Biosensor for the in vivo monitoring of analytes

Abstract

A functional ion-selective sensor (ISS) is provided having an ion-selective membrane (ISM). The ISM includes an ion specific ionophore (I); a cation exchanger (CE) selected from the group consisting of: sodium tetraphenylborate (NaTPB), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), and potassium tetrakis(4-chlorophenyl)borate (KTCIPB); a base membrane polymer (BMP) comprising polyvinyl chloride (PVC) and / or polyurethane (PU), and a plasticiser (P) selected from the group consisting of: bis(2-etheylhexyl) sebacate (DOS); and 2-nitrophyloctyl ether (NPOE). The components are present in the membrane in the following wt% ranges: I between 0.02 - 20 wt%, inclusive; CE between 0.05 - 4 wt%, inclusive; P between 10 - 90 wt%, inclusive; and BMP between 5 - 80 wt%, inclusive. The mole ratio of I and CE is between 0.1 - 10, inclusive. The wt% ratio of BMP and P is between 0.1 – 10, inclusive.

Description

[0001] Title:

[0002] BIOSENSOR FOR THE IN VIVO MONITORING OF ANALYTES

[0003] Cross-Reference to Related Applications:

[0004] The present application is a non-provisional PCT application of and claims priority to US Prov. App. Ser. No. 63 / 556,008 filed on February 21, 2024. The present application is also related to PCT / US2022 / 037198 (PROT.P-OOl-WO), PCT / US2022 / 052927 (PROT.P-002-WO), PCT / IB2023 / 061417 (PROT.P-003-WO), and PCT / US2024 / 034271 (PROT.P-004-WO), which are incorporated herein by reference for all purposes.

[0005] Background of the Invention:

[0006] Selective detection of ions can be achieved by means of a sensor having an ion-selective membrane (ISM) disposed on a transducer / electrode. The sensor must be durable and long lasting to be suitable for in vivo applications. Furthermore, ISMs for in vivo applications, such as transdermal ion monitoring, require the ISM to be biocompatible whilst maintaining a sensor performance that allows for an accurate and reliable ion detection. Prior ISMs, such as hydrophobic ISMs are not suitable for in vivo use without modification, mainly because of two reasons; i) leaching of cytotoxic ISM components into the tissue, and ii) high biofouling caused by cells and proteins attached to the ISM surface. Moreover, leaching of ISM components and biofouling result in the loss of sensor lifetime in addition to impaired response accuracy, sensitivity, and reliability. Furthermore, some transducers when optionally employed with specific ISMs have herein been found to be problematic in that a water layer forms between the ISM and transducer reducing sensor performance, durability, and lifespan. Improvements in all of these areas and provision of various sensor components when used alone or in combination with other sensor components herein described to provide a sensor capable of detecting various ions of interest are strongly desired.

[0007] Brief Summary of the Invention:

[0008] The present invention provides ion (e.g. K++and Na++) selective sensor membrane compositions, new hydrophobic transducer layers, and new coating layers individually or combinations thereof capable for use with continuous in vivo analyte sensing meters and methods which show decreased formation of a water layer between the transducer and ISM as well as superior sensor response and performance.

[0009] In a first embodiment, the present invention provides a functional ion-selective sensor (ISS) having an ion-selective membrane (ISM). The ISM includes an ion specific ionophore (I); a cation exchanger (CE) selected from the group consisting of: sodium tetraphenylborate (NaTPB), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), and potassium tetrakis(4- chlorophenyl)borate (KTCIPB); a base membrane polymer (BMP) comprising polyvinyl chloride (PVC) and / or polyurethane (PU), and a plasticiser (P) selected from the group consisting of: bis(2-etheylhexyl) sebacate (DOS); and 2-nitrophyloctyl ether (NPOE). The components are present in the membrane in the following wt% ranges: I between 0.02 - 20 wt%, inclusive; CE between 0.05 - 4 wt%, inclusive; P between 10 - 90 wt%, inclusive; and BMP between 5 - 80 wt%, inclusive. The mole ratio of I and CE is between 0.1 - 10, inclusive. The wt% ratio of BMP and P is between 0.1 - 10, inclusive.

[0010] In a second embodiment, the present invention provides a layered biosensor for the in vivo monitoring of electrolytes, the biosensor comprising, in order: a hydrophilic coating layer, a hydrophobic ISM layer, a hydrophobic transducer layer, an electrode layer, and non-conductive substrate.

[0011] In a third embodiment, the present invention provides A method of forming an ISS of claim 1, the method comprising the steps of: (a) forming an ISM on an underlying transducer / electrode / substrate layer; (b) optionally forming a coating layer on the ISM and encapsulating an underlying layers; (c) contacting the coating layer with the ISM and an underlying non-conductive layer, thereby forming a layered composition for use with a biosensor for the in vivo monitoring of electrolytes.

[0012] Brief Description of the Figures:

[0013] Fig. 1 shows hydration of an ion selective sensor (ISS) and formation of a water layer.

[0014] Fig. 2 shows an exemplary layered ISS configuration. Fig. 3 shows an exemplary layered ISS configuration.

[0015] Fig. 4 shows an exemplary layered ISS configuration.

[0016] Fig. 5 shows an exemplary layered ISS configuration.

[0017] Fig. 6 shows water layer test results from Example 1 of the Example Section conducted with sensors having A) PEDOT:PSS or B) PHT as the solid-contact transducer layer.

[0018] Fig. 7A shows a sensor response profile to changing K+concentrations ranging from 2 to 8 mM from Example 1 of the Example Section.

[0019] Fig. 7B shows sensor response sensitivity (KA) and offset (Kc) for the first 2, 6, 4, and 8 mM K+, and MARD for concentrations tested after 6 h of testing from Example 1 of the Example Section.

[0020] Fig. 8 shows a demonstration of selectivity of the sensor when at 2 mM K+in the presence of 10 mM NaCl, 50 mM NaHCCh, 5 mM LiCl, and 5 mM MgCL from Example 1 of the Example Section.

[0021] Figs. 9 and 10 show experimental results from Example 2 of the Example Section.

[0022] Fig. 11 shows long term sensor response of a K+ sensor based on two different base polymers of the Example Section.

[0023] Fig. 12 demonstrate simulated dialysis of two different K+ sensor compositions in presence of interfering ions containing 30 mM NaHCCL, 10 mM NaCl, 4 mM MgCL and 5 mM LiCl of the Example Section.

[0024] Fig. 13 demonstrates sensor response to K+ in a complex biological matrix of the Example Section.

[0025] Fig. 14 shows K+ Sensor selectivity on flexible substrate when initially at 2mM K+in the presence of 30mM NaHCCh, lOmM NaCl, 5mM LiCl, 4mM MgCh, and two separate additions of 2mM KC1 of the Example Section.

[0026] Fig. 15 shows evaluation of raw electrical signal utilising ISM 1 composition in vivo and blood potassium concentration reference of the example section. Fig. 16 shows estimated potassium concentration in ISF in comparison to blood potassium concentration reference of the Example Section.

[0027] Detailed Description of the Invention:

[0028] The present Inventors have discovered that a water layer can form between the transducer layer and ion-selective membrane (ISM) of a biosensor during sensor hydration. Fig. 1 shows formation of such a water layer between an ISM and transducer layer during hydration of an ion- selective electrode (ISE).

[0029] The presence of the water layer has been found to decrease the usefulness of the biosensor, increase signal drift, increase equilibration time, decrease selectivity of the sensor and ISM, increase degradation of the respective layers, increase leaching of the components to the surroundings, decrease biocompatibility of the sensor, and decrease the usefulness, reliability of measurements, and overall lifespan of the sensors. The present Inventors have discovered causes of formation of the water layer and solutions to these problems. The present invention provides biosensors, related compositions thereof, and solves the problems of the prior art. In preferred embodiments, the present invention provides compositions for layer(s) of a biosensor for the in vivo monitoring of electrolytes, layered compositions useful for such biosensors, and methods of forming and / or using the same. The sensors are capable of the selective detection of various ions, with a specific focus herein toward selective detection of potassium and sodium. In preferred embodiments, the compositions of the respective layers, the respective layers themselves, and / or the formed biosensors of the present invention are compliant with ISO 10993-5 cytotoxicity validation.

