Biosensors for in vivo monitoring of electrolytes

Abstract

In one embodiment, the present invention provides a layered composition for use with a biosensor for in vivo monitoring of electrolytes. The layered composition comprises a hydrophilic coating layer and a hydrophobic ISM layer. In other embodiments, the present invention also provides a biosensor comprising such a layered composition, as well as a method for producing the layered composition and biosensor described herein.

Description

Technical Field

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[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 523,060 (PROT.P - 004 - PV), filed on Jun. 24, 2023, which can be used in combination with any of the compositions, devices and / or methods described in PCT / US2022 / 037198 (PROT.P - 001 - WO); PCT / US2022 / 052927 (PROT.P - 002 - WO); PCT / IB2023 / 061417 (PROT.P - 003 - WO); U.S. Provisional Patent Application No. 63 / 605,425 (PROT.P - 005 - PV); U.S. Provisional Patent No. 63 / 556,008 (PROT.P - 006 - PV); and U.S. Provisional Patent No. 63 / 651,839 (PROT.P - 009 - PV), which are incorporated by reference in their entirety for all purposes.

Background Art

[0002] The selective detection of ions is typically achieved by hydrophobic ion - selective membranes (ISMs). The use of hydrophobic ISMs for in - vivo applications such as transdermal ion monitoring requires the ISM to be biocompatible while maintaining sensor performance that enables accurate and reliable analyte detection. Hydrophobic ISMs are not suitable for in - vivo use without modification for mainly two reasons: i) the leaching of cytotoxic ISM components into tissues, and ii) high biofouling caused by cells and proteins adhering to the ISM surface. Furthermore, the leaching of ISM components and biofouling result in loss of sensor lifetime in addition to decreased response accuracy, sensitivity, and reliability.

Summary of the Invention

[0003] The present invention solves problems in the art and provides a specific coating layer for use with a specific ISM layer that can be used in combination with a specific ISM layer for a biosensor for continuous measurement of analytes such as potassium and sodium. In a first embodiment, the present invention provides a layered composition for use with a biosensor for in vivo monitoring of electrolytes, comprising a hydrophilic coating layer and a hydrophobic ISM layer, preferably in direct contact without the presence of an intervening layer.

[0004] In another embodiment, the present invention provides a layered biosensor for in vivo monitoring of electrolytes, the biosensor comprising, in order, a hydrophilic coating layer, a hydrophobic ISM layer, a transducer layer, and an electrode layer, preferably each layer being in direct contact with its adjacent layer.

[0005] In further embodiments, the present invention provides a method for forming a layered composition for use with a biosensor for in vivo monitoring of electrolytes. The method includes (a) forming or providing a hydrophobic ISM layer, and (b) optionally oriented onto and in contact with the ISM layer, forming and applying a hydrophilic coating layer composition to the hydrophobic ISM layer formed or provided in step (a), thereby forming a layered composition for use with a biosensor for in vivo monitoring of electrolytes.

[0006] In further embodiments, the present invention provides a method for forming a biosensor for in vivo monitoring of electrolytes. The method includes (a) forming or providing an electrode layer; (b) forming or providing a transducer layer on the electrode layer formed or provided in step (a); (c) forming or providing a hydrophobic ISM layer on the transducer layer formed or provided in step (b); (d) applying a hydrophilic coating layer composition on the hydrophobic ISM layer formed or provided in step (c); and (e) applying the hydrophilic coating layer composition formed in step (b) to the hydrophobic ISM layer formed or provided in step (a), wherein the layer is optionally formed in direct contact with an adjacent layer formed in a previous step. [Brief explanation of the drawing]

[0007] [Figure 1] This figure shows the results from sections of examples where valinomycin levels were detected after 24 hours, 48 ​​hours, and 72 hours, with and without an outer coating layer (OCL). [Figure 2] The figure shows results from the example section where cell viability data are provided, demonstrating that the addition of OCL reduces silver ink toxicity by limiting the diffusion of the component into the external culture medium. [Figure 3] This figure shows the results from the example section, comparing the overall performance of the two OCLs with the base ISM itself. [Figure 4] This figure shows the results from the example section. [Figure 5] This figure shows aging degradation data obtained from the section on examples of various ISM and coating layer configurations. [Figure 6] This figure shows aging degradation data obtained from the section on examples of various ISM and coating layer configurations. [Figure 7] This figure shows aging degradation data obtained from the section on examples of various ISM and coating layer configurations. [Modes for carrying out the invention]

