which consist of crosslinked polyacrylate (MMA and nBA only) without the zwitterionic unit. By comparing R2 (FIG. 10(b), -0.09 ± 0.08 mV / decade) with R6 (FIG. 10(f), -0.21 ± 1.24 mV / decade), R6 exhibited larger sensitivity and significant drift and instability. Hence, this shows that the zwitterionic functional groups plays and important role in stabilizing the potentiometric signals. This stability is probably due to the ionic interactions between the charged groups and the KCI within the membrane, which helps to maintain the equilibrium of KCI across the membrane when exposed to an external analyte. The role of the crosslinker was then investigated by preparing R7, which is composed of linear zwitterionic copolymer without crosslinker. Comparing R2 (FIG. 10(b), -0.09 ± 0.08 mV / decade) with R7 (FIG. 10(g), -14.54 ± 3.98 mV / decade), it was evident that the sensitivity slope was significantly larger in the absence of a crosslinker. This indicates that the reference membrane R7 was responding to the change in concentration in the external analyte solution. Hence, while the presence of the zwitterionic component aided in maintaining potential stability, it was insufficient to effectively retain KCI within the reference membrane as leaching could still occur.

[0134] Another important characteristic of the reference membrane is its resistance towards interfering ions. This was investigated by placing the electrodes in a series of 1 mM solutions each containing KNO3, NaNOs, Na2SC>4, MgCl2, CaCh, LiCI, NaCI, NaBr and Nal salts. In the optimized membrane, good resistance to interfering ions was also demonstrated as the change in potential was limited to less than ±5mV. On the other hand, the control membranes exhibited significantly more drift and response towards interfering ions with the change in potential difference more than ±5 mV (FIG. 10). pH studies were conducted on optimized electrodes R2 and R4 (FIG. 11 ). pH stability study was conducted by preparing a background solution of 1 mM KCI. The initial pH was measured, followed by adding aliquots of concentrated hydrochloric acid or potassium hydroxide to achieve pH 1 to 13.

[0135] Results and Discussion:

[0136] From the results, R2 exhibited good potential stability across a broad pH range, ranging from pH 3 to 11 . In contrast, electrode R4 showed stable potential within a narrower pH range, from pH 4 to 10. Beyond the usable pH, a sharp decrease in potential difference was observed for acidic pH, while sharp increase in potential difference was observed for alkali pH. This sharp change in potential indicates possible degradation of the ester bonds within the membrane as well as significant leaching of KCI due to breaking down of the mesh-like structures.

[0137] Example 4: Lifespan testing

[0138] Lifetime testing was conducted to ascertain the durability of the electrodes. The electrodes were soaked in 1 M KCI at room temperature over 32 days, followed by 50 °C for 18 days and the potentiometric response was measured at regular intervals.

[0139] Results and Discussion:

[0140] The initial sensitivity of R2 and R4 were -0.09 ± 0.08 mV / decade and -0.38 ± 0.23 mV / decade respectively. At the end of 32 days at room temperature, no significant changes were observed in the sensitivity slope of R2 and R4 (0.13 ± 0.52 mV / decade and -0.18 ± 0.26 mV / decade respectively). Hence, lifetime testing was performed at 50°C to accelerate its degradation by approximately 5.6 times. After heating at 50°C for 18 days, the slight changes in the sensitivity slope was observed for R2 and R4 ( -0.82 ± 1.13 mV / decade and -0.73 ± 0.37 mV / decade respectively). The response declined as it might be due to the leaching of components from the membrane into the solution, as this kind of behaviour has been seen for most membrane sensors (Tajik, S., Taher, M. A. & Sheikhshoaie, I. Potentiometric determination of trace amounts of aluminium utilizing polyvinyl chloride membrane and coated platinum sensors based on E-N'-(2-Hydroxy-3-methoxybenzylidene) benzohydrazide. J AOAC Int96, 204-211 (2013)). While the sensitivity slope appears to be relatively unaffected, it was observed that the electrode experienced significantly more drift after heating at 50°C for 18 days. (FIG. 12). This could be due to corrosion of the silver connectors of the screen-printed electrodes. (FIG. 13) It was observed that 2 out of 3 chips had corroded silver connectors for R2 and 1 out of 3 chips had corroded connectors in R4, which corroborated with the potentiometry results obtained. Hence, this indicates that the degradation is mainly due to the corrosion of the silver connectors rather than the reference membrane. Through accelerated lifespan testing, the lifespan of the reference electrode is expected to be approximately 132 days.

