Non-enzymatic potassium sensor

Abstract

The present invention relates to an analyte sensor for detecting the concentration of potassium ions in body fluids, and to a method for using the sensor.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 493,595, filed on March 31, 2023, and U.S. Provisional Patent Application No. 63 / 493,600, filed on March 31, 2023, the disclosures thereof being incorporated herein by reference in their entirety. The present invention relates to an analyte sensor for detecting the concentration of potassium ions in body fluids, and to a method for using the sensor. [Background technology]

[0002] The detection of various analytes within an individual can sometimes be crucial for monitoring their health status as deviations from normal analyte levels that may indicate physiological conditions. For example, monitoring glucose levels allows individuals with diabetes to take appropriate corrective measures, including medication or consumption of specific foods or beverages, to avoid significant physiological harm. Other analytes, such as potassium, may be desirable for monitoring certain physiological conditions. In some cases, particularly when an individual suffers from a concomitant condition involving simultaneous dysregulation of two or more analytes in combination, monitoring one or more analytes to monitor single or multiple physiological conditions may be desirable. Monitoring of analytes in an individual can be performed periodically or continuously over a set period. Periodic monitoring of analytes can be performed by taking samples of bodily fluids, such as blood or urine, at set time intervals and performing ex vivo analysis. Periodic ex vivo monitoring of analytes can be sufficient to determine the physiological conditions of many individuals. However, ex vivo monitoring of analytes can be inconvenient or painful in some cases. Furthermore, if analyte measurements cannot be obtained at the appropriate time, there is no way to recover the lost data. Continuous monitoring of analytes can be performed using one or more sensors that remain at least partially implanted in the individual's tissue, for example, in the skin, subcutaneously, or intravenously, so that analysis can be performed in vivo. The implanted sensors can collect analyte data on demand or continuously according to a set schedule, depending on the individual's specific health needs and / or previously measured levels of analytes. Monitoring of analytes using in vivo implanted sensors may be a more desirable approach in individuals exhibiting severe analyte dysregulation and / or rapidly fluctuating analyte levels, but it can be equally beneficial in other individuals.

[0003] Many analytes represent interesting targets for physiological analysis, provided that suitable detection chemistry can be identified. For this purpose, enzyme-based amperometric sensors configured to continuously assay glucose in vivo have been developed and improved in recent years to assist in monitoring the health of diabetic individuals. Other analytes that typically undergo co-regulation with glucose in diabetic individuals include, for example, potassium. Monitoring potassium independently of glucose regulation can also be desirable. For example, potassium levels may be important to monitor in individuals with various renal or heart diseases. Implanted analyte sensors configured to detect potassium in vivo are currently unavailable. Experimental systems used for in vitro detection of nanomolar potassium concentration in fluids using ion transfer stripping voltammetry are unsuitable for in vivo potassium detection for a variety of reasons. For example, some systems cannot detect higher potassium concentrations found in body fluids and do not have a sufficient sensor lifespan for in vivo applications. The aforementioned systems are also too bulky for in vivo implantation and use, making them more difficult to manufacture. Therefore, there is a need in the industry for sensors to detect potassium in vivo. Since implanted analyte sensors often remain in the tissues of an individual for extended periods, it is often highly desirable that such analyte sensors be fabricated from stable materials exhibiting a high degree of biocompatibility. [Overview of the project]

[0004] This invention provides a potassium sensor that can be used in vivo for continuous monitoring and detection of potassium concentration in body fluids. The potassium sensor is enzyme-free and based on non-enzymatic sensor chemistry. In some embodiments, the potassium sensor includes a working electrode having a potassium-responsive active region located on a portion of the electrode's outer surface. In some embodiments, the working electrode includes a transition metal ion in the form of a salt or complex. In some embodiments, the potassium-responsive active region includes a potassium-selective transport layer that forms the outer surface of the potassium-responsive active region. In some embodiments, the potassium-selective transport layer includes a hydrophobic polymer and a potassium ionophore. In some embodiments, the sensor finds use in in vivo monitoring of potassium concentration in body fluids. In some embodiments, when the sensors mentioned above are used with a wide range of electrochemical detection techniques, such as amperometry, chronoamperometry, and voltammetry, and to improve the service life of the sensors for in vivo use, it is beneficial for the sensors to further include an electrolyte gel layer between the outer surface of the working electrode and the potassium-selective transport layer. While not limited by theory, the electrolyte gel layer is thought to improve the service life of the sensor. The electrolyte gel layer is also thought to be important for providing effective means to facilitate reduction and oxidation reactions at the electrode surface that occur during the operation of the potassium sensor.

[0005] Accordingly, in some embodiments, systems, devices, and methods using a sensor are provided herein, comprising a first working electrode and a potassium-responsive active region disposed on at least a portion of the outer surface of the first working electrode, wherein the portion of the outer surface of the first working electrode comprises a transition metal ion in the form of a salt or complex, and may also comprise a transition metal, and the potassium-responsive active region comprises (i) a potassium-selective transport layer forming the outer surface of the potassium-responsive active region and comprising a polymer, a potassium ionophore, a plasticizer, and an electrolyte, and (ii) an electrolyte gel layer disposed between the potassium-selective transport layer and the portion of the outer surface of the first working electrode. However, in some embodiments, sensors without an electrolyte gel layer, as described above, are provided and used in in vivo detection methods, and there is no significant accompanying sacrifice of sensor lifespan or functionality due to the omission of the electrolyte gel layer. Including an electrolyte gel layer in a sensor increases manufacturing cost and complexity during sensor production. Omitting the electrolyte gel layer from the sensor simplifies manufacturing and reduces costs, which is desirable in some cases. This invention is partly based on the surprising finding that, when using certain electrochemical detection techniques, the electrolyte gel layer does not need to be included in vivo in the above-mentioned type of potassium sensor. Specifically, it was surprisingly found that when chronoamperometry techniques with short potential durations are used with the above-mentioned type of sensor, the electrolyte gel layer can be omitted from the sensor without significantly degrading its functionality. This is quite surprising, but it has been found that when using other electrochemical detection techniques or when using chronoamperometry with long potential durations, the presence of the electrolyte gel layer is essential to obtain good sensor functionality and service life that is effective for use in in vivo potassium concentration detection and monitoring in body fluids.

[0006] Therefore, in some embodiments, the present invention relates to a system, device and method for detecting potassium ions in a fluid, (a) A step of preparing an analyte sensor including a first working electrode and a potassium-responsive active region disposed on at least a portion of the outer surface of the first working electrode, wherein the portion of the outer surface of the first working electrode contains a transition metal ion in the form of a salt or complex, and may contain a transition metal, and the potassium-responsive active region includes a potassium-selective transport layer forming the outer surface of the potassium-responsive active region, the potassium-selective transport layer comprising a polymer, a potassium ionophore, a plasticizer, and an electrolyte; (b) A step of applying a potential to the first working electrode; (c) A step of oxidizing the transition metal ion in the form of a salt or complex, and optionally the transition metal of the first working electrode. The present invention provides a system, device, and method comprising the steps of: (d) obtaining a signal indicating dilution and / or reduction, wherein the signal indicates the concentration of potassium ions in the fluid; and (c) determining the concentration of potassium ions in the fluid from the signal obtained in step (c), wherein steps (b) and (c) include obtaining a signal indicating the concentration of potassium ions in the fluid using chronoamperometry, wherein chronoamperometry includes the sequential application of first and second potentials, the first potential being applied for a duration of 10 milliseconds to 60 seconds, the second potential being applied for a duration of 10 milliseconds to 60 seconds, and the first potential being different from the second potential.

[0007] When chronoamperometry is used, involving the application of potentials of from for durations of 10 milliseconds to 60 seconds, it has been found that the inclusion of an electrolyte gel layer positioned between potassium-selective transport layers (or electrolyte gel layers, if present) is not necessary. Continuous application of a positive potential to the electrode then causes oxidation (i.e., a reversal of the process that occurs with negative potential application), relocating counterions (e.g., chloride anions) from the potassium-selective transport layers (or electrolyte gel layers, if present) back to the electrode surface, regenerating the original transition metal salt. While not limited by theory, it is believed that with longer durations of negative potential application, counterions (e.g., chloride ions) migrate from the electrode surface into the potassium-selective transport layers or electrolyte gel layers, and also passively diffuse from the electrode surface. The longer the negative potential is applied, the more counterions diffuse from the electrode surface. This means that when a subsequent positive potential is applied, it becomes more difficult to recover the counterions from the electrode surface. Over time, this leads to incomplete recovery of counterions at the electrodes and incomplete regeneration of transition metal salts during positive potential. This incomplete generation may impair the sensor's function over time. Surprisingly, we found that using the positive potential for longer periods than negative potential mitigates this problem and improves the sensor's lifespan.

[0008] In some embodiments, both the applied negative and positive potentials include potentials with durations of 10 milliseconds to 5 seconds, and the applied positive and negative potentials have different durations. In some embodiments, the positive potential is applied for a longer duration than the negative potential. In some embodiments, the positive potential is applied for a longer duration than the negative potential, with the positive potential being applied for a duration of 2 to 5 seconds and the negative potential being applied for a duration of 1 to 2 seconds. In some embodiments, chronoamperometry includes the continuous application of alternating positive and negative potentials, with the potentials applied for durations of 10 milliseconds to 60 seconds. In some embodiments, chronoamperometry includes the continuous application of alternating positive and negative potentials, with the potentials applied for durations of 500 milliseconds to 60 seconds. In some embodiments, chronoamperometry includes the continuous application of alternating positive and negative potentials, with the potentials applied for durations of 500 milliseconds to 20 seconds.

[0009] In some embodiments, the duration of the positive potential differs from the duration of the negative potential. In some embodiments, the positive potential is applied for a longer duration than the negative potential. In some embodiments, the duration of the positive potential is 2 to 5 seconds, and the duration of the negative potential is 1 to 2 seconds. In some embodiments, chronoamperometry includes the continuous application of alternating positive and negative potentials, where the continuous negative or positive potentials include potentials of 50mV to 500mV, for example 100mV to 300mV, or for example 150mV to 250mV. In some embodiments, chronoamperometry includes the continuous application of alternating positive and negative potentials, where the negative or positive potential includes potentials of 175mV to 225mV, for example, 200mV. In some embodiments, a positive potential is applied for a duration of 3 seconds, and a negative potential is applied for a duration of 1 second. In some embodiments, for example, the negative potential is 200 mV and the positive potential is 200 mV.