[0008] The present invention provides novel compositions and methods that solve problems of the prior art. In preferred embodiments, the present invention provides compositions for layers(s) of biosensors for in vivo monitoring of electrolytes, layered compositions useful as such biosensors, and methods for forming them. The sensors can selectively detect ions. In some embodiments, to solve problems in the art related to the in vivo use of hydrophobic ISMs, the present invention provides hydrophilic coating compositions and / or layers for biosensors, and in other embodiments, a biosensor comprising at least one layer of conductive electrodes, an electrochemical transducer layer, a hydrophobic ISM, and an electrode (e.g., a working electrode and a reference electrode) including a hydrophilic coating layer (e.g., preferably where the ISM covers only the working electrode). The sensor preferably comprises three interfaces: i) a tissue-ISM interface formed by the hydrophilic coating layer, ii) a coating layer-transducer layer interface formed by the ISM, and iii) an ISM-conductive electrode interface formed by the transducer layer.

[0009] Definition: conductive electrode A conductive material used to facilitate electron transfer and enable the measurement of electron signals. Such a conductive material can be formed from a variety of conductive materials, but in some preferred embodiments, it includes gold.

[0010] Transducer layer A layer that undergoes a reversible chemical change in response to changing analyte concentrations, resulting in a conversion between chemical and electrical energy.

[0011] Hydrophobic ISM A membrane comprising a polymer matrix that may contain selective analyte transport molecules such as ion-selective ionophores, lipophilic components that exchange target analytes with the analyte transport molecules, and plasticizers. An example of such an ISM is a potassium-selective membrane composed of potassium ionophore I, a lipophilic salt of tetraphenyl borate, and polyvinyl chloride, as a membrane polymer plasticized with dioctyl sebacate. In certain embodiments, the hydrophobicity of the ISM is defined in comparison to the hydrophilicity of the coating layer. In these embodiments, the term hydrophobicity applied to the properties of the ISM means that the ISM is more hydrophobic and / or less hydrophilic than the coating layer.

[0012] Hydrophilic coating A coating layer applied over a hydrophobic ISM that enables the biosensor to be biocompatible, limits the diffusion and light transmission of the analyte, and reduces the overall biosensor impedance without impairing the analytical performance of the biosensor. In certain embodiments, the hydrophilicity of the coating is defined in comparison to the hydrophilicity of the coating layer. In these embodiments, the term hydrophilicity applied to the properties of the coating layer means that the coating layer is more hydrophilic and / or less hydrophobic than the ISM.

[0013] Embodiment Throughout this specification, references to “one embodiment,” “another embodiment,” “embodiment,” “several embodiments,” etc., mean that certain elements (e.g., functions, structures, properties, and / or features) described in relation to an embodiment are included in at least one embodiment described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that any described element(s) and / or feature(s) of any embodiment can be combined with any other described embodiment in any appropriate manner.

[0014] numerical values Numerical values ​​in this specification and in the claims herein reflect average values. Furthermore, unless otherwise indicated, numerical values ​​should be understood to include numerical values ​​that are the same when reduced to the same number of significant figures, and numerical values ​​that differ from the stated values ​​by less than the experimental error of the type of conventional measurement technique described in this application for determining the value.

[0015] Preferred coating layer: 1. Hydrophilic coating layer: Structure-ISM interface a. Composition Hydrophilic polymers (polyurethanes and / or others) are a class of polymers that exhibit a strong affinity for water, making them highly suitable for applications in the biomedical industry. These materials possess inherent properties that allow them to absorb and retain significant amounts of water without losing their structural integrity. They typically present hydrophilic surfaces, enabling interaction with biological fluids and tissues and reducing biofouling. These properties make them particularly suitable for a variety of biomedical applications in the development of medical devices and implants, drug delivery systems, and tissue engineering. In the biomedical industry, hydrophilic polymers (polyurethanes, etc.) are used to create flexible, biocompatible materials that can mimic the properties of natural tissues.

[0016] Preferred coating polymers have hydrophilic (soft) and hydrophobic (hard) segments that, when in an aqueous medium, have the ability to absorb water, swell, and form a hydrogel. Isocyanates are important components in polyurethane synthesis. Either aliphatic or aromatic types of isocyanates can be used, but it has been found herein that the choice of isocyanate can affect the reactivity and properties of the final polymer. Aliphatic isocyanates are less reactive than their aromatic counterparts. Some isocyanates that can be used in the synthesis of hydrophilic polyurethanes include hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), toluene diisocyanate (TDI), xylene diisocyanate, and methylenediphenyl diisocyanate (MDI).