[0030] A focus of the present invention is directed to at least three separate embodiments which can be used separately, or in any combination thereof, herein to decrease the propensity of formation of the discovered water layer AND to provide a robust sensor with superior properties capable of detecting various analytes / ions, with a focus directed toward potassium and sodium ion detection. In a first embodiment, the present invention provides a new transducer layer which has herein been discovered to demote formation of the discovered water layer. In a second embodiment, the present invention provides new ISM layers containing base polymers and other components tuned in various ranges and ratios with respect to other components that likewise demote formation of the discovered water layer while providing superior ion selectivity. In a third embodiment, the present invention provides a specific coating layer that has been discovered to allow for ingress of ion to the base layer of the sensor stack, while providing rigidity to resist degradation of the base layers of the stack, prevent leaching, improve biocompatibility and bond securely with the support substrate and ISM of the sensor to enclose, support, protect and provide structure to the intervening layers of the sensor. In a fourth embodiment, the present invention provides preferred combinations of the first, second, and / or third embodiments and / or various aspects thereof.

[0031] The ion-selective sensors (ISS) of the present invention can take various forms as shown in Figs. 2-4. As shown in Fig. 2, within the measurement zone, the ISS is formed from multiple layers including a coating layer, an ISM layer, a transducer layer (such as a single conductive layer of graphite), and substrate layer. Not shown in Fig. 2, but shown in Fig. 4, a separate conductive electrode layer (such as a conductive electrode layer of gold, silver, copper, carbon / graphite, platinum, and the like) may be present and disposed between the substrate and transducer layer. Furthermore, as shown in Figs. 3 and 4, a further insulating layer may be present to pattern and / or protect the transducer layer. In these embodiments the ISM layer and coating layer can be patterned directly on the surface of the transducer and likewise be formed to be in contact with the insulating layer to fully overlay the transducer and underlying electrode and likewise chemically interact and / or bond with the insulating layer (as further explained below). Fig. 5 shows another embodiment of an ISS demonstrating layers of both the working ISE and a silver / silver chloride reference electrode, and capable of distinguishing and determining various ions, such as sodium and / or potassium.

[0032] Definitions:

[0033] Electrically Conductive Electrode layer

[0034] A conductive material used to facilitate electron transfer, enabling electronic signal measurements. Such conductive materials can be formed from various conductive materials however in some preferred embodiments comprise gold.

[0035] Transducer layer A layer that undergoes reversible chemical changes with respect to changing analyte concentrations resulting in conversion between chemical and electrical energy.

[0036] ISM layer

[0037] An ion selective membrane layer that contains selective analyte transport molecules such as ionophore selective to an ion, a lipophilic component that exchanges the target analyte with the analyte transport molecule, and a plasticized polymer matrix.

[0038] Coating layer

[0039] A coating layer applied over the hydrophobic ISM that may enable the biosensor to be biocompatible, antibiofouling, limit analyte diffusion, mitigate light penetration, increase the structural integrity of the sensor layers, and / or decrease the total biosensor impedance, all without compromising the analytical performance of the biosensor.

[0040] Embodiments and Aspects of Invention

[0041] Reference throughout the specification to “one embodiment,” “another embodiment,” “an embodiment,” “some embodiments,” “aspect”, and so forth, means that a particular element (e.g., feature, structure, property, and / or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described element(s) and / or feature(s) of any embodiment may be combined in any suitable manner with any other described embodiments.

[0042] Numerical Values

[0043] Numerical values in the specification and claims of this application reflect average values. Furthermore, unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value. First Embodiment - Transducer Layer

[0044] It has herein been discovered that the transducer layer of the present invention, and respective components thereof, can affect formation of the discovered water layer between the ISM and transducer, particularly when combined with other sensor layers as herein described. In particular, the present Inventors have discovered that a transducer layer containing hydrophilic entities can lead to water being entrapped at the transducer layer. The present Inventors have further found that carbon-based transducer materials, such as nanomaterials, can be a suitable alternative to the widely used conductive (and hydrophilic) polymers for obtaining a solidcontact ISE, and which is less prone to the water layer formation. The preferred carbon-based electrically conductive materials include carbon nanotubes (single and multi-walled), graphene, graphite, and carbon-black.

[0045] Excellent solid-contact transducer layer for an ISE would have a few properties including high hydrophobicity, high electrical conductivity, high redox capacitance, insensitivity to pH, light, and oxygen, good adhesion to the WE, and being non-cytotoxic for an in vivo sensor. Such properties can be produced by the carbon-based transducer layer herein proposed. The transducer layer is preferably hydrophobic and is formed at least partially, if not entirely, from a conductive hydrophobic carbon ink.

[0046] In one example a solid-contact transducer is composed of a carbon-based ink which may contain electrically conductive materials such as carbon black, carbon nanotubes, metal nanoparticles, conductive polymers, and alike; polymeric resin acting as a binding, stabilization, and / or agglomeration agent such as polyvinyl derivatives, polyacrylates, polyurethanes, polyglycols, and alike; solvent to optimize volatility and viscosity type of physicochemical properties such as alcohols, aromatic solvents, and alike; and other additive materials to control properties such as adhesion, chemical stability, photosensitivity, and alike.

[0047] In another example a solid-contact transducer is composed of an ink that contains carbon materials such as carbon nanotubes, graphite, graphene, carbon black, carbon dots, fullerenes, and alike; a binder such as polyvinyl butyral, polyvinyl acetate, chitosan, polystyrene, cellulose derivatives, polylactic acid, polyvinylpyrrolidone, polyurethane, and alike; stabilizer such as glycerol; and one or a combination of the solvents such as water, dimethylformamide, tetrahydrofuran, cyclohexane, and alike.

[0048] A particularly preferred example of such carbon-based ink material for the transducer layer has been surprisingly discovered to be commercially available graphite ink developed for flexographies printing by Sun Chemical Inc.. The product is sold by Sigma- Aldrich under the product number # 901970 and the product name of SunTronic®.

[0049] In addition to carbon-based ink, the transducer layer may optionally further include transducer materials which include self-assembled monolayers, metal and metal oxides nano and microstructures such as platinum nanostructures and molybdenum dioxide microparticles, and conductive polymers which are obtained by polymerizing one or a combination thereof when using materials including, but not limited to pyrrole, l-hexyl-3,4-dimethylpyrrole, 3- octylthiophene, 3,4-ethylenedioxythiophene, 3 -methylthiophene, aniline, indole, a- naphthylamine, o-anisidine, o-aminophenol, possibly in the presence of ?-toluene sulfonate, polystyrene sulfonate, perfluorates, metallic complexes, and alike, and lipophilic ions such as tetrakis[3,5-bis(trifluoromethyl)phenyl borate and (4-chlorophenyl)borate, carbon containing materials such as carbon nanotubes, graphite, graphene, carbon black, carbon dots, fullerenes, and alike, which can facilitate redox-activity and / or double layer capacitance and / or ion-to- electron transduction. It is noted that in many preferred embodiments, addition of conductive polymers does not occur as it is believed that addition of such polymers could increase the chances of water ingress, or otherwise promote formation of said undesirable water layer. a. Method of deposition of transducer on an electrode (if present)

[0050] The transducer layer can be deposited on an electrode (e.g. a gold coating disposed on an insulating surface) by galvanostatic or voltammetric methods, where the conductive material is attracted to the electrode surface with or without doping agents and counter ions by means of applied currents or potentials to the electrode. Examples include electrodeposition methods including but not limited to electrochemical polymerization of poly(3,4-ethylenedi oxythiophene) doped with polystyrene sulfonate, polypyrrole doped with chloride ion, and alike. In another example, the transducing material can be chemically attached to the electrode surface via covalent bonding, it - it interactions, electrostatic interactions, Lennard-Iones and Coulomb interactions. In another example, a mixture of the transducing material with a volatile solvent such as tetrahydrofuran, ethanol, methanol, acetone, and alike can be obtained and deposited on the electrode surface by means of drop-casting, spin-coating, spray-coating and dip-coating. In another example, the transducing material can be obtained by mixing a photosensitive material that allows the photodefinable patterning of the transducer layer on the electrode and substrate material. In another preferred embodiment, a transducer layer can be formed on an electrode (e.g. gold electrode) surface using the compositions and methods as described in US Prov. Pat. App. Ser. No. 63 / 425,658 (PROT. P-003 -PV) and the electrode can be formed from the compositions and methods as described in PCT / US2022 / 037198 (PROT.P-OOl-WO) which are incorporated in their entireties herein by reference for all purposes. b. Method of deposition of transducer on a base substrate (if no separate electrode is present)