[0141] Example 5: Real sample testing

[0142] To demonstrate the potential of our solid-state reference electrode R2 in real applications, the nitrate (Table 4, FIG. 14(a)) and potassium ion (Table 5, FIG. 14(b)) concentration in hydroponic samples (basal medium, basal medium inoculated with C. sorokiniana microalgae and hydroponics medium) was measured.

[0143] Results and Discussion:

[0144] Firstly, a 5-point calibration curve was obtained by measuring KNO3solutions with concentration ranging from 10uM to 0.1 M. The calibration curve obtained using solid-state reference electrode was compared with that of the liquid double junction reference electrode. (FIG. 14) Gratifyingly, the sensitivity achieved using our solid-state reference electrode was - 54.0 mV / decade, which is comparable to the liquid double junction reference (-54.5mV / decade). Similarly, for K+-ISE, our reference electrode demonstrated a sensitivity slope of 53.5 mV / decade which is comparable to using liquid double junction reference electrode (51 .6 mV / decade).

[0145] Table 4. Real sample testing against NOs ISE (n=3) aThe expected concentration of NO3ions is measured by Ion Chromatography (IC).bOnly 1 measurement was conducted for double junction Ag / AgCI electrode.

[0146] Table 5. Real sample testing against K+ISE (n=3) aThe expected concentration of K+ions is measured by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES).bOnly 1 measurement was conducted for double junction Ag / AgCI electrode.

[0147] After establishing the calibration plot, K+ and NOs' ion concentration was measured, and the results was compared with the liquid double junction electrode. From the results obtained, our electrode demonstrated comparable performance to the conventional liquid junction electrode. In addition, triplicate measurements conducted using proprietary solid-state reference electrodes exhibited good reproducibility. The calculated error was within ±6% on the logarithmic scale. Noteworthy, the basal medium inoculated with C. sorokiniana microalgae was measured “as-is” demonstrating the robustness of the reference electrodes when measured in samples without pre-treatment (i.e. filtration to remove algae particles).

[0148] Conclusion

[0149] In conclusion, we have fabricated a solid-state reference electrode using zwitterionic polymer and azide crosslinker. We also have demonstrated that the design of a mesh-like structure aids in effective retention of KCI within the membrane. This strategy could be used with crosslinkers with various functional groups such as disulfide, acetal and phosphate.

[0150] At optimal composition, a sensitivity slope of -0.09 ± 0.08mV / decade was obtained, indicating that the membrane was not responsive to concentration changes in the sample solution. Lifespan studies shown that the membrane was able to maintain its performance up to 132 days. While it appears that the electrode has deteriorated in performance, it can be attributed to corrosion of silver connectors on the screen-printed substrate rather than the degradation of the membrane itself. The solid-state reference electrode also demonstrated good pH stability, with a wide operational pH of 3 to 11 . More significantly, we have fabricated a solid-state reference electrode which possess similar performance as that demonstrated by the conventional liquid double junction reference electrode. In addition, the potential of the solid-state reference electrode for real applications was demonstrated through quantification of K+and NO3 concentration in hydroponic samples.

[0151] The results were in good agreement with the expected concentration determined.

Claims

CLAIMS1 . A membrane for a solid-state reference electrode, the membrane formed from a crosslinked zwitterionic polymeric matrix loaded with an inorganic salt, the inorganic salt being dispersed in the cross-linked zwitterionic polymer matrix.

2. The membrane of claim 1 , wherein the cross-linked zwitterionic polymeric matrix is formed from a zwitterionic acrylate copolymer and a crosslinking agent.

3. The membrane of claim 2, wherein the zwitterionic acrylate copolymer comprises constitutional units containing a quaternary ammonium group and a sulfonate group, and / or comprises constitutional units containing a phosphorylcholine group.

4. The membrane of claim 2 or claim 3, wherein the zwitterionic acrylate copolymer is a co-polymer of:(a) one or more zwitterionic monomers according to formula (I):wherein,R1is H or methyl,R2and R3are each independently C1-20alkylene; andA is NH, O, or S, for example, NH or O;X is -N+(Ra)2- and Y is -SO3-, or X is -O-P(O-)(O)-O- and Y is -N+(Ra)3; and each Rais independently C1-12alkyl; and(b) one or more of additional monomers selected from the group consisting of C1-20alkyl acrylateC,1-20alkyl methacrylate, and methacrylate, optionally wherein the C1-20alkyl acrylate and C1-20alkyl methacrylate are substituted with one or more substituents selected from halo, C1-20alkoxy and Het, wherein halo represents F, Cl, Br or I and Het represents a 4- to 10- membered heterocyclic groups containing one or more heteroatoms selected from O, S and N.