[0017] The hydrophilicity of the synthesized polymer (e.g., polyurethane) depends on the selection of the hydrophilic polyol. Some examples of polyols can include, but are not limited to, polyethylene glycol, polyvinyl alcohol, polypropylene glycol, diethylene glycol, ethylene glycol, or glycerol. In another example, the hydrophilicity can be increased by incorporating hydrophilic additives such as polyvinyl pyrrolidone, polyethylene oxide, polyacrylic acid, and crosslinking agents or chain extenders such as diamines, diacids, diols, etc.

[0018] In another example, the surface hydrophilicity can be increased by plasma treatment or grafting of hydrophilic functional groups such as amines, oxygen-containing functional groups, etc.

[0019] In another example, the polymer contains a photoinitiator to enable photosensitivity. Some examples of photoinitiators used in polyurethane can be 1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-2-methyl-1-phenyl-propan-1-one, bis(2,4,6-trimethylbenzoyl)phenyl phosphine oxide, or diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, in combination with some additives such as polyfunctional acrylates and some additives such as plasticizers and stabilizers, but are not limited to these. This polymer can be obtained by using diisocyanate in combination with a photosensitive polyol and a hydroxyl-terminated acrylate. In another example, polysaccharides such as norbornene and polyurethane or polygelatin modified with thiol groups can be used to induce a thiol-ene click reaction when exposed to UV or near-UV light (320 nm - 500 nm). Subsequently, light of a predetermined wavelength is irradiated onto the target area using an appropriate photomask to initiate the polymerization process.

[0020] In another example, acridine-containing polyurethanes can be used to enable photodegradation of the polymer. The polymer can be obtained using an acridine diol monomer together with polyethylene glycol (PEG) for the preparation of hydrophilic polyurethanes. This enables the application of the coating polymer and subsequent selective removal of the polymer from unwanted areas, which is enabled by exposure with UV or blue light (λ = 320 nm - 500 nm) using an appropriate photomask.

[0021] In other preferred embodiments, both the ISM and the coating layer contain the same or a similar base polymer (e.g., polyurethane). In this embodiment, the base polymer of the ISM is adjusted or selected to be more hydrophobic / less hydrophilic than the polymer of the coating layer. Similarly, in other embodiments, the base polymer of the coating layer is adjusted or selected to be less hydrophobic / more hydrophilic than the polymer of the ISM.

[0022] b. Obtaining a hydrophilic layer on the ISM The hydrophilic polymer can be dissolved in a volatile organic solvent such as cyclopentanone, tetrahydrofuran, or chloroform to form a concentrated solution that can be used to deposit the polymer onto the ISM layer by spin coating, spray coating, casting (e.g., drop casting), inkjet printing, electrodeposition, or screen printing. Also, by immersing the electrode in the polymer solution, the electrode can be coated with the polymer by immersion coating. The thickness of the polymer should not reduce the sensor performance when it limits the transmitted light to the ISM and prevents leaching of the membrane components into the medium.

[0023] In another example, the coating layer is obtained from multiple layers of hydrophobic or hydrophilic polymers. ISMs generally consist of hydrophobic materials for essential hydrophobic components such as ionophores, cation exchangers, and plasticizers. To overcome adhesion problems that arise between the hydrophobic surface and the hydrophilic material, a multi-stage film coating process is carried out such that the hydrophobicity of the coating polymer gradually decreases from the sensor film toward the surface of the coating polymer.

[0024] In another example, to improve the adhesion of a hydrophilic coating to the ISM, the surface of the hydrophobic ISM is functionalized using, for example, an organosilane.

[0025] c. Sensuality i. Biocompatibility For example, a hydrophilic coating layer can have minimal surface roughness along with mechanical properties comparable to the embedded tissue environment, helping to mimic the surface roughness of the natural extracellular matrix. These properties help limit protein adsorption and counteract mechanical mismatches that can contribute to the development of local inflammation and subsequent immune responses. To achieve this, preferred coating layers can incorporate gelatin, methacrylate gelatin, polyurethane, poly(ethylene glycol), or collagen-based hydrogels, whose natural biocompatibility, low immunogenicity, and highly tunable mechanical properties make them well-suited for limiting xenobiotic responses.

[0026] As an alternative to stealth strategies, preferred coating polymers can consist of proteins that actively reduce innate immunogenic responses. One example is the addition of a surface layer of osteopontin to reduce xenobiotic reactions at the transplantation site. In addition, anti-inflammatory molecules / factors (e.g., corticosteroids, tyrosine kinase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), or dexamethasone) can be directly incorporated into hydrophilic coating polymers to regulate their release to the surrounding microenvironment, helping to directly inhibit both immune responses and biofouling. This control over molecular release can be achieved by time-dependent targeted degradation of the active molecule-polymer coating bond, or alternatively, by the incorporation of nanocomplexes into the polymer matrix, from which diffusion can be regulated by diffusion-based mechanisms.