[0051] In some situations, the transducer layer can be used separately, without an electrode. In these situations, the transducer has electrical properties and patterning with conductive lead(s) to attach directly to a meter, without the use of a separate electrode and its conductive patterns and meter connectivity. In these aspects, the transducer layer can be deposited and patterned directly on a base substrate material by various methods including screen printing, transfer printing, lithographic deposition, and thin film deposition methods including but not limited to physical vapour deposition (PVD) , chemical vapour deposition (CVD), and plasma enhanced chemical vapour deposition (PECVD) among others. c. Properties

[0052] The preferred transducer layer of the present invention facilitates electron transduction in the presence of the target analyte, enabling a correlation between the analyte concentration with the recorded signal. The transducer layer is preferably highly hydrophobic, improving the adhesion of the ISM and reducing and / or eliminating the accumulation of aqueous media at the interface of the transducer and ISM, which is detrimental to the reliability of the acquired data and durability of the ISS. Other preferred properties of the present transducer include high electrical conductivity, high redox capacitance, insensitivity to pH, light, and oxygen, good adhesion to the WE, and being non-cytotoxic for an in vivo sensor. Second Embodiment - Ion-Selective Membrane (ISM) layers

[0053] It has herein been found that use of prior art base polymer materials for an ion-selective membrane (ISM) promote, or in the alternative do not dissuade, formation of the herein discovered water layer between the transducer and the ISM. Without being bound by an intended mechanism of action, the present Inventors believe use of certain base polymers, which include hydrophilic base polymers in the ISM create hydrophilic and / or porous conditions within the ISM which promote ingress of water across the ISM, can lead to leaching of ISM components into surrounding environments, have poor adhesion characteristics to the transducer layer, lead to formation of the herein discovered water layer, cause eventual failure of the ISM and sensor for intended purposes, and lead to leaching of sensor components including cytotoxic components to surrounding fluid. Again, without being bound by an intended mechanism of action the present Inventors believe that use of hydrophobic polymers, such as plasticized polyvinyl chloride (PVC), as a base polymer in the ISM (and in specific ratios with respect to other components and / or the ISM itself) decrease porosity of the ISM and / or bias the ISM toward hydrophobic conditions and away from hydrophilicity. As shown herein, decreasing the porosity of the ISM to water and / or increasing hydrophobicity of the ISM decreases water transfer across the ISM and decreases water layer formation between the ISM and transducer. See Fig. 1. Such benefits and actions increase the usefulness, durability, and lifespan of the ISM and associated sensor. The present Inventors propose several different ISMs in various aspects of the present ISM embodiment which produce a robust ISM and durable ion selective sensor which meets cytotoxicity standards and produces accurate and long-lasting measurements when employed in vivo. See Fig. 11.

[0054] The transducer layer can be formed on a supporting substrate, the ISM can be formed on the transducer layer, and a coating (if present) can be formed on the ISM by any known method of deposition, which are not limited herein. In preferred embodiments, any and / or all of individual deposition methods are selected from the group consisting of: drop-casting, spin-coating, spraycoating, ink-jet printing, screen-printing, blade coating, brush-coating, dip-coating, slot-die coating, bar-coating, and stamping. To enable better adhesion of two sensor layers contacting each other, surface modification methods may be used to facilitate covalent, ionic, and metallic bonding, and / or intermolecular interactions such as hydrogen bonding, van der Waals interactions, electrostatic interactions, dipole-dipole interactions, n-n interactions, and alike.

[0055] Other preferred properties of the ISM of the present invention include high hydrophobicity, low porosity, ability to adhere well to the sensor substrate, have high enough flexibility when mixed with a plasticizer to allow ion-transfer in the presence of lipophilic salt and ionophore, have high chemical stability when exposed to temperature of 4 - 40°C (especially when exposed to light at temperature of 4 - 40°C), and at physiological pH, and being non-cytotoxic for an in vivo sensor.

[0056] A First Aspect of the ISM -A Potassium Selective ISM:

[0057] In this first aspect of the present ISM embodiment, a potassium (K+) ion selective membrane composition for use with K+selective sensors is provided. The present Inventors have unexpectedly found that use of such membrane compositions with K+selective sensors, shows superior sensor response and performance in combination with decreasing water layer formation between the transducer and ISM. In this first aspect of the present embodiment, the present invention provides a functional K+sensor membrane composition for use with a K+specific sensor. The membrane composition includes: a K+ionophore (I), a cation exchanger (CE), a base membrane polymer (BMP), and a plasticizer (P). The components I, CE, BMP, and P are not particularly limited herein. However it is noted that in particularly preferred embodiments, the components include any or all of the following.

[0058] The K+ionophore is not particularly limited herein. The word ionophore is derived from Greek (ion and phore, "ion carrier") and is a chemical species that reversibly binds ions and is capable of transporting ions across membranes. Synthetic potassium ionophores include (I=valinomycin, II, III, IV) and all can potentially be employed in a hydrophobic membrane. The most commonly used K+ionophores are ranked I>II>III, and the least common is type IV.

[0059] Biologically derived molecules that act as ionophores also exist and can be employed which include other biological functions such as antimicrobial, anti-inflammatory, and antibiotic functions etc. These biologically derived ionophores can be produced using

[0060] Streptomyces. Examples of such ionophores include: 1) Salinomycin has a high preference to K+, in addition to other alkali metal ions such as Na+, Ca2+and Mg2+; 2) Bafilomycin Al; 3) Nagericin, reactive with both K+and H+ions; 4) Nonactin, reactive with both K+and NH4+; and 5) Gramicidins, type D is formed from type A, B, and C. Gramicidins form transport channels in the cell membrane through which K+and Na+can pass and can be derived from Bacillus.

[0061] In some preferred embodiments the K+ionophore can be K ionophore I (KI-I) (valinomycin) which has the structure:

[0062] In other preferred embodiments the K+ionophore is ionophore II (KI-II) (bis[(benzo-15-crown- 5)-4'-ylmethyl] pimelate); (CAS: 69271-98-3) which is also known as bis[(benzo-15-crown-5)- 15-ylmethyl] pimelate or bis(2,5,8,l l,14-pentaoxabicyclo[13.4.0]nonadeca-l(15),16,18-trien-17- ylmethyl) heptanedioate and has the structure:

[0063] In other preferred embodiments the K+ ionophore is: ionophore III (2-dodecyl-2-m ethyl- 1,3- propanediyl bis[N-[5'-nitro(benzo-15-crown-5)-4'-yl]carbamate]); ionophore IV (dioxacalix[4]arene-tetraacetic acid tetra-tert-butyl ester); salinomycin which has a high preference to K+, in addition to other alkali metal ions such as Na+, Ca2+and Mg2+; bafilomycin Al; nagericin, reactive with both K+and H+ions; nonactin, reactive with both K+and NH4+; and gramicidin, type D is formed from type A, B, and C. Other type of ionophores can be incorporated to detect other ions such as tridodecylamine, ETH 1907, and alike to detect hydrogen; ETH 9033 and alike to detect chloride; ETH 2137 and alike to detect lithium, ETH 5234 and alike to detect calcium; K22B5, ETH 4030, and alike to detect magnesium; carbonate ionophore VII (N,N-dioctyl-3a,12a-bis(4-trifluoroacetylbenzoyloxy)-5P-cholan-24-amide), and alike; silver ionophore IV (2-(octadecyloxymethyl)pyridine), and alike.

[0064] The cation exchanger (CE) is not particularly limited and can include for example lipophilic salts such as tetraphenylborate-based lipophilic salts. In preferred embodiments, the CE preferably comprises, consists of, or consists essentially of potassium tetrakis(4-chlorophenyl)borate) (KTCIPB), which has the following structure:

[0065] C24H16BC14K

[0066] In other preferred embodiments the CE is selected from tetraphenyl derivatives including sodium tetraphenyl borate (NaTPB), which has the structure: tetrakis(4-chlorophenyl)borate, tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, and alike, N- (2,3,5,6,8,9,l l,12-octahydro-16-nitro-l,4,7,10,13-benzopentaoxacyclopentadecin-15-yl)-, 2- dodecyl-2-methyl- 1,3 -propanediyl ester (BME 44), tridodecylmethylammonium salts, and alike. The plasticizer is not particularly limited but preferably comprises, consists of, or consists essentially of a compound or compounds selected from the group consisting of succinic acid derived plasticizers and / or compounds having a long hydrocarbon chain with nitrophenyl ether group (e g. 2-nitrophenyl octyl ether (NPOE), dodecyl 2-nictrophenyl ether), phthalate derivatives (e.g. dibutyl phthalate, dioctyl phthalate), and bis(l-butylpentyl)decane-l,10-diyl di glutarate. NPOE is often selected and preferred and has the structure of:

[0067] In some preferred embodiments, the plasticizer comprises, consists of, or consists essentially of Dioctyl sebacate (di(2-ethylhexyl) sebacate) (DOS).