[0010] The above-mentioned advantages related to a longer duration of positive potential than negative potential are also observed when both the first and second potentials are positive, or when both the first and second potentials are negative. Therefore, in one embodiment, the first potential is applied for a different duration than the second potential. In some embodiments, the potential associated with reduction at the electrode is applied for a shorter duration than the potential associated with oxidation at the electrode. In some embodiments, chronoamperometry includes pulse amperometry. As used herein, the term pulsed amperometry is used to refer to any electrochemical detection technique known in the art, which is encompassed by the terms pulsed amperometry or pulsed amperometry. In pulsed amperometry, chronoamperometry is performed using very short potential steps (e.g., potential steps of duration according to the method of the present invention). Each potential step generates a current signal that can be measured over time. At each potential step, the current signal generated during the latter half of the duration of each potential step indicates the potassium concentration of the fluid in which the sensor is placed. At each potential, pulsed amperometry essentially samples the current generated during this latter half of the duration of each potential step. Pulsed amperometry may include a step of determining the average current value over time for the latter half of the duration of the currents generated for the relevant potentials. Pulsed amperometry therefore shows a single current value (e.g., average current) for each potential. The advantage of using pulsed amperometry in this way is that it makes it easier to compare the currents generated for potential steps at different potassium concentrations.

[0011] In some embodiments of any of the above configurations, the fluid containing the detected potassium ions includes body fluids. In some embodiments, body fluids include dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. In some embodiments, the sensor, or at least a portion of the sensor (the outer surface of the first working electrode), is located in vivo. In some such embodiments, the method includes the step of determining the concentration of potassium ions in an in vivo body fluid. The terms "external" and "internal" as used herein with respect to each surface and layer are used in relation to the first working electrode. The term "internal" as used herein is used to mean nearer to the first working electrode. Thus, if present, the electrolyte gel layer of the potassium-responsive active region may be described in some embodiments as being located inside the potassium-selective transport layer of the potassium-responsive active region, because it is between the potassium-selective transport layer and the working electrode. The term "external" as used herein is used to mean further away from the first working electrode and closer to the analyte containing the fluid in which the sensor is located.

[0012] In some embodiments, the potassium-responsive active region comprises only the potassium-selective transport layer, and no further layers are present. Therefore, in some embodiments, the potassium-responsive active region does not include an electrolyte gel layer. However, in other embodiments, the potassium-responsive active region may include one or more further layers, for example, one or more layers inside, outside, or both inside and outside the potassium-selective transport layer, provided, of course, that the presence of any further layers does not impede the functionality of the sensor. As discussed in more detail below, in some embodiments the potassium-responsive active region may further include an electrolyte gel layer. The potassium-responsive active region therefore, in some embodiments, includes an external potassium-selective transport layer and an internal electrode gel layer between the potassium-selective transport layer and the working electrode. In some embodiments, there are no further layers between these layers, and the potassium-selective transport layer is directly adjacent to the electrolyte gel layer, and the electrolyte gel layer is directly adjacent to the first working electrode. However, this is not essential, and the sensor may include further layers between the first working electrode and the electrolyte gel layer and / or further layers between the electrolyte gel layer and the potassium-selective transport layer, provided, of course, that the presence of any further layers does not impede the functionality of the sensor.

[0013] The polymer in the potassium-selective transport layer can be any suitable polymer. In some embodiments, the polymer is resistant to the bodily fluids in which the sensor is placed. Suitable polymers will be apparent to those skilled in the art and include polyvinyl chloride (PVC), polyethylene, polypropylene, polybutylene, polyurethane, and other suitable hydrophobic polymers. In some embodiments, the polymer includes polyvinyl chloride (PVC). In some embodiments, the polymer has a weight-average molecular weight of 50,000 Da to 500,000 Daltons. In some embodiments, the polymer includes polyvinyl chloride with a weight-average molecular weight of 50,000 Daltons to 500,000 Daltons. In some embodiments, polyvinyl chloride with a weight-average molecular weight of 200,000 Da is used.

[0014] The potassium-selective transport layer further comprises a plasticizer. In some embodiments, the plasticizer is a hydrophobic plasticizer. In some embodiments, the plasticizer comprises a phthalate compound, an adipate compound, a glutarate compound, a sebacate compound, a phosphate compound, a trimellitate compound, an epoxy compound, or a combination thereof. In some embodiments, the plasticizer comprises 2-nitrophenyloctyl ether (NPOE). In some embodiments, the polymer comprises polyvinyl chloride, and the plasticizer comprises a PVC plasticizer, e.g., one discussed above. In some embodiments, the polymer comprises polyvinyl chloride, and the plasticizer comprises 2-nitrophenyloctyl ether (NPOE). Other specific examples of plasticizers that may be used include dinonyl adipate (DNA), tris(2-ethylhexyl) phosphate (TEHP), tris(ethylhexyl) phosphate (TEHP), bis(2-ethylhexyl) adipate (DOA), dioctyl phthalate (DOP), and bis(2-ethylhexyl) sebacate (DOS). The plasticizer should be compatible with the electrode polymer and components. They should also be soluble in the solvent used to prepare the potassium-selective transport layer. The plasticizer functions to soften the polymer, thereby making available the potassium ions present in the fluid in which the sensor is located.

[0015] The polymer and the plasticizer are present in any suitable ratio. Suitable ratios are recognized by those skilled in the art. In some embodiments, the polymer and the plasticizer of the potassium selective transport layer are present in a weight ratio of 1:1 to 1:10, such as 1:2 to 1:8, such as 1:3 to 1:5. The potassium selective transport layer also includes a potassium ionophore. Any suitable potassium ionophore can be used. Suitable potassium ionophores have a high binding affinity and selectivity for potassium ions over other cations commonly found in body fluids, such as sodium. In some embodiments, the potassium ionophore includes valinomycin, gramicidin A, ridomycin, lasalocid, maduramycin, monensin, narasin, nigericin, nonactin, nystatin, salinomycin, crown ether, cryptand or any combination thereof. In some embodiments, the potassium ionophore is valinomycin, gramicidin A, ridomycin, lasalocid, maduramycin, monensin, narasin, nigericin, nonactin, nystatin, salinomycin, crown ether, cryptand or any combination thereof. In some embodiments, the potassium ionophore can include or be a molecule that includes an ionophore moiety, such as a conjugate or derivative of a particular molecule and the types of molecules listed above. For example, the ionophore can be or include a valinomycin moiety conjugated to one or more separate non-ionophore moieties.

[0016] In some embodiments, the potassium ionophore includes a hydrophobic ionophore that can be dispersed within the polymer of the potassium selective transport layer. In some embodiments, the potassium ionophore includes valinomycin or is valinomycin. As used herein, the term potassium ionophore is used to refer to a chemical species that reversibly binds potassium ions. In some embodiments, the ionophore has a cyclic molecular structure and the potassium ions reversibly bind to the ionophore by being located at the center of the cyclic structure. The potassium ionophore is present in the potassium-selective transport layer at any suitable concentration. In some embodiments, the potassium ionophore is present in the potassium-selective transport layer in an amount of 0.1 wt% to 30 wt%, such as 2 wt% to 20 wt%, such as 5 wt% to 15 wt% of the polymer present in the potassium-selective transport layer. The potassium-selective transport layer further comprises an electrolyte. In some embodiments, the electrolyte comprises a hydrophobic electrolyte. In some embodiments, the electrolyte is insoluble in water. In some embodiments, the electrolyte comprises a salt comprising one or more organic cations and one or more organic anions. In some embodiments, the one or more organic cations and the one or more organic anions are sterically bulky. In some embodiments, an electrolyte comprising sterically bulky cations and anions of organic nature is hydrophobic, which is highly desirable. In some embodiments, the electrolyte is relatively hydrophobic so that, as discussed in more detail below, it remains in the potassium-selective transport layer and does not leach into the body fluid in which the sensor is disposed.

[0017] In some embodiments, the electrolyte is of the formula R1R2R3R4N(wherein R1 to R4 are each independently selected from C1-C4 alkoxy, C2-C8 alkoxyalkoxy, C3-C6 cycloalkyl, -OH, -NH2, -SH, -CO2(C1-C6)alkyl and -OC(O)(C1-C6)alkyl and may be substituted by 1 to 3 groups selected therefrom, and is selected from a straight-chain or branched alkyl or alkenyl group, for example, R1 to R4 are each independently selected from C5-C 30 alkyl groups which may be substituted as described above) and comprises a salt comprising a quaternary ammonium cation. In some embodiments, R1 to R4 are each independently selected from C4-C 15 straight-chain or branched alkyl groups which may be substituted as described above. In some embodiments, the electrolyte comprises a salt comprising a tetraoctylammonium cation.

[0018] In some embodiments, the electrolyte is of the formula BX4 - ​​​or PX6 (where each X is independently selected from 1 to 3 groups selected from C1-C4 alkoxy, C2-C8 alkoxyalkoxy, C3-C6 cycloalkyl, -OH, -NH2, -SH, -CO2(C1-C6)alkyl, F, Cl, and -OC(O)(C1-C6)alkyl, and may be substituted by these groups), and contains a salt containing an anion of 15 selected from aliphatic or aromatic hydrocarbyl groups). In some embodiments, each X is independently a C5-C 15 aromatic group, C5-C 15 alkyl group or C5-C 15 alkenyl group. In some embodiments, each X is independently selected from C5-C9 aromatic groups which may be substituted as described above. In some embodiments, each X is independently selected from C6 aromatic groups which may be substituted as described above. In some embodiments, the electrolyte contains a tetrakis(pentafluorophenyl)borate anion. In some embodiments, the electrolyte contains tetraoctylammonium tetrakis(pentafluorophenyl)borate (TOATB).

[0019] The electrolyte is present in the potassium-selective transport layer at any suitable concentration. In some embodiments, the electrolyte is present in the potassium-selective transport layer at a concentration of 5 mM to 50 mM, such as 5 mM to 20 mM, such as 5 mM to 15 mM. As discussed above, in some embodiments, the sensor and / or the potassium-responsive active region do not include an electrolyte gel layer. However, in some embodiments, the potassium-responsive active region may include an electrolyte gel layer. In some embodiments, the potassium-selective transport layer contains PVC, valinomycin, tetraoctylammonium tetrakis(pentafluorophenyl)borate (TOATB), and 2-nitrophenyl octyl ether (NPOE). In some embodiments, the electrolyte gel layer, if included, comprises an electrolyte hydrogel layer. In some embodiments, the electrolyte gel layer comprises an electrolyte hydrogel layer comprising an aqueous electrolyte solution and a crosslinked hydrophilic polymer. Any suitable hydrophilic polymer that forms a hydrogel when crosslinked may be used. In some embodiments, the crosslinked hydrophilic polymer comprises crosslinked polyvinyl alcohol, crosslinked polyethylene glycol, crosslinked acrylate polymer, crosslinked acrylamide polymer, crosslinked hyaluronic acid polymer, crosslinked chitosan, crosslinked heparin, crosslinked alginate, or crosslinked fibrin. In some embodiments, the crosslinked hydrophilic polymer comprises a crosslinked acrylamide polymer. In some embodiments, the crosslinked hydrophilic polymer comprises a crosslinked N,N-dimethylacrylamide polymer.