[0027] The hydrophilic coating layer facilitates interaction between the natural extracellular matrix and the sensor, enabling signal stabilization within very short timeframes, such as one hour.

[0028] ii. Mass transport restrictions For example, a hydrophilic coating layer restricts the diffusion of the analyte toward the ISM to prevent rapid signal saturation. This is particularly beneficial on a logarithmic scale, as the signal change decreases exponentially with increasing analyte concentration. The mass transport properties of water-soluble molecules can be reduced by decreasing the porosity of the hydrophilic coating polymer, which can be achieved by increasing the amount of chain extender incorporated during the synthesis of the hydrophilic polyurethane.

[0029] In another example, a hydrophilic coating polymer may contain proteins, amino acids such as lysine, or amines that promote crosslinking by aldehyde chemistry, or carbodiimide crosslinking chemistry, or photoinitiators that enable photo-induced crosslinking, where higher crosslinking results in lower mass transport.

[0030] iii.Light transmission limit In one example, a biosensor includes a transducer layer, such as poly(3-hexylthiophene), whose electron transfer properties are affected by the presence of light. To prevent non-selective changes in the biosensor response caused by light, an opaque hydrophilic coating layer is used, such as a cryogel, such as polyacrylamide or polysaccharide hydrogel crosslinked with N,N'-methylenebisacrylamide or glutaraldehyde, or a photoinitiator such as Irgacure 2959, and a crosslinking agent such as N,N'-methylenebisacrylamide.

[0031] In another example, light transmission can be controlled by the molar amounts of diisocyanate and chain extender components used during synthesis.

[0032] iv. Prevention of ISM component exudation The leaching of cytotoxic ISM components can be slowed by increasing porosity and crosslinking density, as illustrated in the given example, to restrict mass transport to the ISM.

[0033] In another example, lipophilic components such as fatty acids covalently bonded to a hydrophilic polymer can be incorporated so that lipophilic toxic compounds leaching from ISM can be captured by the coating layer.

[0034] v. Lowering the impedance of the biosensor Electrochemical sensors generate electrical signals in response to a large set of parameters such as ion concentration, temperature, and pressure. The electrical model includes various layers modeled by resistors, capacitors, inductors, Warburg elements, voltage supplies, current supplies, and other electrical components.

[0035] Hydrophobic ISMs can be modeled by high resistance (hundreds of MOhm). Therefore, from an electrical standpoint, ion-selective electrodes exhibit very high output impedance. This has been found to be problematic for the following reasons:

[0036] During open-circuit potential difference (OCP) measurement, a potential is generated between the electrodes, which correlates with the concentration of the target analyte via the Nernst equation. High output impedance causes unstable signals to become steady and makes the circuit highly sensitive to noise (electromagnetic interference such as 50Hz or wireless communication, sensitivity to static charges such as the presence of the human body). Specific amplification layers, filtering, and shielding are required to reduce / remove this type of electrical noise.

[0037] During electrochemical impedance spectroscopy (EIS), the primary source of the measured impedance is due to the resistance of the sensor film, and not the desired impedance that correlates with the change in target analyte concentration.

[0038] During measurement techniques, including the measurement of current or current flow, high impedance significantly reduces the order of magnitude of such current for a given potential, typically within the range of -15V to +15V.

[0039] In one example, an ISM (Integrated Sensor Microscope) contains a low-impedance conductive polymer. Different materials have different conductivity and ion transport properties. Examples of low-impedance conductive materials that can be incorporated into an ISM include, but are not limited to, polyaniline and polythiophene. These polymers are known to have low impedance and high conductivity. In another example, the impedance of a biosensor can be reduced by using a hydrophilic outer coating material.

[0040] d. Composition The ISM includes a polymer matrix composed of hydrophobic polymers used individually, or combinations of hydrophobic polymers including but not limited to polyvinyl chloride, silicone, fluorosilicone, polyurethane, polyacrylate, and perfluoropolymer. The sensor membrane may also contain plasticizers, but are not limited to, nitrophenyl octyl ether, dioctyl sebacate, dibutyl sebacate, and dioctyl phthalate, to enhance the fluidity of the polymer matrix and membrane permeability to the target analyte. The sensor membrane contains ion transport molecules, such as ionophores, which have a higher affinity for and transport capacity to the target analyte compared to the remaining electrolytes, including other ions via the ISM, such as sodium (e.g., ETH227), hydrogen (e.g., toridodecylamine, ETH1907), chloride (e.g., ETH9033), lithium (e.g., ETH2137), potassium (e.g., valinomycin, BB15C5), calcium (e.g., ETH5234), magnesium (e.g., K22B5, ETH4030), etc. ISM also contains lipophilic salts of tetraphenyl derivatives, N-(2,3,5,6,8,9,11,12-octahydro-16-nitro-1,4,7,10,13-benzopentaoxacyclopentadecine-15-yl)-, 2-dodecyl-2-methyl-1,3-propanediyl ester (BME44), toridodecylmethylammonium salt, and the like.