[0068] DOS is likewise often preferred. DOS is an oily colorless liquid and is an organic compound which is the diester of sebacic acid and 2-ethylhexanol. DOS has the following structure:

[0069] The base membrane polymer preferably comprises, consists of, or consists essentially of polyvinyl chloride (PVC). Additional polymers may be present in the base membrane polymer together with PVC, or in some situations in substitution for PVC. The additional polymers may be selected from the group consisting of: silicones, fluorosilicones, polyurethane, polyacrylates, and perfluoro polymers. Other polymers may likewise be present in the base polymeric composition and selected from the group consisting of: polyurethanes (PU), silicones, polyether ether ketone (PEEK), cycloolefin copolymer (COC), polymethyl methacrylate (PMMA), polystyrene, acrylates, poly(vinyl butyral), polyamide, polyimide, and Teflon. In most preferred embodiments the polymer consists of, or consists essentially of PVC. In preferred embodiments PVC has a molecular weight of 100 kg / mol and has the following structure:

[0070] The membrane compositions may contain additional components added in amounts such that the ratios described above are maintained. These additional components can be added to perform functions that are known in the art. For example, additional components can include materials such as conductive materials including nanomaterials or pyrrole type of monomers that do not significantly affect the membrane performance and selectivity yet might increase the sensitivity. Additional components can also include redox markers introduced to the membrane in order to enable Voltammetry-based (e.g. DPV, SWV, and CV) detection of the ions. These redox markers are well-known in the art and are described inter alia at: https: / / analyticalsciencejournals.onlinelibrary.wiley.eom / doi / 10.1002 / elan.201800080).

[0071] Other additives (e.g. additional components) might be also introduced to modify mechanical or surface chemistry properties of the membrane, such as to increase adhesion, modify the hydrophobicity / hydrophilicity, tune stiffness or enable enhanced anti-biofouling properties. The K+sensor membrane compositions as described herein are particularly suitable for use in biological and biomedical applications (for example in connection with potassium sensors for determining potassium in biological fluids, such as blood and / or interstitial fluid). This is due to the biocompatibility of all membrane components.

[0072] Accordingly, in another embodiment, the present invention provides a method of use of the herein described membrane compositions, alone or in combination with a sensor, in vivo and / or in biomedical applications and / or in biomedical apparatuses. In another embodiment, the present membrane compositions can be used in connection with a potassium sensor in biological samples such as blood and / or interstitial fluid and are useful for determining potassium levels in a range (e.g. a linear range) of 2 to 8 mM K+which cover critically low and high, and normal, blood K levels. Blood K+levels correlatable with interstitial fluid (ISF) levels.

[0073] In this the first aspect of the ISM embodiment, the present Inventors have discovered that to form a durable and robust ISM that resists formation of the water layer between the ISM and transducer layer, the components of ISM should be present in the following ratios and / or ranges with respect one another and / or the ISM as a whole. In most preferred embodiments, the components are present in the following amounts: I: 0.02 - 20.0 wt% (such as .2 to 20 wt%), more preferably 0.1 - 5.0 wt% (such as .2 -5 wt%), and most preferably 8 wt%; CE: 0.05 - 4.00 wt%, more preferably 0.5 - 2.00 wt%, and most preferably 0.5 wt%; P: 10 - 90 wt%, more preferably 20 - 80 wt%, and most preferably 65 wt%; and BMP: 5 - 80 wt%, more preferably 10 - 60 wt%, and most preferably 32.5 wt%. hi preferred embodiments the mole ratio of K / CE is 0.1 - 10, more preferably 0.2 to 3.5, and most preferably 1.2. In other preferred embodiments the wt% ratio of BMP / P is 0.1-10, more preferably 0.2 -1.5, and most preferably 0.5.

[0074] Also, in the present aspect of the ISM embodiment, the present Inventors have discovered that to form the most preferred durable and robust ISM that resists formation of the water layer between the ISM and transducer layer, the individual components of ISM can be narrowed to the following preferred selection of components: Ionophore - potassium ionophore I (KI-I) (e.g. 2 wt%); Cation exchanger - sodium tetraphenylborate (NaTPB) (e g 0.5 wt%); Plasticiser - bis(2- ethylhexyl) sebacate (DOS) (e.g. 65 wt%); and Base Polymer - PVC (e.g. 32.5 wt%).

[0075] Second Aspect of the ISM -A Sodium Selective ISM:

[0076] In a second aspect of the present ISM embodiment, a sodium (Na+) ion selective membrane composition for use with Na+selective sensors is provided. The present Inventors have unexpectedly found and shown herein that use of such membrane compositions with Na+selective sensors, shows superior sensor response and performance in combination with decreasing and / or preventing water layer formation between the transducer and ISM. In particular, the present Inventors demonstrate herein that the ISM of aspect 1 of the present ISM embodiment can be modified to produce an ISM which is specific and selective for sodium rather than potassium. In the present aspect, the same ISM components described above with respect to the first aspect of the ISM can be employed, save for substitution of the selected ionophore. Other components, such as the plasticizer and / or cation exchanger and / or additional components of the base polymer can likewise be substituted and / or the ranges in which they are present can be altered and / or optimized to tune performance and characteristics of the resulting sodium-selective ISM and sensor.

[0077] In the present aspect, the ionophore is preferably selected from the group consisting of: Na+ionophore I (N,N',N''-triheptyl-N,N',N"-trimethyl-4,4',4''-propylidynetris(3-oxabutyramide), ETH 227), Na+ ionophore II (N, N1-dibenzyl -N,N' -diphenyl- 1,2-phenylenedi oxy diacetamide, ETH 157), Na+ ionophore III (N,N,N',N'-tetracyclohexyl- 1,2-phenylenedi oxy diacetamide, ETH 2120), Na+ ionophore IV (2,3:11,12-didecalino-16-crown-5, 2,6,13,16,19- pentaoxapentacyclo[18.4.4.47,12.01,20.07,12]dotriacontane, DD-16-C-5), Na+ionophore V (4- octadecanoyloxymethyl-N,N,N',N'-tetracyclohexyl-l,2-phenylenedioxydiacetamide, ETH 4120), Na+ionophore VI (bis[(12-crown-4)methyl] dodecylmethylmalonate), Na+ionophore X (4-tert- butylcalix[4]arene-tetraacetic acid tetraethyl ester), monensin, in addition to non-selective cation ionophores such as nigericin, monactin, trinactin, tetranactin, salinomycin, and alike. In a preferred embodiment, the preferred ionophore is sodium ionophore VI (Nal-VI), which has the structure:

[0078] In variation of the second aspect, the ISM may contain an ion transport molecule with the ability to transport hydrogen such as tridodecylamine, ETH 1907, and alike, chloride such as ETH 9033 and alike, lithium such as ETH 2137 and alike, potassium such as valinomycin, BB15C5, and alike, calcium such as ETH 5234 and alike, magnesium such as K22B5, ETH 4030, and alike.

[0079] As discussed above, the Cation exchanger (CE) is lipophilic salt and can be selected from those discussed in the first aspect of the ISM. In other embodiments, the CE is selected from the group consisting of tetraphenyl derivatives, N-(2,3,5,6,8,9,ll,12-octahydro-16-nitro-l,4,7,10,13- benzopentaoxacyclopentadecin-15-yl)-, 2-dodecyl-2-methyl-l,3-propanediyl ester (BME 44), tridodecylmethylammonium salts, and alike. In a much preferred embodiment, the CE is sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), having the structure:

[0080] Further as discussed above, the plasticiser (P) can be selected from those discussed in the first aspect of the ISM. In other embodiments, the CE is selected from the group consisting of: nitrophenyl octyl ether, bis(2-ethylhexyl) sebacate (dioctyl sebacate), dibutyl sebacate, dioctyl phthalate, and alike. DOS is often preferred.