[0020] In some embodiments, the crosslinked hydrophilic polymer is formed by the reaction of a hydrophilic monomer and a crosslinking agent. Any suitable ratio of crosslinking agent to hydrophilic monomer can be used. In some embodiments, the hydrophilic monomer and crosslinking agent are reacted in amounts of 90 mol% to 99 mol% of the hydrophilic monomer and 1 mol% to 10 mol% of the crosslinking agent, for example, 95 mol% to 99 mol% of the hydrophilic monomer and 1 mol% to 5 mol% of the crosslinking agent. The hydrophilic monomer and crosslinking agent react to form a crosslinked hydrophilic polymer. In this respect, the monomers react with each other in the polymerization reaction to form polymer chains, and the crosslinking agent crosslinks the polymer chains. Suitable crosslinking agents are obvious to those skilled in the art. In some embodiments, the crosslinking agent includes ethylene glycol dimethacrylate. Therefore, in some embodiments, the crosslinked hydrophilic polymer includes N,N-dimethylacrylamide crosslinked with ethylene glycol dimethacrylate. The aqueous electrolyte in the electrolyte gel layer may be any suitable electrolyte or a suitable combination of electrolytes. In some embodiments, the aqueous electrolyte includes a solution of an aqueous solution of a Group 1 metal salt, for example, an aqueous solution of a Group 1 metal halide salt, for example, an aqueous solution of lithium chloride. Alternatively, the aqueous electrolyte solution includes a quaternary ammonium halide salt. In some embodiments, the aqueous electrolyte solution includes (i) an aqueous solution of a Group 1 metal salt, for example, an aqueous solution of a Group 1 metal halide salt, for example, an aqueous solution of lithium chloride, and (ii) a quaternary ammonium halide salt. In some embodiments, the quaternary ammonium halide salt is tetraC4-C 12 This includes alkylammonium halide salts, such as tetraoctylammonium chloride.

[0021] In some embodiments, the aqueous electrolyte solution contains an aqueous solution of a Group 1 metal salt, as discussed above, and the Group 1 metal salt is present in the solution at a concentration of 50 mM to 150 mM, for example, 75 mM to 125 mM. In some embodiments, when the aqueous electrolyte solution contains both the above-mentioned Group 1 metal salt and the above-mentioned quaternary ammonium halide salt, the quaternary ammonium halide salt is present in the aqueous electrolyte solution at a concentration of 1 mM to 5 mM. The aqueous electrolyte solution may also contain a buffer. Any suitable buffer may be used. An example of a buffer that may be used is (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). The buffer may be present at any suitable concentration. In some embodiments, if present, the buffer is present in the electrolyte solution at a concentration of 1 mM to 5 mM.

[0022] The aqueous electrolyte solution and the crosslinked hydrophilic polymer are present in any suitable amount in the electrolyte gel layer to form a hydrogel layer. In some embodiments, the aqueous electrolyte solution is present in the electrolyte hydrogel layer in an amount of 60% to 95% by mass, and the crosslinked hydrophilic polymer is present in the electrolyte hydrogel layer in an amount of 5% to 40% by mass. In some embodiments, the aqueous electrolyte solution is present in the electrolyte hydrogel layer in an amount of 70% to 90% by mass, and the crosslinked hydrophilic polymer is present in the electrolyte hydrogel layer in an amount of 10% to 30% by mass. The first working electrode includes a portion of its outer surface, the portion of which includes a transition metal ion in the form of a salt or complex, and may also include a transition metal. In some embodiments, the transition metal ions in salt or complex form, and optionally the transition metal, include silver, gold, osmium, iron, cobalt, nickel, copper, ruthenium, platinum, their ions, or any combination thereof. In some embodiments, the transition metal ions in salt or complex form, and optionally the transition metal, include silver, gold, osmium, their ions, or any combination thereof. In some embodiments, the transition metal ions in salt or complex form, and optionally the transition metal, include silver or osmium or their ions. In some embodiments, when a portion of the outer surface of the first working electrode contains a transition metal ion and a transition metal in the form of a salt or complex, both the transition metal and the transition metal ion are of the same element. For example, the outer surface portion may contain silver metal and silver ions in the form of a salt or complex. In some embodiments, the outer surface portion contains silver metal and silver salts, such as silver halides. In some embodiments, the outer surface portion contains silver metal and silver chloride.

[0023] In some embodiments, if a portion of the outer surface of the first working electrode contains a transition metal ion in the form of a salt or complex, but does not contain a transition metal, the electrode portion contains a first transition metal ion in a first oxidation state and a second transition metal ion of the same element in a second oxidation state. For example, the electrode portion may contain osmium ions Os(II) and Os(III). In such embodiments, both the first and second transition metal ions are in the form of a salt or complex. Some of the transition metal ions on the outer surface of the first working electrode may be in salt form. The salt may include any suitable salt. In some embodiments, the salt includes a halide salt, such as a chloride salt. For example, the salt may include a chloride salt of silver chloride or any of the other metals listed above. Some of the transition metal ions on the outer surface of the first working electrode may be in complex form. As used herein, the term "complex form" refers to a transition metal ion coordinated to one or more ligands. The resulting complex may have a positive, negative, or neutral static charge depending on the charge of the transition metal ion and the sum of its associated ligands. In some embodiments, the complex has a positive static charge. If the complex has a positive or negative static charge, it also has one or more associated counterions. In this respect, the complex is a salt containing a complex ion and counterions. In some embodiments, if the complex ion has a positive charge, the complex is in salt form and associated with one or more anionic counterions. Examples of preferred anionic counterions include halide ions and other preferred anions. In some embodiments, the counterions include chloride ions.

[0024] Examples of suitable complexes of transition metal ions include, [ka] (In the formula, M is one of the metals discussed above, for example, osmium.) L is

[0025] [ka] Selected from the group consisting of, R1, R2, and R'1 are independently substituted or unsubstituted alkyl, alkenyl, or aryl groups. For example, R1, R2, and R'1 are unsubstituted C1-C12 alkyl groups. R3, R4, R5, R6, R'3, R'4, R a , R b , R c and R dc is independently selected from H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, NHNH2, SH, OH, NH2 or substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino, arylcarboxamide, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl or alkyl, and c is an integer selected from -1 to -5 or +1 to +5 indicating a positive or negative charge. X represents at least one counterion, d is an integer between 1 and 5 that represents the number of counterions X. L1, L2, L3, and L4 are ligands. L1 contains a heterocyclic compound linked to the polymer backbone, and L1 and L2 together form a first bidentate ligand. Examples include:

[0026] In some embodiments, L3 and L4 combine to form a second bidentate ligand. In some embodiments, at least one of the first or second bidentate ligand is selected from the group consisting of substituted or unsubstituted 2,2'-bipyridine, 2,2'-biimidazole, and 2-(2-pyridyl)imidazole. In some embodiments, the second bidentate ligand is selected from the group consisting of substituted or unsubstituted 2,2'-bipyridine, 2,2'-biimidazole, and 2-(2-pyridyl)imidazole.

[0027] In some embodiments, the first bidentate ligand is selected from the group consisting of substituted 2,2'-bipyridine, 2,2'-biimidazole, and 2-(2-pyridyl)imidazole. In some embodiments, the substituted 2,2'-bipyridine, 2,2'-biimidazole, or 2-(2-pyridyl)imidazole is linked to the polymer backbone via substituents. In some embodiments, these substituents include substituted C1-C12 alkyl groups, substituted C1-C12 alkoxy groups, C1-C12 alkylthio groups, C1-C12 alkylamino groups, C2-C24 dialkylamino groups, or C1-C12 alkyl groups. In some embodiments, the polymer backbone is selected from the group consisting of styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer, poly(4-vinylbenzyl chloride) copolymer, poly(allylamine) copolymer, poly(4-vinylpyridine) copolymer, poly(4-vinylpyridine), poly(N-vinylimidazole), and poly(4-styrene sulfonate). Further details of such complexes are disclosed in US6605201, which is incorporated herein by reference in its entirety.

[0028] For example, the complex may be in the form of a redox polymer, including a polymer backbone grafted onto the complex, although not limited to those discussed above. In some embodiments, the redox polymer includes a polymer backbone linked to a transition metal ion complex as described above. In some embodiments, the redox polymer includes a polymer backbone linked to a transition metal ion complex as described above, where L1 is a ligand containing a heterocyclic compound linked to the polymer backbone. In some embodiments, the redox polymer is crosslinked and includes a crosslinking agent. Further details of such redox polymers are disclosed in US2012 / 0132525, which is incorporated herein by reference in its entirety.

[0029] In some embodiments, the polymer backbone is poly(vinylpyridine). For example, poly(vinylpyridine) having the following general structure may be used. [ka]

[0030] In some embodiments, in the structure described above, n is 2, n' is 17, and n'' is 1. In some embodiments, the polymer backbone is crosslinked. For example, the polymer backbone may be crosslinked by poly(ethylene glycol) diglycidyl ether (PEGDGE), for example, PEGDGE of the following formula (wherein n is an integer from 1 to 500). [ka]

[0031] The purpose of including transition metal ions in complexes, or redox polymers grafted onto complexes, such as those discussed above, is, firstly, to alter the oxidation / reduction potential of the ions so that oxidation / reduction is more easily facilitated by applying a potential from the electrode. The presence of complexes / redox polymers therefore promotes the oxidation and / or reduction of ions. Furthermore, complexes and redox polymers are useful in that they have the function of retaining ions in appropriate locations on the surface of the working electrode so that they are more easily reduced and / or oxidized by applying a potential from the electrode. It is not essential that the transition metal ions are in complex form. For example, as discussed above, in some embodiments, the portion of the outer surface of the working electrode contains silver in the form of silver salts, such as halides (e.g., chlorides), or silver ions (i.e., silver ions are not in complex form).

[0032] In some embodiments, where a transition metal ion in the form of a salt or complex, and optionally the transition metal, includes an osmium ion, the osmium ion is in the form of a complex or redox polymer, such as those discussed above. For example, in some embodiments, the electrode portion includes osmium(II) and / or osmium(III) ions, such as osmium(II) and osmium(III). In some embodiments, the osmium ions are in complex form. In some embodiments, the osmium ions are in the form of a redox polymer linked to a ligand complexed with osmium(II) and / or osmium(III) ions. In some embodiments, the redox polymer is as described above. In some embodiments, the counterion for the redox polymer is a chloride anion.

[0033] The sensor may further include a support layer on which a first working electrode is disposed, for example, the support layer being a polymer, such as PET. In some embodiments, if the portion of the outer surface of the first working electrode contains silver and silver chloride (AgCl) as described above, the first working electrode includes a silver / silver chloride electrode layer. In some embodiments, the silver / silver chloride is located along the length of the first working electrode (for example, along the length of the silver / silver chloride electrode layer). In such embodiments, if the potassium-responsive active region is located not along the entire length of the outer surface of the first working electrode but in a portion of the outer surface of the first working electrode, the silver / silver chloride may be located in both the portion where the potassium-selective transport layer is located and the portion of the outer surface of the first working electrode where the potassium-responsive active region is not located. If the first working electrode includes a silver / silver chloride electrode layer, the sensor may further include a support layer on which the silver / silver chloride electrode layer is disposed. In some embodiments, the support layer includes a polymer, such as PET. In some embodiments, if a portion of the first working electrode includes an osmium(II) complex, an osmium(III) complex, or a combination thereof, the first working electrode may include a carbon electrode layer, and the portion of the outer surface of the first working electrode includes a layer of one or more osmium ion complexes disposed on a portion of the outer surface of the carbon electrode layer. In some embodiments, the sensor further includes a support layer on which the carbon electrode layer is disposed. In some embodiments, the support layer includes a polymer, such as PET.