[0041] For example, when dissolved in solvents such as tetrahydrofuran and cyclohexanone, ISM contains potassium ionophore II, potassium tetrakis(4-chlorophenyl)borate, dioctyl sebacate, and polyurethane.

[0042] In another example, photocurable ISM can be obtained by direct photopolymerization of the electrode surface, in which case a solution containing a polymer monomer such as methacrylate or acrylate, and a photoinitiator such as 2,5-dimethyl-3'-methoxybenzophenone, in addition to one or a combination of the plasticizer, lipophilic salt, and ionophore mentioned above, is deposited on the electrode surface using a solvent such as dichloromethane or tetrahydrofuran and exposed to UV light.

[0043] e. Deposition method Solutions of ISM are obtained in volatile solvents such as tetrahydrofuran, chloroform, cyclohexanone, and dichloromethane, and are used to obtain at least one layer of ISM on a transducer layer by drop casting, spin coating, spray coating, or immersion coating.

[0044] In another example, a photocurable sensor film is selectively defined on an electrode by photolithography, the ISM solution is exposed to a UV or near-UV light source, or a laser light source with a micron-sized spot size, via a photomask, and then the non-crosslinked regions are removed by dissolving them in a suitable solvent that does not remove the crosslinked regions. The photocurable film can be obtained using azobisisobutyronitrile as a photoinitiator for polyvinyl chloride (PVC) polymer films.

[0045] In another example, a non-toxic photosensitive o-nitrobenzyl moiety can be introduced into a dextran skeleton having an acrylate group to function as a photosensitive masking layer. Using a suitable photomask, UV light is irradiated onto the desired film region to initiate the decomposition and removal of the modified dextran layer. Subsequently, ISM is applied to the substrate, and the modified dextran layer is dissolved in water, leaving ISM only in the desired region.

[0046] In another example, the ISM is deposited on the electrode area by spatially and volumetrically controlled deposition, which is enabled by, but is not limited to, inkjet printing, ultra-low volume dispensers, etc. Furthermore, controlled film application is used by defining the region of interest using a spin coater and doctor blade in conjunction with a stencil or screen mesh, after which the stencil is removed and the sensor film remains only in place, similar to screen printing.

[0047] Preferred ISMs can be formed from compositions and methods described in PCT / US2022 / 052927 (PROT.P-002-WO), which is incorporated herein by reference for all purposes.

[0048] f. Function ISM enables selective analyte transport across the membrane toward the transducer layer. Its hydrophobicity creates a barrier between the aqueous medium and the transducer layer, preventing non-selective signal generation.

[0049] 2. Transducer layer: ISM-electrode interface a. Composition The transducer layer can be obtained using conductive polymers obtained by polymerizing one or a combination thereof when using materials including but not limited to pyrrole, 1-hexyl-3,4-dimethylpyrrole, 3-octylthiophene, 3,4-ethylenedioxythiophene, 3-methylthiophene, aniline, indole, α-naphthylamine, o-anisidine, and o-aminophenol, possibly in the presence of p-toluenesulfonate, polystyrenesulfonate, perfluorolate, metal complexes, etc., and using lipophilic ions such as tetrakis[3,5-bis(trifluoromethyl)phenyl borate and (4-chlorophenyl) borate, which can promote redox activity, bilayer capacitance, and ion-electron transfer.

[0050] b. Deposition method The transducer layer can be deposited on the electrode surface by constant current or voltammetry, and the conductive or semiconducting polymer is attracted to the electrode surface by the applied current or potential, regardless of the presence or absence of doping agents and counterions. Examples include electrochemical polymerization of poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate and polypyrrole doped with chloride ions.

[0051] In another example, the conversion material can be chemically bonded to the electrode surface via covalent bonds, π-π interactions, electrostatic interactions, and Lennard-Jones and Coulomb interactions.