[0081] In the present aspect of the ISM, the preferred amounts, ranges, and ratios of ISM components in the ISM and with respect to one another are similar to those discussed above with respect to the first aspect of the ISM. In particular the components are present in the following ranges / ratios. The Na+ionophore (I) is preferably sodium ionophore VI (NaLVI) and is present in an amount of 0.2 - 15 wt%, more preferably 0.1 - 5 wt%, more preferably 2 wt%. The Cation exchanger is preferably NaTFPB and is present in an amount of 0.05 - 4 wt%, more preferably 0.5 - 2 wt%, such as 0.5 wt%. The plasticizer is preferably DOS and is present in an amount of 10 - 90 wt%, more preferably 20 - 80 wt%, such as 65 wt%. The based polymer is preferably PVC, and is present in an amount of 5 - 80 wt%; more preferably 10 - 60 wt%, for example preferably 32.5 wt%. The mole ratio of ionophore and cation exchanger (Nal-VI / NaTFPB) is preferably 0.1 - 15; more preferably 0.2 - 7.5, such as 5.3. The wt% ratio of ISM polymer and plasticizer (PVC / DOS) is preferably in a range 0.1 - 10, more preferably a range of 0.2 - 1.5, such as 0.5. Without being bound by an intended mechanism of action, it is herein believed that highly accurate and long-lasting measurements can be observed when employed in vivo.

[0082] A Third Aspect of the ISM -A Potassium Selective ISM:

[0083] In a third aspect of the present ISM embodiment, a particularly preferred potassium (K+) ion selective membrane composition for use with K+selective sensors has been discovered. The present Inventors have unexpectedly found that use of such membrane compositions with K selective sensors, shows superior sensor response and performance in combination all the while decreasing, and even eliminating, water layer formation between the transducer and ISM. Like the first and second aspects of the ISM embodiment, the present potassium selected ISM composition employs PVC as a base polymer in the ISM for its decreased porosity and increased hydrophobic properties as discussed above.

[0084] The ISM of the present aspect of the invention employs the following materials in the stated amounts in the composition: potassium ionophore II (KI-II), in an amount between 0.02 - 20.0 wt%, more preferably between 0.1 - 10.0 wt%, for example at 0.9 wt%; KTCIPB as the CE in an amount between 0.05 - 4.00 wt%, more preferably 0.1 - 2.0 wt%, for example at 0.3 wt%; NPOE as the plasticizer in an amount of 10 - 90 wt%, for example 20 - 80 wt%, for example at 65.9 wt%; PVC as the base polymer component in an amount of 5 - 80 wt%, more preferably 10 - 60 wt%, for example at 32.9 wt%. The mole ratio of ionophore and cation exchanger (KI-II / KTCIPB) are preferably in a range of 0.1 - 10, more preferably 0.2 - 3.5, for example 2.0. The wt% ratio of ISM polymer and plasticizer (PVC / NPOE) are preferably in a range of 0.1 - 10, more preferably 0.2 - 1.5, for example at 0.5.

[0085] As discussed below, in preferred embodiments the ISM and underlying sensor layers / components are coated with a coating layer. In the present aspect polyurethane is a suitable coating material, however in preferred embodiments a coating layer comprising polyvinyl acetate has been found to produce superior results. Third Embodiment - Coating Layers for ISS (ISM and Underlying Sensor Layers / Components);

[0086] It has herein been found that use of coating on prior art sensors and more particularly on the ISSs of the present invention, which include the transducer layer of embodiment 1 and / or the ISM layers of the various aspects of embodiment 2, can further and unexpectedly improve sensor performance, robustness, and durability all while decreasing leaching of cytotoxic sensor materials into surround fluid. Furthermore, the coating layers of the present invention and the methods in which they are applied quite unexpectedly significantly increase the lifespan of the ISS.

[0087] The coating layer of the present invention preferably comprises polyurethane (PU) and / or polyvinyl acetate (PVA). The coating layer is formed over the ISM and is preferably in direct contact with the ISM and one or more of the non-conductive layers, as shown in Figs. 3 and 4, and / or the transducer / el ectrode support substrate, as shown in Fig. 5. In these embodiments, the coating layer would not only serve to provide an additional barrier against one-dimensional degradation / transport from the top surface of the ISM, but when in contact with the non- conductive layer and / or the transducer / electrode support substrate, the coating layer completely surrounds the ISM, transducer, and underlying electrode protecting and encapsulating the entirety of the ISS components and layers from directly being in contact with the sample. Furthermore, the present Inventors have found that the coating layer materials of PVA and / or PU have preferable chemical interactions with the non-conductive layer and / or the transducer / electrode support substrate such that the coating layer bonds with these materials unexpectedly forming a durable and long-lasting structure and resulting sensor which lasts significantly longer than an ISS without such coating.

[0088] The coating layer preferably has the following properties: excellent adhesion to both ISM and sensor substrate, water permeability to allow aqueous media to reach the ISM, have a high chemical stability when exposed to light at 4 - 40°C and at physiological pH, and being non- cytotoxic for an in vivo sensor.

[0089] In the present embodiments, adhesion of the ISM to the sensor substrate can be useful to further prevent water layer formation and allow a stable sensor construction, which contributes to the sensor response stability and lifetime. PVC does not have readily available functional groups which can be utilized to obtain significant chemical bonding to the sensor substrate and / or non- conductive layers (shown in Figs 2-5) which include polyimide (PI) and / or PVC. Accordingly, the outer coating layer (OCL) of PU and / or PVA can be applied on top of ISM and in contact with non-conductive substrate material to prevent ISM lift off from the substrate when in aqueous solution. A strong adhesion of PVA and / or PU to PI and / or PVC substrate materials can be obtained due to the dipole-dipole interactions, hydrogen bonds forming between oxygen from the PI and amide hydrogen from the PU etc., and n-n interactions occurring between benzene rings present in both PI and PU. The adhesion of PU to PVC is satisfactory presumably because of the dipole-dipole and van der Waals interactions.

[0090] PU and PVA are also suitable polymers for the coating layer due in part to their porosity and thus water permeability to the surface of the ISM all the while adhering very well to the ISM, PI and / or PVC non-conductive substrate materials, as shown in Figs. 3, 4, and 5 etc.

[0091] Fourth Embodiment - Preferred Combinations of Embodiments 1. 2, and / or 3:

[0092] The transducer layer of Embodiment 1, with or without an underlying electrode such as gold (Au), is preferably employed in an ISS in combination with either or both the ISM of any of the aspects of Embodiment 2 and / or the coating layer of any of the aspects of Embodiment 3. Likewise, the ISM of any of the aspects of Embodiment 2 is preferably employed in an ISS in combination with the coating layer of any of the aspects of Embodiment 3.

[0093] Additional preferred combinations of embodiments 1, 2, and 3 include various ISSs having the following layers:

[0094] Transducer layer of Embodiment 1 + ISM of Aspect 1 of Embodiment 2 + PU coating layer of Embodiment 3;

[0095] Transducer layer of Embodiment 1 + ISM of Aspect 3 of Embodiment 2 + PVA coating layer of Embodiment 3; AND

[0096] Transducer layer of Embodiment 1 + ISM of Aspect 2 of Embodiment 2 + PU coating layer of Embodiment 3. Examples:

[0097] Having described the invention herein in detail, the present invention will now be further described with respect to the following non-limiting examples.

[0098] WORKING EXAMPLE #!: K ISE

[0099] COMPONENTS:

[0100] A new ISM composition is as follows (Components and their wt%):

[0101] Ionophore: potassium ionophore I (KI-I), 2 wt%; Cation exchanger: sodium tetraphenylborate (NaTPB), 0.5 wt%; Plasticizer: bis(2-ethylhexyl) sebacate (DOS), 65 wt%; Polymer: polyvinyl chloride (PVC), 32.5 wt%.

[0102] RESULTS:

[0103] Experimental Conditions and Materials

[0104] All ISM components are dissolved in anhydrous tetrahydrofuran (THF). Working electrode for K+sensor is obtained by drop-casting the desired amount of ISM solution on graphite ink-coated gold microelectrode and evaporating the THF solvent (overnight at room temperature). The tested transducer layers other than graphite ink include:

[0105] Electrochemically polymerized poly(3,4-ethylenedioxythiophene) polystyrene sulfonate PEDOT:PSS

[0106] Drop-casted poly (3 -hexylthiophene-2 5-diyl) (PHT)

[0107] The reference electrode is a Ag / AgCl electrode having 3 M NaCl as the internal liquid junction unless otherwise specified. All experiments are conducted in 50 mM phosphate-buffered saline containing 140 mM NaCl solution at a pH of 7.4 at room temperature. Calibration plot experiments were conducted using KC1 solution, and selectivity tests were conducted using NaCl, LiCl, MgCh, and NaHCC solutions. Water layer test experiments were conducted in aqueous solutions of 100 mM KC1 and 100 mM NaCl.