5. The membrane of claim 4, wherein each Rais methyl.

6. The membrane of claim 4 or claim 5, wherein R2is C1.4 alkylene and R3is Cv 12 alkylene.

7. The membrane of claim 4 or claim 5, wherein R2is C1-4 alkylene and R3is C1-4 alkylene.

8. The membrane of claim 7, wherein the zwitterionic monomer according to formula (I) is selected from the group consisting of:[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1 -sulfonate, [2-(acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [3-(methacryloylamino)propyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1 -sulfonate, 3-[(3-acrylamidopropyl)dimethylammonio]propane-1 -sulfonate, 2-methacryloyloxyethyl phosphorylcholine, and a combination of two or more thereof.

9. The membrane of any one of claims 4 to 8, wherein the one or more of additional monomers are selected from the group consisting of n-butyl acrylate, s-butyl acrylate, n-hexyl acrylate, n-octyl acrylate, n-dodecyl acrylate, 2-ethylhexyl acrylate, 2,2,2-trifluoroethyl acrylate, 2-methoxyethyl acrylate, tetrahydrofurfuryl acrylate, methyl methacrylate, n-hexyl methacrylate, n-octyl methacrylate, n-dodecyl methacrylate, octadecyl methacrylate and methyl methacrylate, for example the one or more additional monomers are n-butyl acrylate and methyl methacrylate.

10. The membrane of any one of claims 4 to 9, wherein the monomer according to formula (I) is 2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, and the one or more of additional monomers are n-butyl acrylate and methyl methacrylate.11 . The membrane of any one of claims 2 to 10, wherein the crosslinking agent comprises two or more groups selected from the groups consisting of aryl azide, azidoformate, a- diazoester, a-diazoamide, and diazirine, for example the crosslinking agent may comprise two or more aryl azide groups.

12. The membrane of claim 1 1 , wherein the crosslinking agent comprises two or more perfluorophenyl azide groups.

13. The membrane of claim 12, wherein the crosslinking agent is selected from the group consisting of:and a combination of two or more thereof.

14. The membrane of any one of claims 2 to 13, wherein the cross-linked zwitterionic polymeric matrix is formed by reacting the zwitterionic acrylate copolymer with from about 1 wt% to about 10 wt% of the crosslinking agent based on the combined mass of the crosslinking agent and zwitterionic acrylate copolymer, for example the cross-linked zwitterionic polymeric matrix may be formed by reacting the zwitterionic acrylate copolymer with from about 1 wt% to about 5 wt% (e.g. about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt% or about 5 wt%) of the crosslinking agent based on the combined mass of the crosslinking agent and zwitterionic acrylate copolymer.

15. The membrane of any one of claims 2 to 14, wherein the zwitterionic acrylate copolymer is formed by the polymerisation of the zwitterionic monomer and the one or more of additional monomers at a molar ratio of the zwitterionic monomer to the one or more of additional monomers of from about 1 :20 to about 1 :250, for example about 1 :90.

16. The membrane of claim 15, wherein the one or more of additional monomers are n- butyl acrylate and methyl acrylate at a molar ratio of n-butyl acrylate to methyl methacrylate of from about 3:1 to 20:1 , for example about 7:1 .

17. The membrane of any one of the preceding claims, wherein the inorganic salt is provided in a particle size of less than or equal to about 45 pm, for example, less than or equal to about 30 pm or less than or equal to about 25 pm.

18. The membrane of any one of the preceding claims, wherein the inorganic salt is a chloride salt.

19. The membrane of any one of the preceding claims, wherein the chloride salt is selected from the group consisting of potassium chloride, sodium chloride, lithium chloride, and a combination of two or more thereof, for example, the chloride salt is potassium chloride.