[0034] The potassium-responsive active region layer may have any suitable thickness. In some embodiments, the potassium-selective transport layer has a thickness of 1 to 200 μm. In some embodiments, the electrolyte gel layer, if included, has a thickness of 1 to 50 μm. In some embodiments, the first working electrode has a thickness of 1 nm to 20 μm. In some embodiments, the sensor further includes a layer of dielectric material located on a second portion of the outer surface of the first working electrode to define the exposed region of the working electrode where the potassium-responsive active region is located. For in vivo use, any suitable dielectric layer known to be suitable for use in analyte sensors may be used. An example of a dielectric material that may be used is Dupont 5018. In some embodiments, the potassium-selective transport layer forms the outer layer of the sensor and is in direct contact with the bodily fluid in which the sensor is located. In these embodiments, the dielectric material also forms a portion of the outer surface of the sensor, if present.

[0035] In other embodiments, the sensor further includes a mass transport restriction membrane. For example, the first working electrode and the potassium-responsive active region may be fully or partially encapsulated by the mass transport restriction membrane. In some embodiments, the mass transport restriction membrane includes a polymer membrane. The polymer membrane may include any suitable polymer. In some embodiments, the polymer membrane includes a copolymer of polyvinylpyridine, polyvinylimidazole, vinylpyridine, and styrene, or a combination thereof. In some embodiments, the polymer of the polymer membrane is crosslinked. Any suitable crosslinking agent may be used. In some embodiments, the polymer of the polymer membrane is crosslinked by reaction with polyethylene glycol tetraglycidyl ether. The mass transport restriction membrane is, of course, permeable to potassium so that potassium from the fluid in which the sensor is placed can reach the potassium-responsive active region. The sensor may further include one or more additional active regions. These additional active regions may be as described above. Alternatively, the additional active regions may include various chemical substances and can be adapted for the detection and concentration measurement of other analytes. Any suitable detection chemistry known in the industry may be used to detect other analytes.

[0036] The sensor may further include one or more additional working electrodes and / or one or more additional active regions. Optionally, one or more additional working electrodes or one or more additional active regions may be as described above. Alternatively, one or more additional working electrodes or one or more additional active regions may contain a variety of chemical substances and be adapted for the detection and concentration measurement of other analytes. Any suitable detection chemistry known in the industry may be used to detect other analytes. Therefore, one or more additional active regions may be configured to respond to potassium, or to respond to one or more additional analytes. In some embodiments, the sensor is an in vivo analyte sensor, and the sensor or at least a portion of the sensor is suitable for in vivo implantation. In some embodiments, the sensor includes a sensor tail, if any, which includes a first working electrode and any other working electrodes.

[0037] In some embodiments, the sensor is suitable for detecting the concentration of potassium in body fluids, for example, body fluids including skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. The analyte sensor may have any suitable design and may be set up for in vivo detection of potassium and / or determination of potassium concentration, as described in further detail below. Further details of the analyte sensor design will be apparent to those skilled in the art from the interest of this disclosure. In some embodiments, in addition to the sensor tail, the sensor further includes a sensor housing, where an adhesive layer in contact with the sensor housing is adapted to adhere to the tissue surface of the sensor housing, and the sensor tail protrudes from the sensor housing through the adhesive layer. However, alternative designs and modifications of this design may also be used, provided that the sensor is still suitable for in vivo potassium detection. The analyte sensor may further include a counter electrode, a reference electrode, or both. In some embodiments, the sensor has a length of 5 mm to 10 mm, a width of 0.23 mm to 0.5 mm (e.g., 0.3 mm to 0.5 mm), and a thickness of 0.1 mm to 0.4 mm (e.g., 0.2 mm to 0.4 mm).

[0038] In some embodiments, the resulting signal indicates oxidation of a transition metal atom or ion to a higher oxidation state, or reduction of a transition metal ion to a lower oxidation state. For example, the signal may indicate oxidation of a transition metal atom with an oxidation state of 0 to an oxidation state of +1, oxidation of a transition metal atom with an oxidation state of +1 to an oxidation state of +2, or oxidation of a transition metal atom with an oxidation state of +2 to an oxidation state of +3. Alternatively, the signal may indicate reduction of a transition metal ion with an oxidation state of +1 to 0, reduction of a transition metal ion with an oxidation state of +2 to +1, or reduction of a transition metal ion with an oxidation state of +3 to +2. In some embodiments, the signal may indicate the oxidation of a silver atom with an oxidation state of zero to a silver ion with an oxidation state of +1, and / or the reduction of a silver ion with an oxidation state of +1 to a silver atom with an oxidation state of zero. Alternatively, the signal may indicate the oxidation of an osmium ion with an oxidation state of +2 to an osmium ion with an oxidation state of +3, and / or the reduction of an osmium ion with an oxidation state of +3 to an osmium ion with an oxidation state of +2. In some embodiments, the transition metal ions in salt or complex form on a portion of the outer surface of the first working electrode, and optionally the transition metal, include silver and / or silver chloride (AgCl) as described above, and the signal indicates the reduction of silver chloride (AgCl) to silver and / or the oxidation of silver to silver chloride (AgCl).

[0039] Alternatively, the transition metal ions in salt or complex form, and optionally the transition metal on the outer surface of the first working electrode, include osmium(II) complexes, osmium(III) complexes, or both, as described above, and the signal indicates the reduction of osmium(III) chloride to osmium(II) chloride and / or the oxidation of osmium(II) chloride to osmium(III) chloride. For example, the use of either the above-mentioned or herein-described sensors for detecting the concentration of potassium ions in a fluid is provided herein. In some embodiments, the fluid is a body fluid. In some embodiments, the body fluid and analyte sensors are in vivo. In some embodiments, the body fluid includes skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. The detection method may include any suitable electrochemical detection method. For example, in some embodiments, the detection method includes the step of applying a fluctuating potential to a first working electrode, and the detected signal includes a voltammetry method including peak current measurement. Alternatively, the detection method may include an amperometric method, which includes the steps of applying a constant potential to a first working electrode and measuring the resulting current. Alternatively, the detection method may include a chronoamperometric method, which includes the steps of applying a potential that alternately repeats two different values ​​and measuring the resulting current. [Brief explanation of the drawing]

[0040] [Figure 1] This is a schematic diagram of an illustrative sensing system that can incorporate the analyte sensor of this disclosure. [Figure 2A-2C] This is a cross-sectional view of an analyte sensor containing a single active region. [Figure 3A-3C] This is a cross-sectional view of an analyte sensor containing two active regions. [Figure 4] This is a cross-sectional view of an analyte sensor containing two active regions. [Figure 5A-5C] This is a perspective view of an analyte sensor containing two active regions on separate working electrodes. [Figure 6-7] This figure shows an exemplary sensor chemistry of the present invention. Figures 6a and 7a show embodiments using a gel layer, and Figures 6b and 7b show embodiments without a gel layer. [Figure 8]This is a graph comparing currents observed at different voltages. The potassium concentration in the fluid is determined using voltammetry with the sensor of this disclosure and a comparison sensor that does not contain potassium ionophores. [Figure 9-10] This graph shows the difference between current and peak current when the potassium concentration of the fluid in which the sensor of this disclosure is placed is determined using voltammetry. [Figure 11] This graph shows the effect of voltage on current in a voltammetry experiment using a comparative sensor that does not contain potassium ionophores. [Figure 12] This is a graph showing the current as a function of time in an amperometry method performed using the sensor of this disclosure. [Figure 13-14] This is a graph showing the current as a function of time in a chronoamperometry method performed using the sensor of this disclosure. [Figure 15-16] This is a graph showing the current as a function of time in a chronoamperometry method performed using the sensor of this disclosure. [Figure 17-18] This is a graph showing the current as a function of time in the chronoamperometric method of the present invention using the sensor disclosed herein. [Figure 19] This graph shows the data sampled using pulse amperometry (Figure 18). [Figure 20] This figure shows the sensors of the present disclosure tested using pulsed amperometry at different potassium concentrations. [Figure 21-22] This graph shows the current as a function of time in the pulse amperometry method of the present invention using the sensor disclosed herein. The sensor is tested daily. [Modes for carrying out the invention]

[0041] This disclosure provides sensor chemistry, particularly detection chemistry utilizing non-enzymatic systems, that is suitable for monitoring potassium levels over a range of physiologically relevant potassium concentrations. definition The terms used herein generally have the common meaning in the industry within the context of this disclosure and in the specific context in which each term is used. Certain terms used in the descriptions of the compositions and methods of this disclosure, and how they are prepared and used, are discussed below or elsewhere in this specification for the purpose of providing further guidance to practitioners. As used herein, the use of the words “a” or “an,” when used in the claims and / or in conjunction with the term “comprising,” may mean “one,” but also coincide with the meanings of “one or more,” “at least one,” and “one or more than one.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” and “contain(s),” and their variations as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude additional functions or structures. This disclosure also assumes, whether expressly or not, other embodiments “including,” embodiments or elements “consisting of,” and “consisting essentially of” those present herein.

[0042] The terms "approximately" or "about" mean a range of acceptable error for a particular value as determined by those skilled in the art, and depend in part on how that value is measured or determined, i.e., the limitations of the measuring system. As used herein, the term “body fluid” refers to any body fluid or humoral derivative on which the analyte can be measured. Non-limiting examples of body fluids include skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sweat, and tears. In certain embodiments, the body fluid is skin fluid or interstitial fluid. As used herein, the term “reference electrode” may refer to either a reference electrode or an electrode that functions as both a reference electrode and a counter electrode. Similarly, as used herein, the term “counter electrode” may refer to either a counter electrode or a counter electrode that also functions as a reference electrode. As used herein, the term “homogeneous membrane” refers to a membrane containing a single type of membrane polymer. As used herein, the term “multicomponent membrane” refers to a membrane containing two or more types of membrane polymers.

[0043] Analytical Sensor & Sensor Configuration Before further detailing the analyte sensors and their components of this disclosure, a schematic of a preferred in vivo analyte sensor configuration and a sensor system using the analyte sensor is provided to better understand the embodiments of this disclosure. Figure 1 shows a diagram of an illustrative sensing system into which the analyte sensor of this disclosure may be incorporated. As shown, sensing system 1 includes a sensor control device 102 and a reader device 120 configured to communicate with each other over a local communication path or link 140 which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 may be a component of an output medium for visualizing the analyte concentration and alerts or notifications determined by the sensor 104 or an associated processor, and for enabling one or more user inputs according to a particular embodiment. The reader device 120 may be a multipurpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, there may be multiple reader devices 120 in a particular embodiment. The reader device 120 may communicate with the remote terminal 170 and / or the trusted computer system 180 via wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted communication paths / links 141 and / or 142, respectively. The reader device 120 may also, or alternatively, communicate with a network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. The network 150 may further be communicably connected to the remote terminal 170 via communication path / link 152 and / or the trusted computer system 180 via communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of the intervening reader device 120.For example, but not limited to, the sensor 104 may, according to a particular embodiment, communicate with a remote terminal 170 and / or a trusted computer system 180 via a direct communication link to the network 150, as described in U.S. Patent Application Publication No. 2011 / 0213225, the whole of which is incorporated herein by reference. Any suitable electronic communication protocol may be used for the communication path or link, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth® or Bluetooth® Low Energy Protocol, or Wi-Fi. The remote terminal 170 and / or trusted computer system 180 may, according to a particular embodiment, be available to an individual other than the primary user who is interested in the user's analyte level. The reader device 120 may include a display 122 and an optional input component 121. The display 122 may, according to a particular embodiment, include a touchscreen interface.