[0108] Figure 6 shows water layer test results of working electrodes containing either PEODT:PSS or PHT as solid-contact transducer layer. Figure 7 shows sensor calibration results conducted with different K+concentrations ranging from 2 to 8 mM. Figure 8 shows selectivity test results towards K+.

[0109] The presence of a water layer can be verified by recording the sensor response in a solution of target ion, K+in the current invention, then in the primary interfering ion, which is Na+in the current invention, and then target ion again over a few hours of test time. Unstable sensor response particularly in the primary interfering ion solution indicates non-selective sensor response which is caused by the ions present in the water layer reaching the transducer. A significant improvement in the signal stability can be achieved by replacing PEDODPSS with a more hydrophobic polymer PHT as shown in Figure 1. The PSS portions of the PEDOT :PSS are hydrophilic and thus encourage water entrapment under the ISM.

[0110] The current invention describes the use of graphite ink as the solid-contact transducer combined with the PVC-based ISM and PU as a coating layer on the ISM, which results in outstanding sensor performance due to the suppression of water layer formation. Figure 6 demonstrates the sensor response over 24 h of testing time to K+concentrations varying within 2 and 8 mM.

[0111] The current sensor invention has high selectivity to K+such that the spiking of 10 mM NaCl, 5 mM LiCl, 5 mM MgCh, or 50 mM NaHCCE does not induce a significant signal change as shown in Figure 7. This is attributed to high selectivity of ionophore in addition to excellent ISM adhesion to the substrate and the inhibition of water penetration towards the transducer.

[0112] Figure 8 shows a demonstration of selectivity of the sensor when at 2 mM K+in the presence of 10 mM NaCl, 50 mM NaHCO3, 5 mM LiCl, and 5 mM MgCl2.

[0113] WORKING EXAMPLE #2: Na+ISE

[0114] COMPONENTS:

[0115] A new ISM composition is as follows (components and their wt%):

[0116] Ionophore: sodium ionophore VI (Nal-VI), 2 wt%; Cation exchanger (lipophilic salt): sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), 0.5 wt%; Plasticizer: bis(2-ethylhexyl) sebacate (DOS), 65 wt%; Polymer: polyvinyl chloride (PVC), 32.5 wt%.

[0117] Transducer layer: Identical to Working Example #1. RESULTS:

[0118] Experimental Conditions and Materials

[0119] All ISM components are dissolved in anhydrous tetrahydrofuran (THF). Working electrode for Na+sensor is obtained by drop-casting the desired amount of ISM solution on graphite ink- coated gold microelectrode and evaporating the THF solvent (overnight at room temperature). The reference electrode is a Ag / AgCl electrode having 3 M NaCl as the internal liquid junction. All experiments are conducted in 100 mM 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffer solution at a pH of 7.2 at room temperature. Calibration plot experiments were conducted using NaCl solution, and selectivity tests were conducted using LiCl, KC1, and MgCh solutions.

[0120] Figure 9 shows sensor response to Na+within 1 - 300 mM (A) and the corresponding calibration plot (B) of three sensors.

[0121] Figure 10 shows selectivity test results when using 10 mM K+, 10 mM Li+, and 10 mM Mg2+. The potential signal is not significantly affected by the addition of the tested interferent ions. Moreover, the sensitivity of the sensor is not significantly affected (5% variation) by the presence of the tested interferent ions.

[0122] Graphite ink was used as the exemplary transducer layer throughout the experiments, however others might be used with potential similar results.

[0123] Figure 9. A) Demonstration of Na+sensing at different sodium concentrations (1 mM - 300 mM). B) Linear plot of log(Na+) versus EMF (potential) signal. Theoretical maximum sensitivity (slope) at 25°C: 59 mV / decade. Obtained: 49.8 ± 0.8 mV / decade.

[0124] Figure 10. A) Selectivity to Na+when adding Li+, Mg2+, and K+. Response to interfering ions is insignificant. B) EMF signal changes with interfering ions showing variation less than 5%.

[0125] WORKING EXAMPLE #3: K+ ISE

[0126] COMPONENTS:

[0127] A new ISM composition is as follows (components and their wt%): Ionophore: potassium ionophore II (KI-II), 0.9 wt%; Cation exchanger: potassium tetrakis(4- chlorophenyl)borate (KTC1PB), 0.3 wt%; Plasticizer: 2-nitrophenyl octyl ether (NPOE), 65.8 wt%; Polymer: polyvinyl chloride (PVC), 32.9 wt%.

[0128] Transducer layer: Identical to Working Example #1 and #2.

[0129] Coating Layer 1 : PU

[0130] Coating Layer 2: PVA

[0131] Preamble to Working Examples #4 and #5:

[0132] Working Examples #1 to #3, and Figs. 1 to 10, show in some situations utilisation of a specific ISM formulation on a firm substrate. When an ISS is employed for in vivo applications flexible substrates are often desired having minimal thickness (e.g. for example less than 1000 microns, less than 800 microns, such as 200-500 microns, etc.) while maintaining, or even increasing, sensor sensitivity and functionality of the K+. The compositions of working examples #4 and #5 are capable of producing such ISSs. Figures 11-14 exhibit data acquired with the new ISM formula containing a potassium based cation exchanger on a flexible substrate. All ISM compositions are non-cytotoxic, confirming that the ISM components are not leaching into the surrounding environment.

[0133] WORKING EXAMPLE #4: K ISE

[0134] COMPONENTS:

[0135] A new ISM composition is as follows (Components and their wt%):

[0136] Ionophore: potassium ionophore I (KI-I), 2 wt%; Cation exchanger: potassium tetrakis (4- chlorophenyl) borate (KTC1PB) 0.6 wt%; Plasticizer: bis(2-ethylhexyl) sebacate (DOS), 64.7 wt%; Polymer: polyvinyl chloride (PVC), 32.7 wt%.

[0137] RESULTS:

[0138] Experimental Conditions and Materials

[0139] All ISM components are dissolved in anhydrous tetrahydrofuran (THF). Both working and reference electrodes are prepared on a polyimide flexible substrate. A working electrode for K+sensor is obtained by dip-coating the desired amount of ISM solution on graphite ink-coated gold microelectrode and evaporating the THF solvent at 60°C. The reference electrode is an electroplated Ag|AgCl electrode. All experiments are conducted in 50 mM phosphate-buffered saline containing 140 mM NaCl solution at a pH of 7.4 at room temperature. Calibration plot experiments were conducted using KC1 solution, and selectivity tests were conducted using NaCl, LiCl, MgCh, and NaHCCh solutions.

[0140] Figure 11(A) shows a long term experiment conducted in phosphate-buffered saline to measure the potential change when a specified amount of K+ is spiked into the electrochemical cells containing a working electrode and an electroplated Ag|AgCl reference electrode made on a polyimide flexible substrate. The data was recorded by spiking 2 mM, 6 mM, 4 mM and 8 mM K+ in a series for 3 consecutive rounds over a 12 h time frame. On average, the K+sensitivities obtained were ~60 mV / decade with <10% change over the 12 h period, demonstrating satisfactory results in vitro; acquired sensitivity values are adequately close to expected theoretical values, indicating the ability of the K+sensor to accurately measure fluctuations in K+concentration in solution.

[0141] Figure 12 (A) shows a long term simulated dialysis experiment conducted in phosphate-buffered saline to measure the potential change when a specified amount of K+was spiked into the electrochemical cells. The data was recorded by gradually decreasing the K+ions from 7 mM, 6 mM, 5 mM and 4.5 mM followed by increasing the K+ions to 5.5 mM and 6 mM in a series over a 6 h time frame. Retrospective sensitivity analysis value was 57 mV / decade, in alignment with the expected theoretical range of values. Results demonstrated the successful ability of the K+sensor to record the varying levels of K+over a period of 17 hours in vitro.