20. The membrane of any one of the preceding claims, wherein the membrane comprises from about 10 wt% to about 60 wt% of the inorganic salt based on the total mass of the membrane, for example the membrane may comprise from about 10 wt% to about 55 wt% (e.g. about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt%) of the inorganic salt based on the total mass of the membrane.21 . A solid-state reference electrode comprising a reference electrode with the membrane of any one of claims 1 to 20 disposed on a surface of the reference electrode.

22. The solid-state reference electrode of claim 21 , wherein the reference electrode is selected from the group consisting of a silver-silver chloride electrode, a calomel electrode, silver-silver sulfate electrode, mercury-mercury sulfate electrode, and copper-copper(ll) sulfate electrode.

23. The solid-state reference electrode of claim 22, wherein the reference electrode is a silver-silver chloride electrode.

24. The solid-state reference electrode of claim 23, wherein the silver-silver chloride electrode comprises a solid electron-conducting material coated with a layer of silversilver chloride.

25. The solid-state reference electrode of claim 24, wherein the solid electron-conducting material is silver, gold, platinum, copper, steel, carbon (or carbon nanomaterials), metal oxide (e.g. indium-tin-oxide, titanium dioxide, fluorine-doped tin oxide) or aluminium.

26. The solid-state reference electrode of claim 24 or claim 25, wherein the solid electronconducting material is disposed on a substrate, optionally wherein the substrate is a ceramic substrate or a polyimide substrate.

27. The solid-state reference electrode of any one of claims 22 to 26, wherein the reference electrode is a screen-printed electrode, for example a screen-printed silver electrode.

28. A method of producing the solid-state reference electrode of any one of claims 21 to 27, the method comprising: i) preparing a mixture of the zwitterionic acrylate copolymer, the crosslinking agent, and the inorganic salt in an organic solvent by dissolving the zwitterionic acrylate copolymer and the crosslinking agent in the organic solvent, followed by adding the inorganic salt to the organic solvent; ii) applying the mixture from step i) to a surface of the reference electrode followed by evaporating the organic solvent; iii) subjecting the reference electrode to a temperature of at least 100 °C for a time period sufficient to form a cross-linked zwitterionic polymeric matrix of the surface from the zwitterionic acrylate copolymer and the crosslinking agent; iv) submersing the reference electrode following step iii) in an aqueous solution of the inorganic salt, thereby producing the solid-state reference electrode of any one of clauses 21 to 27.

29. The method according to claim 28, wherein the zwitterionic acrylate copolymer used in step i) is prepared by a polymerisation reaction between (methacryloyloxy)ethyl]dimethyl- (3-sulfopropyl)ammonium hydroxide, n-butyl acrylate and methyl methacrylate.

30. The method according to claim 28 or claim 29, wherein the inorganic salt is KCI.31 . The method according to any of claims 28 to 30, wherein the reference electrode is a silver-silver chloride electrode.

32. The method according to claim 31 , wherein the silver / silver chloride electrode comprises a screen-printed silver electrode coated with a layer of silver-silver chloride.

33. The method according to any one of claims 28 to 32, wherein the crosslinking agent is selected from the group consisting of:and a combination of two or more thereof.

34. The method according to claim 33, wherein step iii) comprises subjecting the reference electrode to a temperature of about 140 °C for about 4 to 5 hours under vacuum, optionally wherein step iii) comprises subjecting the reference electrode to a temperature of about 140 °C for about 4 to 5 hours under vacuum at a pressure of from 100 Pa to 5,000 Pa, such as from 133 Pa to 5,000 Pa, such as from 2,000 Pa to 5,000 Pa, such as from 4,000 Pa to 5,000 Pa, such as from 100 Pa to about 4,000 Pa, such as from 100 Pa to 2,000 Pa, such as from 100 Pa to 133 Pa or such as from 133 Pa to 4,000 Pa.

35. The method according to any one of claims 28 to 34, wherein step iv) comprises submersing the reference electrode following step iii) in a 1 M KCI solution for about 48 hours.

36. A solid-state electrode obtained by the method according to any one of claims 28 to 35.

37. A potentiometric sensor comprising the solid-state reference electrode of any one of claims 21 to 27 or 36 and an ion-selective electrode.

38. Use of the solid-state reference electrode of any one of claims 21 to 27 or 36 in an electrochemical analysis.

39. The use according to claim 38 comprising determining a concentration of a solute in a solution using the solid-state reference electrode and an ion-selective electrode.

40. The use according to claim 38 comprising determining a pH of a solution using the solid-state reference electrode and an ion-selective electrode.

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