[0044] The sensor control device 102 includes a sensor housing 103 capable of housing the circuitry and power supply for operating the sensor 104. Optionally, the power supply and / or active circuitry may be omitted. A processor (not shown) may be communicatively connected to the sensor 104, and the processor is physically located within the sensor housing 103 or the reader device 120. According to a particular embodiment, the sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105 adapted to adhere the sensor housing 103 to a tissue surface, such as skin. The sensor 104 is adapted to be at least partially inserted into the target tissue, for example, into the skin or the subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to be inserted to a desired depth in the given tissue. The sensor tail may include at least one working electrode. In some configurations, the sensor tail may include an active region for detecting an analyte placed on the working electrode. A counter electrode may be present in combination with at least one working electrode. Specific electrode configurations in the sensor tail are described in more detail below.

[0045] The active region may be configured to monitor a specific analyte, such as potassium or potassium ions. In certain embodiments, the active region may be configured to detect two or more analytes. In certain embodiments of the Disclosure, one or more analytes may be monitored in any body fluid of interest, such as skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. In certain detailed embodiments, the analyte sensors of the Disclosure may be adapted to skin fluid or interstitial fluid assays for determining the concentration of one or more analytes in vivo. In certain embodiments, the body fluid is interstitial fluid.

[0046] Referring further to Figure 1, the sensor 104 can automatically transfer data to the reader device 120. For example, but not limited to, analyte concentration data (e.g., potassium ion concentration) can be communicated automatically and periodically, for example, when the data is obtained or at regular intervals after a certain period has passed, and the data is stored in memory until transmission (e.g., every minute, every 5 minutes, or other predetermined periods). In another embodiment, the sensor 104 can communicate with the reader device 120 by a non-automatic method and without following a set schedule. For example, but not limited to, data can be communicated from the sensor 104 using RFID technology when the sensor electronic components are brought within the communication range of the reader device 120. The data may remain stored in the sensor 104's memory until it communicates with the reader device 120. Thus, the user does not need to constantly maintain proximity to the reader device 120, but rather can upload data at a convenient time. In another embodiment, a combination of automatic and non-automatic data transfer can be practiced. For example, and not limited to, data transfer may continue automatically until the reader device 120 is no longer within the communication range of the sensor 104.

[0047] An introducer may be transiently present to facilitate the introduction of sensor 104 into the tissue. In an embodiment of the illustration, the introducer may include a needle or a similar sharp object. As will be readily apparent to those skilled in the art, other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be transiently present in close proximity to sensor 104 before tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the insertion of sensor 104 into the tissue by opening a pathway for sensor 104 to follow. For example, and not limited to, the needle may, according to one or more embodiments, facilitate penetration of the epidermis as a pathway to the dermis, thereby enabling the implantation of sensor 104. After opening the pathway, the needle or other introducer may be withdrawn in a manner that does not pose a sharp object hazard. In certain embodiments, a suitable needle may have a solid or hollow cross-section, beveled or non-beveled, and / or circular or non-circular. In more detailed embodiments, a suitable needle may be comparable to an acupuncture needle in terms of cross-sectional diameter and / or tip design, which may have a cross-sectional diameter of about 250 μm. However, a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0048] In certain embodiments, the tip of the needle (while present) may be angled across the end of the sensor I04 so that the needle first penetrates the tissue and opens a path for the sensor 104 to reach it. In certain embodiments, the sensor 104 may be located within the lumen or groove of the needle, and the needle similarly opens a path for the sensor 104 to reach it. In either case, the needle is subsequently withdrawn after facilitating the insertion of the sensor. Sensor configurations featuring a single active region configured to detect a single corresponding analyte can utilize two-electrode or three-electrode detection motifs further described herein with respect to Figures 2A-2C. Sensor configurations featuring two different active regions for detecting the same or different analytes, either on separate working electrodes or on the same working electrode, are described separately later with respect to Figures 3A-5C. Sensor configurations with multiple working electrodes may be particularly advantageous for incorporating two different active regions within the same sensor tail, as the signal contribution from each active region can be more easily determined. When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, where the second electrode may function as both a counter electrode and a reference electrode (i.e., counter electrode / reference electrode). The various electrodes may be at least partially stacked (layered) with respect to each other and / or laterally spaced apart at the sensor tail. A preferred sensor configuration may be substantially flat in shape, substantially cylindrical in shape, or any other preferred shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator.

[0049] Figure 2A shows a diagram of an illustrative two-electrode analyte sensor configuration suitable for use in the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 positioned between a working electrode 214 and a counter electrode / reference electrode 216. Alternatively, the working electrode 214 and the counter electrode / reference electrode 216 may be located on the same side of the substrate 212, with a dielectric material between them (configuration not shown). An active region 218 is positioned as at least one layer on at least a portion of the working electrode 214. The active region 218 may include multiple spots or a single spot configured to detect an analyte, as further discussed herein. Referring further to Figure 2A, the film 220 overcoats at least the active region 218. In certain embodiments, the film 220 may also overcoat part or all of the working electrode 214 and / or the counter electrode / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be overcoated with the film 220. The film 220 may comprise one or more polymer film materials having the ability to restrict the analyte flux to the active region 218 (for example, the film 220 is a material transport restriction film with some permeability to the analyte of interest). According to the disclosures herein and further described below, the film 220 may be crosslinked with a branched crosslinking agent in certain sensor configurations. For example, but not limited to, the film 220 may be crosslinked with a branched glycidyl ether. The composition and thickness of the film 220 may be varied to facilitate the desired analyte flux to the active region 218, thereby obtaining the desired signal intensity and stability. The analyte sensor 200 may be operable for analyte assays using any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0050] Figures 2B and 2C show diagrams of illustrative three-electrode analyte sensor configurations, which are also suitable for use in the disclosure herein. The three-electrode analyte sensor configuration may be similar to that shown in analyte sensor 200 in Figure 2A, except for the inclusion of an additional electrode 217 in analyte sensors 201 and 202 (Figures 2B and 2C). With the additional electrode 217, the counter electrode / reference electrode 216 may then function as the counter electrode or reference electrode, and the additional electrode 217 may perform other electrode functions not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 may be placed on the working electrode 214 or electrode 216, with a dielectric material isolation layer between them. For example, and not limited to, as depicted in Figure 2B, dielectric layers 219a, 219b, and 219c isolate and electrically insulate electrodes 214, 216, and 217 from each other. Alternatively, at least one of electrodes 214, 216, and 217 may be located on the opposite side of the substrate 212, as shown in Figure 2C. Thus, in certain embodiments, electrodes 214 (working electrode) and 216 (counter electrode) may be located on the opposite side of the substrate 212, and electrode 217 (reference electrode) may be located on one of electrodes 214 or 216, spaced apart from it by the dielectric material.

[0051] A reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the location of the reference material layer 230 is not limited to those depicted in Figures 2B and 2C. Similar to the sensor 200 shown in Figure 2A, the active region 218 in the analyte sensors 201 and 202 may consist of multiple spots or a single spot. Furthermore, the analyte sensors 201 and 202 may be operable for analyte assays using any of the coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques. Like the analyte sensor 200, the film 220 can also overcoat the active region 218 and other sensor components in the analyte sensors 201 and 202, thereby functioning as a material transport restriction film. In certain embodiments, an additional electrode 217 may be overcoated with film 220. Figures 2B and 2C depict electrodes 214, 216, and 217 as being overcoated with film 220, although it should be noted that in certain embodiments, only the working electrode 214 may be overcoated. Furthermore, the thickness of film 220 on each of electrodes 214, 216, and 217 may be the same or different. As in the two-electrode analyte sensor configuration (Figure 2A), one or both sides of the analyte sensors 201 and 202 may be overcoated with film 220 in the sensor configurations of Figures 2B and 2C, or the entirety of the analyte sensors 201 and 202 may be overcoated. Therefore, the three-electrode sensor configuration shown in Figures 2B and 2C should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of this disclosure.

[0052] Figure 3A illustrates the configuration of an illustrative sensor 203 having a single working electrode with two distinct active regions. Except for the presence of two active regions on the working electrode 214: a first active region 218a and a second active region 218b, these respond to the same or different analytes and are laterally spaced apart from each other on the surface of the working electrode 214. The active regions 218a and 218b may comprise multiple spots or a single spot configured to detect each analyte. The composition of the film 220 may vary or be compositionally identical in the active regions 218a and 218b. The first active region 218a and the second active region 218b may be configured to detect their corresponding analytes at different working electrode potentials, as further discussed below.

[0053] Figures 3B and 3C show cross-sectional views of three-electrode sensor configurations illustrating sensors 204 and 205, respectively, each characterizing a single working electrode having a first active region 218a and a second active region 218b positioned on top. Figures 3B and 3C are otherwise similar to Figures 2B and 2C and can be better understood by referring to them. As with Figure 3A, the composition of the film 220 may vary or be compositionally identical in the active regions 218a and 218b. Sensor configurations in examples having multiple working electrodes, specifically two working electrodes, are described in more detail with respect to Figure 4-SC. While the following description primarily focuses on sensor configurations with two working electrodes, it should be recognized that through the expansion of the disclosure herein, more than two working electrodes may be incorporated. Additional working electrodes may be used to impart additional sensing capability to the analyte sensor, for example, to detect a third and / or fourth analyte, in addition to the first and second analytes.

[0054] Figure 4 shows a cross-sectional view of an illustrative analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode, and suitable for use in the disclosure herein. As shown, the analyte sensor 300 includes working electrodes 304 and 306 positioned on opposite sides of the substrate 302. A first active region 310a is located on the surface of the working electrode 304, and a second active region 310b is located on the surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. External dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. A film 340 may, according to various embodiments, overcoat at least the active regions 310a and 310b, and other components of the analyte sensor 300 or the entire analyte sensor 300 may be overcoated with film 340. In certain embodiments, the film 340 may be continuous but compositionally varied in the active region 310a and / or active region 310b to obtain different permeability values ​​that separately adjust the analyte flux at each location. For example, different film formulations may be sprayed and / or printed on opposing surfaces of the analyte sensor 300. Immersion coating techniques may also be particularly suitable for depositing at least a portion of a bilayer film on one of the active regions 310a and 310b. In certain embodiments, the film 340 may be identical or compositionally varied in the active regions 310a and 310b. For example, but not limited to, the film 340 may include a bilayer film overcoating the active region 310a and a homogeneous film overcoating the active region 310b, or the film 340 may include a bilayer film overcoating the active region 310b and a homogeneous film overcoating the active region 310a. In certain embodiments, the analyte sensor may include more than one membrane 340, for example, two or more membranes. For example, but not limited to, the analyte sensor may include a membrane overcoating one or more active regions, for example, 310a and 310b, and an additional membrane overcoating the entire sensor as shown in Figure 4.