[0052] In another example, a mixture of conversion materials with volatile solvents such as tetrahydrofuran, ethanol, methanol, and acetone can be obtained and deposited on the electrode surface by drop casting, spin coating, spray coating, and immersion coating.

[0053] In another example, the conversion material can be obtained by mixing a photosensitive material that enables photosensitive patterning of the transducer layer on the electrode and substrate material.

[0054] In another preferred embodiment, the transducer layer may be formed on the surface of an electrode (e.g., a gold electrode) using a composition and method as described in U.S. Provisional Patent Application Publication No. 63 / 425,658 (PROT.P-003-PV), and the electrode may be formed from a composition and method as described in PCT / US2022 / 037198 (PROT.P-001-WO), and these are incorporated herein by reference in whole for all purposes.

[0055] c.Characteristics The transducer layer facilitates electron transfer in the presence of the target analyte, enabling a correlation between the analyte concentration and the recorded signal.

[0056] The transducer layer is preferably hydrophobic to improve adhesion of the hydrophobic ISM and reduce / eliminate the accumulation of aqueous media at this interface, which is detrimental to the reliability of acquired data. [Examples]

[0057] Without being constrained by a specific mechanism of action, the present invention is further described in the following sections of examples illustrating some of the embodiments described herein.

[0058] Material description: Hydrophobic ISM1: A polyurethane-based ISM containing potassium ionophore 1 (valinomycin).

[0059] Hydrophobic ISM2: A polyvinyl chloride-based ISM containing potassium ionophore 1 (valinomycin).

[0060] Hydrophilic OCL1: A polyurethane-based hydrophilic polymer with a water absorption rate of approximately 15%.

[0061] Hydrophilic OCL2: A siloxane-based hydrophilic polyurethane with a water absorption rate of approximately 20%.

[0062] 1. Valinomycin leaching Two sensor configurations were prepared: 1. Hydrophobic PVC ISM without OCL, and 2. Hydrophobic PVC ISM with hydrophilic OCL. Ten sensors from each configuration were immersed in buffer for different periods of 24, 48, and 72 hours. This yielded six test solutions. The extracts were analyzed for valinomycin levels using liquid chromatography-tandem mass spectrometry. All batches of samples were analyzed using a calibration curve (R2 ≥ 0.99) ranging from 1 ng / mL to 1000 ng / mL. Figure 1 shows the valinomycin levels detected after 24, 48, and 72 hours with and without OCL.

[0063] 2. OCL inhibits RE leaching. A 1 μL silver ink droplet was placed on a PI-based substrate. The sample was then divided into two groups: one containing only PI+Ag ink, and the other with 10 μL of OCL1 added. The triplicate samples were then incubated in cell culture medium at 37°C for 24, 48, and 72 hours, after which indirect cytotoxicity was evaluated against L929 cells for 24 hours using the CCK-8 cytotoxicity assay. Figure 2 shows cell viability data demonstrating that the addition of OCL reduces silver ink toxicity by limiting the diffusion of components into the external medium.

[0064] 3. ISM with OCL Six configurations will be tested in a self-made flow cell for 12 hours. The configurations are: hydrophobic PVC ISM; hydrophobic PVC ISM with OCL1; hydrophobic PVC ISM with OCL2; hydrophobic PU ISM; hydrophobic PU ISM with OCL1; and hydrophobic PU ISM with OCL2. The KCl concentration in the flow cell will be sequentially changed to 2 mM, 6 mM, 4 mM, and 8 mM, and each concentration will be maintained for 1 hour.

[0065] Hydrophobic PVC ISM with OCL Different combinations of PVC ISMs with two different OCLs were constructed, and their sensor response to K+ was evaluated. Long-term experiments were performed in phosphate-buffered saline to measure the potential when a specified amount of K+ was spiked into an electrochemical cell containing a working electrode (fabricated using a hydrophobic ISM with OCL1 or OCL2) and a reference electrode. Data were recorded by sequentially adding 2 mM, 6 mM, 4 mM, and 8 mM K+ over a 12-hour time frame three times.

[0066] Performance: As shown in Figure 3, all components respond to K+ changes overall, but the PVC ISM with OCL1 and OCL2 exhibits superior performance compared to the PVC ISM counterpart. This performance may be due to reduced leaching of the active ingredient, namely valinomycin, from the ISM. Of the two OCLs, OCL2 exhibits superior performance compared to OCL1 due to its hydrophilicity.

[0067] Hydrophobic PU ISM with OCL Different combinations of PU ISMs with two different OCLs were constructed, and their sensor response to K+ was evaluated. Long-term experiments were performed in phosphate-buffered saline to measure the potential when a specified amount of K+ was spiked into an electrochemical cell containing a working electrode (fabricated using a hydrophobic ISM with OCL1 or OCL2) and a reference electrode. Data were recorded by sequentially adding 2 mM, 6 mM, 4 mM, and 8 mM K+ over a 12-hour time frame three times.