[0142] Figure 13 evaluates K+sensor performance in a complex biological matrix containing approximately 3.2 mM K+. The data was recorded by spiking 2 mM, 4 mM, 6 mM. On average, the concentration of K+ measured was 3.1 mM, demonstrating the successful ability of the K+ sensor to respond to varying concentrations of K+ in a complex biological matrix. This indicates the K+ sensor is capable of distinguishing target electrolytes irrespective of interference from other components in the surrounding matrix such as other proteins or lipids and other interfering ions such as Na+ Figure 14 demonstrates sensor selectivity, when the solution is spiked with 2 mM K+, 30 mM NaHCOa, 10 mM NaCl, 5 mM LiCl, 4 mM MgCL, followed by an additional 2 mM K+. On average, after the addition of interfering ions, the total potential change when translated into the potassium domain was demonstrated to be 0.18 mM, which is within the defined acceptable range (between 0.3 to 0.5 mM). This indicates sensor's ability to distinguish target ions from other potentially interfering ions, demonstrating selectivity in a complex solution, as there is minimal fluctuation between potential values measured when spiked by ions excluding potassium.

[0143] Figure 15 depicts the evaluation of a raw electrical signal (left axis) and blood potassium concentration reference (right axis) during a clinical trial (6h). Measurements conducted 1 hour before and after dialysis. The estimated potassium concentration was calculated from an electrical signal (Fig. 15) using a retrospective method as shown in Figure 16. The retrospective analysis was conducted via the generation of a single electrical value paired with each reference measurement: this was done by averaging the electrical signal around the reference measurement (time window from -7.5 to +7.5min centered on the reference measurement). Following this, sensor parameters were evaluated via fitting a Nernst equation model including a linear drift to the data in order to match the reference data as closely as possible. Some data points are excluded because of the warm-up period and hemolyzed samples. Results indicate the potential of the K+ sensor to accurately track potassium variations in vivo, as a likely correlation is exhibited between potassium concentrations measured in the blood versus via the K+ sensor in interstitial fluid.

[0144] The current invention describes the use of graphite ink as the solid-contact transducer combined with the PVC-based ISM and a PU-based hydrophilic coating layer on the ISM, which results in outstanding sensor performance due to the suppression of water layer formation. The current sensor invention has high selectivity to K+such that the spiking of 10 mM NaCl, 5 mM LiCl, 5 mM MgCh, or 50 mM NaHCCL does not induce a significant signal change as shown in Figure 14. This is attributed to high selectivity of the ion selective membrane in addition to excellent ISM adhesion to the substrate, attributed to the hydrophobicity of the ISM and the inhibition of water layer formation at the transducer / membrane interface WORKING EXAMPLE #5: K ISE

[0145] A new ISM composition is as follows (Components and their wt%):

[0146] Ionophore: potassium ionophore I (KI-I), 2 wt%; Cation exchanger: potassium tetrakis (4- chlorophenyl) borate (KTC1PB) 0.5 wt%; Plasticizer: bis(2-ethylhexyl) sebacate (DOS), 64.5wt%; Polymer: polyurethane (PU) 33 wt%.

[0147] RESULTS:

[0148] Experimental Conditions and Materials

[0149] All ISM components are dissolved in anhydrous tetrahydrofuran (THF). Both working and reference electrodes are prepared on a polyimide flexible substrate. A working electrode for K+sensor is obtained by dip-coating the desired amount of ISM solution on graphite ink-coated gold microelectrode and evaporating the THF solvent at 60 °C. The reference electrode is an electroplated Ag|AgCl electrode. All experiments are conducted in 50 mM phosphate-buffered saline containing 140 mM NaCl solution at a pH of 7.4 at room temperature. Calibration plot experiments were conducted using KC1 solution, and selectivity tests were conducted using NaCl, LiCl, MgCh, and NaHCCh solutions. Figure 11(B) shows a long term experiment to measure the potential change when a specified amount of K+ is spiked into the electrochemical cells . The data was recorded by spiking 2 mM, 6 mM, 4 mM and 8 mM K+ in a series for 3 consecutive rounds over a 12 h time frame. On average, the K+ sensitivities obtained were ~61 mV / decade with <10% change over the 12 h period, demonstrating satisfactory results in vitro; acquired sensitivity values are adequately close to expected theoretical values, indicating the ability of the K+ sensor to accurately measure fluctuations in K+ concentration in solution.

[0150] Figure 12 (B) shows a long term simulated dialysis experiment to measure the potential change when a specified amount of K+ was spiked into the electrochemical cells c. The data was recorded by gradually decreasing the K+ ions from 7 mM, 6 mM, 5 mM and 4.5 mM followed by increasing the K+ ions to 5.5 mM and 6 mM in a series over a 6 h time frame. Retrospective sensitivity analysis value was 60mV / decade, in alignment with the expected theoretical range of values. Results demonstrated the successful ability of the K+ sensor to record the varying levels of K+ over a period of 17 hours in vitro.

Claims

Claims:

1. A functional ion-selective sensor (ISS) comprising: an ion-selective membrane (ISM) comprising: an ion specific ionophore (I); a cation exchanger (CE) selected from the group consisting of: sodium tetraphenylborate (NaTPB), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), and potassium tetrakis(4-chlorophenyl)borate (KTCIPB); a base membrane polymer (BMP) comprising polyvinyl chloride (PVC) and / or polyurethane (PU), and a plasticiser (P) selected from the group consisting of: bis(2-etheylhexyl) sebacate (DOS); and 2-nitrophyloctyl ether (NPOE); wherein the components are present in the membrane in the following wt% ranges:I between 0.02 - 20 wt%, inclusive;CE between 0.05 - 4 wt%, inclusive;P between 10 - 90 wt%, inclusive; andBMP between 5 - 80 wt%, inclusive, wherein the mole ratio of I and CE is between 0.1 - 10, inclusive, and wherein the wt% ratio of BMP and P is between 0.1 - 10, inclusive.

2. The ISS of claim 1, wherein: the components of the ISM comprise:I: potassium ionophore I (KI-I);CE: sodium tetraphenylborate (NaTPB);P: bis(2-ethylhexyl) sebacate (DOS); andBMP: PVC. the components are present in the following amounts:I: 0.02 - 20,0 wt% (for example 0.2 wt% to 20.0 wt %), more preferably 0.1 - 10,0 wt%, and most preferably 8.00 wt%;CE: 0.05 - 4.00 wt%, more preferably 0.5 - 2.00 wt%, and most preferably 0.5 wt%;P: 10 - 90 wt%, more preferably 20 - 80 wt%, and most preferably 65 wt%; andBMP: 5 - 80 wt%, more preferably 10 - 60 wt%, and most preferably 32.5 wt%; the mole ratio of K / CE is 0.1 - 10, more preferably 0.2 to 3.5, and most preferably 1.

2. the wt% ratio of BMP / P is 0.1-10, more preferably 0.2 -1.5, and most preferably 0.5.

3. The ISS of claim 1, wherein: the components of the ISM comprise the following components in the amounts of:I: sodium ionophore VI (Nal-VI) in an amount of 0.2 - 15 wt%, more preferably 0.1 - 5 wt%, more preferably 2 wt%;CE: NaTFPB in an amount of 0.05 - 4 wt%, more preferably 0.5 - 2 wt% such as 0.5 wt%;P: DOS in an amount of 10 - 90 wt%, more preferably 20 - 80 wt%, such as 65 wt%;BMP: PVC in an amount of 5 - 80 wt%; more preferably 10 - 60 wt%, for example preferably 32.5 wt%; the mole ratio of I and CE (Nal-VI I NaTFPB) is preferably 0.1 - 15; more preferably 0.2 - 7.5, such as preferably 5.3; and the wt% ratio of ISM polymer and plasticizer (PVC I DOS) is preferably in a range 0.1 - 10, more preferably a range of 0.2 - 1.5, such as 0.5.

4. The ISS of claim 1, wherein:the components of the ISM comprise the following components in the amounts of:I: potassium ionophore II (KI-II) in an amount between 0.02 - 20.0 wt%, more preferably between 0.1 - 10.0 wt%, for example at 0.9 wt%;CE: KTCIPB in an amount between 0.05 - 4.00 wt%, more preferably 0.1 - 2.0 wt%, for example at 0.3 wt%;P: NPOE in an amount of 10 - 90 wt%, for example 20 - 80 wt%, for example at 65.9 wt%;BMP: PVC in an amount of 5 - 80 wt%, more preferably 10 - 60 wt%, for example at 32.9 wt%. the mole ratio of ionophore and cation exchanger (KI-II I KTCIPB) are preferably in a range of 0.1 - 10, more preferably 0.2 - 3.5, for example 2.0; and the wt% ratio of ISM polymer and plasticizer (PVC / NPOE) are preferably in a range of 0.1 - 10, more preferably 0.2 - 1.5, for example at 0.5.