[0055] Alternative sensor configurations having multiple working electrodes, different from the configuration shown in Figure 4, feature counter / reference electrodes instead of separate counter and reference electrodes 320, 321, and / or feature layer and / or film arrangements that differ from those explicitly depicted. For example, and not limited to, the positioning of the counter electrode 320 and reference electrode 321 may be reversed from those depicted in Figure 4. Furthermore, the working electrodes 304 and 306 of the substrate 302 do not necessarily have to be on opposing surfaces as shown in Figure 4. A preferred sensor configuration may feature substantially planar electrodes, but it should be recognized that sensor configurations featuring non-planar electrodes may be advantageous and particularly preferred for use in the disclosure herein. In detail, substantially cylindrical electrodes arranged concentrically with respect to each other may facilitate the deposition of a mass transport limiting film, as described below herein. Figures 5A–5C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically with respect to each other. It should be recognized that sensor configurations having a concentric electrode arrangement but without a second working electrode are also conceivable in this disclosure.

[0056] Figure 5A shows a perspective view of an illustrative sensor configuration in which multiple electrodes are substantially cylindrical and arranged concentrically with respect to one another around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402, around which all electrodes and dielectric layers are arranged concentrically with respect to one another. In detail, the working electrode 410 is located on the surface of the central substrate 402, and the dielectric layer 412 is located on a portion of the working electrode 410 distal to the sensor tip 404. The working electrode 420 is located in the dielectric layer 412, and the dielectric layer 422 is located distal to a portion of the working electrode 420 at the sensor tip 404. The counter electrode 430 is located in the dielectric layer 422, and the dielectric layer 432 is located on a portion of the counter electrode 430 distal to the sensor tip 404. The reference electrode 440 is located in the dielectric layer 432, and the dielectric layer 442 is located on a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of the working electrode 410, the working electrode 420, the counter electrode 430, and the reference electrode 440 are spaced apart from each other along the vertical axis B of the analyte sensor 400.

[0057] Referring further to Figure 5A, the first active region 414a and the second active region 414b, which respond to different or the same analytes, are located on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby enabling contact with the fluid for sensing. Although the active regions 414a and 414b are depicted as three separate spots in Figure 5A, it should be recognized that several or more spots, including a continuous layer of active regions, may exist in alternative sensor configurations. In Figure 5A, the sensor 400 is partially coated with film 450 over the working electrodes 410 and 420, and the active regions 414a and 414b located thereon. Figure 5B shows an alternative sensor configuration in which substantially the entire sensor 401 is overcoated with film 450. Film 450 may be identical or compositionally varied in the active regions 414a and 414b. For example, film 450 may include a bilayer overcoating the active region 414a and a homogeneous film overcoating the active region 414b.

[0058] It should be further recognized that the positioning of various electrodes in Figures 5A and 5B may differ from those explicitly depicted. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from those depicted in Figures 5A and 5B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly depicted in Figures 5A and 5B. Figure 5C shows an alternative sensor configuration to that shown in Figure 5B, where the sensor 405 includes the counter electrode 430 and the reference electrode 440 located more proximal to the sensor tip 404, as well as the working electrodes 410 and 420 located more distal to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are located more distal to the sensor tip 404 may be advantageous by increasing the surface area for the deposition of active regions 414a and 414b (five separate sensing spots shown as an example in Figure 5C), thereby promoting increased signal intensity in some cases. Similarly, the central substrate 402 can be omitted in any concentric sensor configuration disclosed herein, and the innermost electrode can instead support the subsequently deposited layer.

[0059] Sensor Chemistry & Electrochemical Detection Methods As discussed in detail above, the analyte sensor of the present disclosure includes a potassium-responsive active region, the potassium-responsive active region includes a potassium-selective transport layer forming the outer surface of the potassium-responsive active region, the potassium-selective transport layer includes a polymer, a potassium ionophore, a plasticizer, and an electrolyte. Optionally, the sensor further includes an electrolyte gel layer positioned between the potassium-selective transport layer and a portion of the outer surface of a first working electrode, but as discussed above, a key aspect of some embodiments of the present invention is that the presence of such a layer is not required when the method of the present invention is used. The potassium-responsive active region plays a role in detecting the presence and concentration of potassium ions in the fluid in which the analyte sensor is placed. When the analyte sensor is placed in a potassium-containing fluid, potassium ions migrate into the potassium-selective transport layer by reversibly binding to potassium ionophores. This process occurs during the reduction of transition metal ions at the electrode. The number of potassium ions that migrate from the fluid into the potassium-selective transport layer is proportional to the concentration of potassium ions in the fluid in which the sensor is placed. Therefore, a higher concentration of potassium ions in the fluid results in a higher concentration of potassium ions in the potassium-selective transport layer. The purpose of the potassium ionophore is to allow potassium ions to enter the potassium-selective transport layer. Potassium ions in aqueous solutions are hydrophilic and therefore typically do not migrate into hydrophobic polymer layers, such as PVC. Ionophores are typically hydrophobic molecules and can therefore be efficiently dispersed in the polymer of the potassium-selective transport layer. The reversible binding of the ionophore to potassium ions makes the potassium ions suitably hydrophobic, allowing them to migrate into the polymer layer.

[0060] When an electric potential is applied to the working electrode, transition metal ions present in a portion of the first working electrode are reduced to a lower oxidation state. This causes a charge imbalance at the working electrode due to the presence of anionic counterions. To balance the charge at the working electrode, anions migrate from the portion of the first working electrode to the electrolyte gel layer. For example, if the sensor contains silver and silver chloride in a portion of the first working electrode, silver chloride (AgCl) reduces elemental silver, and the chloride ions migrate into the electrolyte gel layer. Similarly, if a portion of the first working electrode contains a mixture of osmium(II) and osmium(III) ions (e.g., osmium ions in complex form), the osmium(III) ions are reduced to osmium(II) ions upon application of an electric potential to the first working electrode, and the complex counterions migrate into the electrolyte gel layer to balance the charge. The flow of anions into the electrolyte gel layer causes a charge imbalance in the electrolyte gel layer. To balance the charge in the electrolyte gel layer, anions flow from the electrolyte gel layer into the potassium-selective transport layer, thus causing a charge imbalance in the potassium-selective transport layer. To suppress this charge imbalance in the potassium-selective transport layer, potassium ions from the fluid in which the sensor is located are drawn into the potassium-selective transport layer. In the absence of the electrolyte gel layer, ions flow directly from the electrode into the potassium-selective transport layer.

[0061] The electrodes obtain a signal indicating the flow of potassium ions from the fluid in which the sensor is located into the potassium-selective transport layer. In some embodiments, the signal is detected as an electric current. The amount of potassium ions that can flow from the fluid in which the sensor is located into the potassium-selective transport layer is a limited step in the ion flow cascade. Therefore, the magnitude of the detected current is positively correlated with how many potassium ions can flow from the fluid in which the sensor is located into the potassium-selective transport layer. As discussed above, how many potassium ions can enter the potassium-selective transport layer is correlated with the concentration of potassium ions in the fluid in which the sensor is located. The detected signal therefore indicates the concentration of potassium ions in the fluid in which the sensor is located.

[0062] The detected signal also indicates the degree of reduction occurring at the first working electrode. The intensity of the signal is correlated with the degree of reduction occurring at the electrode. The degree of reduction occurring at the electrode is correlated with the number of potassium ions that have migrated from the fluid into the potassium-selective transport layer to balance the charge (and thus correlated with the concentration of potassium ions in the fluid, as discussed above). Since the migration of potassium ions into the potassium-selective transport layer is a limiting factor in the ion flow cascade, the amount of potassium ions that can migrate into the potassium-selective transport layer determines the degree of reduction that can occur at the electrode. Since the amount of potassium ions that can migrate from the fluid into the potassium-selective transport layer is related to the concentration of potassium ions in the body fluid, the intensity of the signal obtained at the electrode is therefore correlated with the concentration of potassium ions in the fluid in which the sensor is located. The sensor signal can therefore indicate the concentration of potassium ions in the fluid in which the sensor is located.

[0063] Once the above process occurs, the transition metal salt can be recovered at the first working electrode by applying a potential to the electrode that causes oxidation of the reduced chemical species. For example, in the above system, the silver atom is Ag +Osmium(II) complex ions can be oxidized to osmium(III) complex ions. This process re-transfers the anions from the electrolyte gel layer into a portion of the first working electrode to balance the charge, yielding a salt of silver chloride or an osmium(III) salt. This process then re-transfers the anions from the potassium-selective transport layer into the electrolyte gel layer to balance the charge in the electrolyte gel layer. To balance the charge in the potassium-selective transport layer, potassium ions re-transfer from this layer into the surrounding fluid in which the sensor is placed. If the electrolyte gel layer is absent, the ions flow directly from the potassium-selective transport layer into a portion of the working electrode. The electrode then obtains a signal indicating the re-flow of potassium ions from the potassium-selective transport layer into the surrounding fluid. In some embodiments, the signal is an electric current. The measured current indicates the concentration of potassium ions in the fluid in which the sensor is placed, as discussed above. This is because the amount of potassium ions released from the potassium-selective transport layer is equal to (or at least very similar to) the amount of potassium ions originally drawn into the layer during the reduction step, and this amount is correlated with the concentration of potassium ions in the fluid in which the sensor is located, as discussed above. The degree of oxidation occurring at the electrode is correlated with the number of potassium ions in the potassium-selective transport layer. Since the number of potassium ions in the potassium-selective transport layer is correlated with the concentration of ions in the analyzed fluid, the degree of oxidation is therefore correlated with the concentration of potassium ions in the fluid in which the sensor is located. An electrode signal for the above oxidation process occurring at the electrode is also obtained, and this signal indicates the concentration of potassium ions in the analyzed fluid.

[0064] The sensor of the present invention can operate continuously due to the reversibility of potassium ions, which can move in and out of the potassium-selective transport layer, and the corresponding oxidation / reduction process that occurs at the electrode and thus generates a signal indicating the potassium ion concentration in the analyzed fluid. Potassium ions can enter the potassium-selective transport layer so that a signal indicating the potassium ion concentration of the analyzed fluid is generated. Potassium ions may then subsequently move out of the potassium-selective transport layer, so that the sensor can be reused in another measurement cycle when potassium ions move back into the potassium-selective transport layer. The sensor can therefore continuously monitor the concentration of potassium ions in the fluid in which the sensor is placed, and also monitor changes in the potassium ion concentration in the fluid over time. If an electrolyte gel layer is included in the sensor of this disclosure, the inclusion of such a layer is useful in extending the service life of the sensor. This is thought to be because the gel layer provides a reservoir for anions (from the electrolyte) that can, if necessary, migrate into the potassium-selective transport layer or, if necessary, into a portion of the working electrode. The electrolyte gel layer can also accept anions migrating from the potassium-selective transport layer during oxidation occurring at the electrode. It has also been found that the electrolyte gel layer is useful in providing an effective medium for promoting reduction and oxidation reactions at the electrode surface. However, as discussed above, the inclusion of an electrolyte gel layer increases the complexity and cost of the process for manufacturing the sensor. Therefore, it is desirable to omit such a layer if the sensor can still have sufficient functionality and service life. As discussed above, it was unexpectedly found that the electrolyte gel layer can be omitted when the method of the present invention is used, and that the sensor can still be used to detect potassium in in vivo body fluids effectively.