[0068] Performance: As shown in Figure 4, in long-term flow cell tests, considering the noise level, the hydrophobic PU ISM-based sensor showed the highest noise, while the sensor with OCL2 showed the lowest noise. Regarding drift, the hydrophobic PU ISM-based sensor had positive drift, while the other two groups showed negligible drift.

[0069] Lifespan experiment up to 72 hours: PU ISM only: Three configurations will be tested: hydrophobic PU ISM, hydrophobic PU ISM with OCL1, and hydrophobic PU ISM with OCL2. They will be evaluated under open-circuit potential (OCP) at K+ concentrations of 2 mM, 4 mM, 6 mM, and 8 mM on day 1. Subsequently, they will be kept in a conditioning solution (2 mM KCl in PBS) for 72 hours, and the OCP test will be repeated on day 3.

[0070] Performance: As shown in Figures 5, 6, and 7, the hydrophobic PU ISM-based sensors exhibited a linear and rapid response (<5 seconds) but showed a significant decrease in sensitivity after 3 days of conditioning (e.g., 55mV to 40mV / decade). The hydrophobic PU ISM with an OCL1-based sensor showed a slower response (5 to 10 minutes) and a moderate decrease in sensitivity after 3 days of conditioning (e.g., 55mV to 50mV / decade). Given our sensitivity requirement of higher than 45mV / decade, the performance of such sensors is acceptable. In the case of hydrophobic PU ISMs with an OCL2-based sensor, they exhibited a linear and rapid response (<5 seconds) and showed no loss of sensitivity after 3 days of conditioning. Figure 5 shows the aging data for a hydrophobic PU ISM without an outer coating layer. Figure 6 shows the improved aging data for the same PU ISM with OCL1. Figure 7 shows improved aging data for the same PU ISM, but with OCL2.

Claims

1. A layered composition for use with a biosensor for in vivo monitoring of electrolytes, Hydrophilic coating layer, Hydrophobic ISM layer, A layered composition for use with a biosensor for in vivo monitoring of electrolytes, including the following:

2. The layered composition according to claim 1, wherein the hydrophilic coating layer comprises a polyurethane formed from an isocyanate and a polyol.

3. The isocyanate is an aliphatic isocyanate selected from the group consisting of hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), toluene diisocyanate (TDI), xylene diisocyanate, methylenediphenyl diisocyanate (MDI), etc. The polyol is a hydrophilic polyol selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polypropylene glycol, diethylene glycol, ethylene glycol, and glycerol, and The hydrophilic coating layer and / or polyurethane may optionally include a hydrophilic additive selected from the group consisting of polyvinylpyrrolidone, polyethylene oxide, polyacrylic acid, etc.; a crosslinking agent and / or chain extender selected from the group consisting of diamines, dio acids, diols, etc.; preparation by plasma treatment or grafting hydrophilic functional groups, such as amines, oxygen-containing functional groups, etc.; and preparation using a photoinitiator. The layered composition according to claim 2.

4. The layered composition according to claim 3, wherein the hydrophilicity of the polyurethane is adjusted by selection of an isocyanate, a polyol, or both an isocyanate and a polyol.

5. The layered composition according to claim 1, further comprising a transducer layer and an electrode layer, wherein the electrode layer comprises a working electrode and a counter electrode, and the transducer layer is in contact with the working electrode.

6. A layered biosensor for monitoring electrolytes in vivo, wherein the biosensor is sequential Hydrophilic coating layer, Hydrophobic ISM layer, Transducer layer, Electrode layer and This includes, in some cases, each layer being in direct contact with its adjacent layer. A layered biosensor for monitoring electrolytes in vivo.

7. The hydrophilic coating layer includes a polyurethane formed from an isocyanate and a polyol. The isocyanate is an aliphatic isocyanate selected from the group consisting of hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), toluene diisocyanate (TDI), xylene diisocyanate, methylenediphenyl diisocyanate (MDI), etc. The polyol is a hydrophilic polyol selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polypropylene glycol, diethylene glycol, ethylene glycol, and glycerol, and The hydrophilic coating layer and / or polyurethane may optionally include a hydrophilic additive selected from the group consisting of polyvinylpyrrolidone, polyethylene oxide, polyacrylic acid, etc.; a crosslinking agent and / or chain extender selected from the group consisting of diamines, diacides, diols, etc.; preparation by plasma treatment or grafting hydrophilic functional groups, such as amines, oxygen-containing functional groups, etc.; and preparation using a photoinitiator. The biosensor according to claim 6.