5. The ISS of claim 1, further comprising a carbon-based transducer layer, preferably wherein the transducer comprises a carbon-based ink, preferably wherein the carbon-based ink is a commercially available graphite ink developed for flexographies printing by Sun Chemical Inc. The product is sold by Sigma-Aldrich under the product number # 901970 and the product name of SunTronic®.

6. The ISS of claim 1, further comprising a coating layer comprising a polymer selected from the group consisting of PU and PVA.

7. The ISS of claim 6, wherein: the ISS further comprises a supporting substrate and / or non-conductive layer; and the coating layer is in contact with the ISM and a supporting substrate and / or non-conductive layer.

8. The ISS of claim 6, wherein the the components of the ISM comprise the following components in the amounts of:I: potassium ionophore II (KI-II) in an amount between 0.02 - 20.0 wt%, more preferably between 0.1 - 10.0 wt%, for example at 0.9 wt%;CE: KTCIPB in an amount between 0.05 - 4.00 wt%, more preferably 0.1 - 2.0 wt%, for example at 0.3 wt%;P: NPOE in an amount of 10 - 90 wt%, for example 20 - 80 wt%, for example at 65.9 wt%;BMP: PVC in an amount of 5 - 80 wt%, more preferably 10 - 60 wt%, for example at 32.9 wt%. the mole ratio of ionophore and cation exchanger (KI-II I KTCIPB) are preferably in a range of 0.1 - 10, more preferably 0.2 - 3.5, for example 2.0; and the wt% ratio of ISM polymer and plasticizer (PVC / NPOE) are preferably in a range of 0.1 - 10, more preferably 0.2 - 1.5, for example at 0.5, and wherein the coating layer comprises PVA.

9. A layered biosensor for the in vivo monitoring of electrolytes, the biosensor comprising, in order: a hydrophilic coating layer, a hydrophobic ISM layer, a hydrophobic transducer layer, an electrode layer, and non-conductive substrate.

10. The biosensor of claim 9, wherein the hydrophilic coating layer is in contact with both the ISM layer and the non-conductive substrate.

11. The biosensor of claim 9, wherein the hydrophobic ISM comprises: an ion specific ionophore (I); a cation exchanger (CE) selected from the group consisting of: sodium tetraphenylborate (NaTPB), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), and potassium tetrakis(4-chlorophenyl)borate (KTCIPB); a base membrane polymer (BMP) comprising polyvinyl chloride (PVC) and / or polyurethane (PU), and a plasticiser (P) selected from the group consisting of: bis(2-etheylhexyl) sebacate (DOS); and 2-nitrophyloctyl ether (NPOE); wherein the components are present in the membrane in the following wt% ranges:I between 0.02 - 20 wt%, inclusive;CE between 0.05 - 4 wt%, inclusive;P between 10 - 90 wt%, inclusive; andBMP between 5 - 80 wt%, inclusive, wherein the mole ratio of f and CE is between 0.1 - 10, inclusive, and wherein the wt% ratio of BMP and P is between 0.1 - 10, inclusive.

12. The biosensor of claim 11, wherein: the components of the ISM comprise:I: potassium ionophore I (KI-I);CE: sodium tetraphenylborate (NaTPB);P: bis(2-ethylhexyl) sebacate (DOS); andBMP: PVC. the components are present in the following amounts:I: 0.02 - 20.0 wt% (for example 0.2 wt% to 20.0 wt %), more preferably 0.1 - 10.0 wt%, and most preferably 8.00 wt%;CE: 0.05 - 4.00 wt%, more preferably 0.5 - 2.00 wt%, and most preferably 0.5 wt%;P: 10 - 90 wt%, more preferably 20 - 80 wt%, and most preferably 65 wt%; and BMP: 5 - 80 wt%, more preferably 10 - 60 wt%, and most preferably 32.5 wt%; the mole ratio of K / CE is 0.1 - 10, more preferably 0.2 to 3.5, and most preferably 1.

2. the wt% ratio of BMP / P is 0.1-10, more preferably 0.2 -1.5, and most preferably 0.5.

13. The biosensor of claim 11, wherein: the components of the ISM comprise the following components in the amounts of:I: sodium ionophore VI (Nal-VI) in an amount of 0.2 - 15 wt%, more preferably 0.1 - 5 wt%, more preferably 2 wt%;CE: NaTFPB in an amount of 0.05 - 4 wt%, more preferably 0.5 - 2 wt% such as 0.5 wt%;P: DOS in an amount of 10 - 90 wt%, more preferably 20 - 80 wt%, such as 65 wt%;BMP: PVC in an amount of 5 - 80 wt%; more preferably 10 - 60 wt%, for example preferably 32.5 wt%; the mole ratio of I and CE (Nal-VI I NaTFPB) is preferably 0.1 - 15; more preferably 0.2 - 7.5, such as preferably 5.3; and the wt% ratio of ISM polymer and plasticizer (PVC I DOS) is preferably in a range 0.1 - 10, more preferably a range of 0.2 - 1.5, such as 0.5.

14. The biosensor of claim 11, wherein: the components of the ISM comprise the following components in the amounts of:I: potassium ionophore II (KI-II) in an amount between 0.02 - 20.0 wt%, more preferably between 0.1 - 10.0 wt%, for example at 0.9 wt%;CE: KTCIPB in an amount between 0.05 - 4.00 wt%, more preferably 0.1 - 2.0 wt%, for example at 0.3 wt%;P: NPOE in an amount of 10 - 90 wt%, for example 20 - 80 wt%, for example at 65.9 wt%;BMP: PVC in an amount of 5 - 80 wt%, more preferably 10 - 60 wt%, for example at 32.9 wt%. the mole ratio of ionophore and cation exchanger (KI-II / KTCIPB) are preferably in a range of 0.1 - 10, more preferably 0.2 - 3.5, for example 2.0; and the wt% ratio of ISM polymer and plasticizer (PVC I NPOE) are preferably in a range of 0.1 - 10, more preferably 0.2 - 1.5, for example at 0.5.

15. The biosensor of claim 9, wherein the transducer layer is a carbon-based transducer layer, preferably wherein the transducer comprises a carbon-based ink, preferably wherein the carbonbased ink is a commercially available graphite ink developed for flexographies printing by Sun Chemical Inc. The product is sold by Sigma-Aldrich under the product number # 901970 and the product name of SunTronic®.

16. The biosensor of claim 9, wherein the coating layer comprises a polymer selected from the group consisting of PU and PVA, and wherein the coating layer is in contact with the ISM and the supporting substrate.

17. The ISS of claim 16, wherein the the components of the ISM comprise the following components in the amounts of:I: potassium ionophore II (KI-II) in an amount between 0.02 - 20.0 wt%, more preferably between 0.1 - 10.0 wt%, for example at 0.9 wt%;CE: KTCIPB in an amount between 0.05 - 4.00 wt%, more preferably 0.1 - 2.0 wt%, for example at 0.3 wt%;P: NPOE in an amount of 10 - 90 wt%, for example 20 - 80 wt%, for example at 65.9 wt%;BMP: PVC in an amount of 5 - 80 wt%, more preferably 10 - 60 wt%, for example at 32.9 wt%. the mole ratio of ionophore and cation exchanger (KI-II / KTC1PB) are preferably in a range of 0.1 - 10, more preferably 0.2 - 3.5, for example 2.0; and the wt% ratio of ISM polymer and plasticizer (PVC I NPOE) are preferably in a range of 0.1 -10, more preferably 0.2 - 1.5, for example at 0.5, and wherein the coating layer comprises PVA.

18. A method of forming an ISS of claim 1, the method comprising the steps of:(a) forming an ISM on an underlying transducer / electrode / substrate layer;(b) optionally forming a coating layer on the ISM and encapsulating an underlying layers,(c) contacting the coating layer with the ISM and an underlying non-conductive layer. thereby forming a layered composition for use with a biosensor for the in vivo monitoring of electrolytes.

19. The method of claim 18, wherein the layers are applied by a process selected from the group consisting of: spin-coating, spray-coating, casting (e.g. drop-casting), ink-jet printing, electrodeposition, and screen printing on an underlying layer.

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