[0065] Figures 6 and 7 illustrate the sensor chemistry and configuration of an exemplary analyte sensor of the present disclosure. Figures 6a and 7a show embodiments using a gel layer, and Figures 6b and 7b show embodiments without a gel layer. In Figure 6a, the first working electrode is a silver / silver chloride electrode layer 501. The electrolyte gel layer 502 is located adjacent to this electrode layer. In this embodiment, a potassium-selective transport layer 503 containing PVC, a plasticizer, an electrolyte, and a potassium ionophore is located adjacent to the electrolyte gel layer. Both the electrolyte gel layer 502 and the potassium-selective transport layer 503 are potassium-responsive active regions. It can be seen that a portion of the silver / silver chloride layer 501 is directly exposed to the electrolyte gel layer 502, while a portion of the silver / silver chloride layer 501 extends further along the length of the working electrode with the dielectric layer 504. The PET electrode support layer 505 is adjacent to the Ag / AgCl layer 501. The working electrode is surrounded by a mass transport limiting membrane 506, as discussed in more detail below. The mass transport restriction membrane 506 limits contact between the potassium-responsive active region and the surrounding fluid in which the sensor is located, but is at least partially permeable to potassium ions so that potassium ions can come into contact with the potassium-responsive active region.

[0066] Figure 7a shows an alternative configuration to the sensor configuration shown in Figure 6a. In the configuration depicted in Figure 7a, the silver / silver chloride electrode layer is replaced by a carbon electrode 601. An external electrode portion 607 containing an osmium(II) / osmium(III) ion complex with a potassium-selective transport layer positioned on top is located on a portion of the external surface of the carbon electrode 601. A dielectric layer 604 is located on the other portion of the carbon electrode 601. The external electrode portion 607 contains a redox polymer with a polymer backbone grafted onto the ligand of the osmium complex (in the figure, the redox polymer is not depicted, and the osmium ion is depicted as a simple osmium chloride salt). An electrolyte gel layer 602 is located adjacent to the external electrode portion 607, and a potassium-selective transport layer 603 is located adjacent to the electrolyte gel layer 602. A PET electrode support layer 605 is adjacent to the carbon electrode 601. The working electrode is surrounded by a mass transport limiting membrane 606. In Figure 6b, the first working electrode is a silver / silver chloride electrode layer 501. In this embodiment, a potassium-selective transport layer 502 containing PVC, a plasticizer, an electrolyte, and a potassium ionophore is located adjacent to the silver / silver chloride layer. The potassium-selective transport layer 502 forms a potassium-responsive active region. Although not shown in the figure, a portion of the silver / silver chloride layer is directly exposed to the potassium-selective transport layer 502, while a portion of the silver / silver chloride layer 501 extends further along the length of the working electrode, directly adjacent to the dielectric layer. The PET electrode support layer is adjacent to the Ag / AgCl layer 501. The working electrode may be surrounded by a mass transport limiting membrane, as discussed in more detail below. The mass transport limiting membrane restricts contact of the potassium-responsive active region with the surrounding fluid on which the sensor is located, but is at least partially permeable to potassium ions, allowing potassium ions to contact the potassium-responsive active region.

[0067] Figure 7b shows an alternative configuration to the sensor configuration shown in Figure 6b. In the configuration depicted in Figure 7b, the silver / silver chloride electrode layer is replaced by a carbon electrode 601. An external electrode portion 603 containing an osmium(II) / osmium(III) ion complex with a potassium-selective transport layer 602 positioned on top is located on a portion of the external surface of the carbon electrode. The external electrode portion contains a redox polymer with a polymer backbone grafted onto the ligand of the osmium complex (in the figure, the redox polymer is not depicted, and the osmium ion is depicted as a simple osmium chloride salt). In addition to step (a) preparing an analyte sensor, the method of the present invention includes step (b) applying a potential to a first working electrode and obtaining a signal indicating oxidation and / or reduction of a transition metal or transition metal salt of the first working electrode, the signal indicating the concentration of potassium ions in the fluid. As discussed above, in some embodiments, the method of the present invention involves the steps of applying a potential that alternately repeats between two different values, and measuring the resulting current. The two different values ​​are selected to correspond to oxidation and reduction, respectively, at the working electrode. For example, one value may be selected to correspond to the electrochemical reduction of AgCl to metallic silver, and the other value may be selected to electrochemically oxidize AgCl to metallic silver to form silver chloride. The resulting current is the obtained sensor signal, indicating the reduction / oxidation that occurred at the working electrode. The potassium concentration in the potassium-selective transport layer, and therefore in the fluid in which the sensor is located, can be determined from this measurement.

[0068] Details of how to perform the steps of the above method and how to obtain the signal will be well known to those skilled in the art in light of the interests of this disclosure. In some embodiments, the method of the present invention also includes the step of determining the concentration of potassium ions in a fluid from the obtained signal. A method for determining the potassium ion concentration from the obtained sensor signal will be apparent to those skilled in the art in light of the interests of this disclosure. It should be recognized that, in the method described above, the sensitivity (output current) of the analyte sensor to each analyte can be varied by changing the coating (area or size) of the active region, the area ratio of the active regions to each other, and the identity, thickness, and / or composition (discussed in further detail below) of the mass transport limiting film overcoating the active region. Variations of these parameters can be readily carried out by those skilled in the art who have been previously granted the benefit of the disclosure herein.

[0069] Material transport limiting membrane In certain embodiments, the analyte sensor disclosed herein further includes an analyte-permeable film that overcoats at least an active region present on the working electrode of the analyte sensor. In certain embodiments, a membrane overcoating the analyte-responsive active region may function as a mass transport limiting membrane and / or to improve biocompatibility. The mass transport limiting membrane may act as a diffusion-limiting barrier that reduces the mass transport rate of the analyte. For example, but not limited to, limiting the reach of an analyte, such as potassium, to the analyte-responsive active region can help avoid sensor overload (saturation) with a mass transport limiting membrane, thereby improving detection performance and accuracy. In certain embodiments, the mass transport limiting membrane may be homogeneous and single-component (containing a single membrane polymer). Alternatively, the mass transport limiting membrane may be multi-component (containing two or more different membrane polymers).

[0070] In certain embodiments, the multicomponent film may exist as a bilayer or as a homogeneous mixture of two or more film polymers. The homogeneous mixture can be deposited by combining two or more film polymers in a solution and then depositing the solution onto the working electrode. In a particular embodiment, the composition of a mass transport restriction film placed on an analyte sensor having two active regions may be the same or different if the mass transport restriction film overcoats each active region. In certain embodiments, the substance transport restriction membrane may include chemically related materials or membranes made of polyvinylpyridine, polyvinylimidazole, a copolymer of vinylpyridine and styrene, polyurethane or polyether urethane, or silicone. In certain embodiments, the membrane, for example, a single-component membrane, may include polyvinylpyridine. In certain embodiments, the membrane, for example, a single-component membrane, may include a copolymer of vinylpyridine and styrene.

[0071] Suitable copolymers of vinylpyridine and styrene may have styrene content ranging from about 0.01 mol% to about 50 mol%, or about 0.05 mol% to about 45 mol%, or about 0.1 mol% to about 40 mol%, or about 0.5 mol% to about 35 mol%, or about 1 mol% to about 30 mol%, or about 2 mol% to about 25 mol%, or about 5 mol% to about 20 mol%. Substituted styrene may be used in a similar manner and in similar amounts. Suitable copolymers of vinylpyridine and styrene may have a weight-average molecular weight of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In non-limiting examples, suitable copolymers of vinylpyridine and styrene may have a weight-average molecular weight ranging from about 5 kDa to about 150 kDa, or about 10 kDa to about 125 kDa, or about 15 kDa to about 100 kDa, or about 20 kDa to about 80 kDa, or about 25 kDa to about 75 kDa, or about 30 kDa to about 60 kDa.

[0072] In some other embodiments, a membrane polymer overcoating one or more active regions can be crosslinked with a branched crosslinking agent containing three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether, thereby reducing the amount of extract obtainable from the substance transport restriction membrane mentioned above. In certain embodiments, the substance transport restriction membrane may comprise polyvinylpyridine or a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinking agent containing three crosslinkable groups, such as polyethylene glycol tetraglycidyl ether. In certain embodiments, the epoxide groups of polyethylene glycol tetraglycidyl ether may form covalent bonds with pyridine or imidazole via ring-opening of the epoxide ring, thereby generating a hydroxyalkyl group that crosslinks the main body of the crosslinking agent to the heterocycle of the membrane polymer. Polydimethylsiloxane (PDMS) may be incorporated into any of the material transport restriction membranes disclosed herein.

[0073] Manufacturing method This disclosure further provides methods for manufacturing the analyte sensors disclosed herein. Preferred methods for manufacturing the analyte sensors disclosed herein will be apparent to those skilled in the art, given the interests of this disclosure. In some embodiments, the manufacturing process begins by screen printing the silver / silver chloride electrode using, for example, a technique known in the art, to screen print the electrode. If desired, an electrolyte gel layer may then be formed on the exposed electrode (e.g., silver / silver chloride) area (defined by a dielectric layer) by photopolymerizing monomers and crosslinking agents with electrolyte ions in a buffer. If an electrolyte gel layer is not included, such a step is omitted. The electrode may then be immersed in a solution mixture of polymer, plasticizer, electrolyte, and ionophore to form a potassium-selective layer. Optionally, the membrane composition may then be cured on the upper surface of the sensor to form a mass transport restriction membrane. [Examples]

[0074] Examples 1-3 described below are included to demonstrate that the sensors of this disclosure, without an electrolyte gel layer, can function and detect the presence of potassium in body fluids when various electrochemical detection techniques are used. However, the methods used in these examples do not involve chronoamperometry using short potential durations, as shown in later examples. These examples are included to demonstrate that potassium can be detected by sensors without an electrolyte gel layer and without chronoamperometry using short potential durations, but the sensors exhibit a short sensor lifespan, which limits their suitability for use in in vivo potassium detection methods.