8. The layered composition according to claim 7, wherein the hydrophilicity of the polyurethane is adjusted by selection of an isocyanate, a polyol, or both an isocyanate and a polyol.

9. The biosensor according to claim 7, wherein the electrode layer includes a working electrode and a counter electrode, and the transducer layer is in contact with the working electrode.

10. A method for forming a layered composition for use with a biosensor for in vivo monitoring of electrolytes, (a) A step of forming or providing a hydrophobic ISM layer, (b) Form a hydrophilic coating layer composition and apply it to the hydrophobic ISM layer formed or provided in step (a), This involves a process of forming a layered composition for use with a biosensor for in vivo monitoring of electrolytes, A method for forming a layered composition for use with a biosensor for in vivo monitoring of electrolytes, including the composition.

11. The hydrophilic coating layer includes a polyurethane formed from an isocyanate and a polyol. The isocyanate is an aliphatic isocyanate selected from the group consisting of hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), toluene diisocyanate (TDI), xylene diisocyanate, methylenediphenyl diisocyanate (MDI), etc. The polyol is a hydrophilic polyol selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polypropylene glycol, diethylene glycol, ethylene glycol, and glycerol, and The hydrophilic coating layer and / or polyurethane may optionally include a hydrophilic additive selected from the group consisting of polyvinylpyrrolidone, polyethylene oxide, polyacrylic acid, etc.; a crosslinking agent and / or chain extender selected from the group consisting of diamines, diacides, diols, etc.; preparation by plasma treatment or grafting hydrophilic functional groups, such as amines, oxygen-containing functional groups, etc.; and preparation using a photoinitiator. The method according to claim 10.

12. The method according to claim 10, wherein the hydrophilicity of the polyurethane is adjusted by selection of an isocyanate, a polyol, or both an isocyanate and a polyol.

13. The method according to claim 10, wherein in step (b), the hydrophilic coater layer is applied to the hydrophobic ISM layer by a process selected from the group consisting of spin coating, spray coating, casting (e.g., drop casting), inkjet printing, electrodeposition, and screen printing.

14. The method according to claim 10, wherein the hydrophobic ISM layer is disposed on a transducer layer, the transducer layer is disposed on an electrode layer, the electrode layer includes a working electrode and a counter electrode, and the transducer layer is in contact with the working electrode.

15. A method for forming a biosensor for in vivo monitoring of electrolytes, (a) A step of forming or providing an electrode layer, (b) A step of forming or providing a transducer layer on the electrode layer formed or provided in step (a), (c) A step of forming or providing a hydrophobic ISM layer on the transducer layer formed or provided in step (b), (d) A step of applying a hydrophilic coating layer composition onto the hydrophobic ISM layer formed or provided in step (c), (e) A step of applying the hydrophilic coating layer composition formed in step (b) to the hydrophobic ISM layer formed or provided in step (a), A method for forming a biosensor for in vivo monitoring of electrolytes, including the presence of electrolytes.

16. The hydrophilic coating layer includes a polyurethane formed from an isocyanate and a polyol. The isocyanate is an aliphatic isocyanate selected from the group consisting of hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), toluene diisocyanate (TDI), xylene diisocyanate, methylenediphenyl diisocyanate (MDI), etc. The polyol is a hydrophilic polyol selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polypropylene glycol, diethylene glycol, ethylene glycol, and glycerol, and The hydrophilic coating layer and / or polyurethane may optionally include a hydrophilic additive selected from the group consisting of polyvinylpyrrolidone, polyethylene oxide, polyacrylic acid, etc.; a crosslinking agent and / or chain extender selected from the group consisting of diamines, diacides, diols, etc.; preparation by plasma treatment or grafting hydrophilic functional groups, such as amines, oxygen-containing functional groups, etc.; and preparation using a photoinitiator. The method according to claim 15.

17. The method according to claim 15, wherein the hydrophilicity of the polyurethane is adjusted by selection of an isocyanate, a polyol, or both an isocyanate and a polyol.

18. The method according to any one of claims 15, wherein in step (d), the hydrophilic coater layer is applied to the hydrophobic ISM layer by a process selected from the group consisting of spin coating, spray coating, casting (e.g., drop casting), inkjet printing, electrodeposition, and screen printing.

19. The method according to any one of claims 15, wherein the electrode layer includes a working electrode and a counter electrode, and the transducer layer is in contact with the working electrode.

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