[0075] (Example 1) We manufactured a potassium sensor. This potassium sensor does not contain an electrolyte gel layer. The potassium-selective transport layer contained 3% by mass of PVC dissolved in an NPOE plasticizer solution. The PVC was dissolved in the plasticizer at a temperature of 120°C. The potassium ionophore was valinomycin, present in the layer at a concentration of 10 mM. The layer also contained tetraoctylammonium tetrakis(pentafluorophenyl) borate (TOATB) as an electrolyte, present at a concentration of 10 mM. The first working electrode contained silver and silver chloride electrode layers. The silver / silver chloride electrode was screen printed and then immersed in a PVC solution containing a potassium ionophore and other components of the potassium-selective transport layer described above. The potassium concentration was then detected using voltammetry, where a potential was applied to the working electrode. The potential was varied between 0.5V and -0.8V at a scan rate of 5mV / sec using a CHI1040C potentiostat. The electrochemical cell used had a three-electrode configuration with a screen-printed carbon counter electrode and a screen-printed silver / silver chloride reference electrode. The generated current was recorded and plotted as a voltammogram. The peak current was used as the sensor signal to determine the potassium concentration.

[0076] Figure 8 compares the sensor response in a PVC polymer layer tested in a 20 mM Tris buffer solution (pH=7.5, temperature=33°C) containing 100 mM NaCl and 10 mM KCl, with and without valinomycin. The results clearly show that the sensor current was much lower without valinomycin than in the presence of valinomycin. The experiment was repeated several times at various potassium concentrations using a sensor with valinomycin in a PVC layer. The resulting voltammogram is shown in Figure 9. This can be seen in both the cathode and anode peak currents, which increased with increasing potassium concentration. Figure 10 plots the anode peak current against potassium concentration. A linear relationship was obtained within the concentration range of 1–10 mM. The peak current therefore indicates the potassium concentration of the fluid. Another comparative experiment was conducted in which valinomycin, which is present in the potassium-selective transport layer, was absent. The results of this experiment are shown in Figure 11. Figure 11 shows that when the potassium-selective transport layer does not contain valinomycin, the obtained peak currents are very similar and remain at very low levels at various potassium or sodium ion concentrations. This indicates that the sensor does not function when the potassium ionophore is not present in the layer.

[0077] (Example 2) A group of four sensors similar to those used in Example 1 was manufactured using the same manufacturing method. The first working electrode contained silver and silver chloride electrode layers. The potassium-selective transport layer contained 20% by mass of PVC in NPOE, along with 10 mM of the same electrolyte as in Example 1 and 10 mM of valinomycin. One sensor without valinomycin in this layer was also fabricated. Conventional amperometry experiments were performed using the same electrochemical cell configuration, where a constant potential of E = -200 mV was applied to the working electrodes of these sensors. The resulting current was continuously recorded when different amounts of KCl were added to a 20 mM Tris buffer solution containing 140 mM NaCl at 33°C. The results shown in Figure 12 indicate that when valinomycin is included in the potassium-selective transport layer, the current increases as the potassium concentration rises. In the absence of valinomycin, the sensor current remained constant as the potassium concentration increased. The sensors can therefore be used to determine the potassium concentration in body fluids.

[0078] (Example 3) The chronoamperometric method was also performed for 15 minutes each, using alternating potential steps between E=200mV and E=-200mV. This method does not involve pulse amperometry and has longer duration potential steps (i.e., 15 minutes each) compared to the method of the present invention. To compare and demonstrate the advantages of this method, constant potentials of E=200mV and E=-200mV were also tested. All sensors used were manufactured using the same method as in Example 2. The first experiment, shown in Figure 13, was performed in a fluid containing 20 mM Tris buffer and 140 mM NaCl, with no potassium present. The second experiment, shown in Figure 14, was then performed in 20 mM Tris buffer, 140 mM NaCl, and 4 mM KCl. Both experiments involved three types of potential profiles applied to the sensor: alternating between +200 and -200 mV, constant at +200 mV, and constant at -200 mV. The results from these three different potential profiles were examined and analyzed as follows. At a constant potential of 200mV, the sensors in both experiments showed no response. This result was expected. At this potential, the silver metal can be oxidized, generating a positive charge on the working electrode surface. However, in order to conduct current, some cations need to be discharged from the organic PVC layer into the aqueous fluid. Since all components in this layer are hydrophobic, their migration into the aqueous phase is prohibited. Consequently, current cannot flow under such conditions.

[0079] At a constant -200mV, the results were similar to those from Example 2. Silver chloride can be reduced on the electrode surface under these conditions. The extra negative charge due to the generation of chloride anions, in the presence of valinomycin, promotes the inhalation of potassium cations from the aqueous solution into the PVC layer, resulting in a continuous current flow, as shown in the trace in Figure 14. The current is much smaller when the aqueous solution does not contain potassium ions, as shown in the case of Figure 13. It is noteworthy that this current, shown in Figure 14, continues for about 3 hours and then rapidly decreases to essentially zero. This is because all the silver chloride present on the exposed electrode surface decreases, causing the sensor to stop working. To extend the sensor's lifespan, alternating potentials between +200mV and -200mV were applied to the working electrode. At these two potentials, the same electrode reactions and ion migration described above can be applied. However, the difference here is that current can flow at +200mV because potassium ions that have migrated from the previous potential period at -200mV are present in the PVC layer. Silver chloride reduced at -200mV can therefore be recovered after the potential is switched to +200mV. The results in Figure 14 demonstrate an extension of the sensor's lifespan. The following embodiment further improves the sensor performance in this respect.

[0080] (Example 4) While the sensors used in Examples 1, 2, and 3 were capable of detecting potassium concentration, their service life was found to be relatively short. The sensor of the present invention, which includes an additional hydrogel layer, was therefore manufactured to address and overcome this problem. The first working electrode contained silver and silver chloride electrode layers. A hydrogel layer was formed on the working electrode following the procedure described below. (1) A monomer solution is prepared by mixing 97 mol% N,N'-dimethylacrylamide and 3 mol% ethylene glycol dimethacrylate (EGDMA). (2) Prepare an aqueous electrolyte solution containing 100 mM LiCl, 1 mM tetraoctylammonium chloride, and 1 mM HEPES buffer at pH=8.0. (3) UV polymerization: Mix the monomer solution and aqueous solution in a ratio of 20 / 80. Transfer approximately 0.1-0.5 μL of such mixture to the working electrode and expose the mixture to UV light at a wavelength of 254 nm for 5 minutes. A potassium-selective transport layer was then prepared using the same composition and procedure as described in Examples 2 and 3.

[0081] The sensor was subjected to chronoamperometric testing using alternating potential steps of +200mV and -200mV for 2400 seconds and 900 seconds, respectively, in a fluid containing 5mM potassium ions, 20mM Tris, and 140mM NaCl at pH 7.5 and 33°C. The results shown in Figure 15 demonstrate that chronoamperometry can be used to determine the potassium concentration in the fluid in which the sensor is placed. The sensor current remained stable for a duration of 7 days at 5mM potassium, which also indicates a significant improvement in sensor lifespan in the presence of a hydrogel layer under this potential step profile.

[0082] (Example 5) The sensors used in Example 4 (both with and without a hydrogel layer) were tested using chronoamperometry. The sensors were tested in a fluid containing 20 mM Tris buffer; 140 mM NaCl; and 6 mM KCl. Potential steps of +200 mV and -200 mV were used. Each potential step lasted only 10 seconds. Graphs showing the currents resulting from the flow of potassium ions into and out of the sensor upon application of potential pulses are depicted in Figures 17 and 18. It can be seen that the currents obtained from negative and positive potential pulses are nearly symmetric in both sensors with and without a hydrogel layer. This indicates nearly complete recovery of AgCl at the electrode and the release of all potassium ions that had previously migrated into the potassium-selective transport layer upon application of a negative potential pulse, from the potassium-selective transport layer upon application of a positive potential pulse. The observed currents are higher in the sensor with the hydrogel layer, but in both sensors, the currents generated from positive and negative potential pulses are nearly equal (symmetric). The results indicate that, when a chronoamperometric method with a shorter potential step duration is used, both sensors with and without a hydrogel layer sufficiently extend the sensor lifespan and are therefore suitable for use in in vivo methods for detecting potassium in body fluids. As shown in Figures 17 and 18, in both sensors, the currents generated from the positive and negative pulses are similar, and the graphs are symmetrical (in contrast to, for example, Figure 14, if the currents generated by the positive and negative potential steps are not equal, it indicates insufficient sensor lifespan).

[0083] In the currents shown in Figure 18, pulse amperometry was used to sample the current generated over the latter half of each potential step's duration. For example, in a potential step applied between 200 and 210 seconds, the current between 205 and 210 seconds was sampled using pulse amperometry. The average current observed over the latter 5 seconds of each 10-second potential step was sampled. The current generated during the latter half of each potential step is sampled because the current generated during the first half does not represent the current generated by potassium ions. The results of the pulse amperometry used to sample the data in Figure 18 are shown in Figure 19. The sensors were retested at different potassium concentrations using the pulsed amperometry method discussed above. The results of these experiments are shown in Figure 20. In both sensors, it can be seen that the currents generated from the positive and negative potential steps were symmetrical at different potassium concentrations of 1–6 mM, and that similar currents were generated in the flow of potassium ions into and out of the sensor. The results therefore indicate that both sensors with and without hydrogels have a long sensor life. A long sensor life is preferable for use in in vivo potassium detection methods. The results also indicate that the sensors can be used to detect a variety of potassium concentrations found in vivo.

[0084] The sensors were retested daily using pulsed amperometry. The currents generated from each applied positive and negative potential step are shown in Figure 21. These experiments used potential steps of +200mV and -200mV. Each potential step lasted only 10 seconds. In Figure 21, it can be seen that in sensors without the hydrogel layer, the current generated during the application of potential steps decreased slightly over time. This indicates a slight decrease in the sensor's stability over time. Over time, the transport of potassium ions into and out of the potassium-selective transport layer decreases, which means a decrease in the sensor's functionality (however, the sensor's lifespan is still suitable for use in in vivo potassium detection compared to when different methods, e.g., chronoamperometry with longer step durations, are used). In an attempt to increase sensor stability (and therefore sensor lifespan), pulse amperometry was performed using a 3-second positive potential step and a 1-second negative potential step. Each potential step was 200 mV. Surprisingly, this change in pulse duration was found to significantly increase sensor stability and lifespan. As shown in Figure 22, even in sensors without a hydrogel layer, no decrease in current generated from the application of positive and negative potential pulses was observed over a 10-day period.

Claims

1. An analyte sensor comprising a first working electrode and a potassium-responsive active region disposed on at least a portion of the outer surface of the first working electrode, wherein the portion of the outer surface of the first working electrode contains a transition metal ion in the form of a salt or complex, and may optionally contain a transition metal, and the potassium-responsive active region is (i) A potassium-selective transport layer that forms the outer surface of a potassium-responsive active region, comprising a polymer, a potassium ionophore, a plasticizer, and an electrolyte, and (ii) an electrolyte gel layer disposed between the potassium-selective transport layer and the portion of the outer surface of the first working electrode. Includes an analyte sensor.

2. The analyte sensor according to claim 1, wherein the polymer of the potassium-selective transport layer comprises polyvinyl chloride (PVC).

3. The potassium ionophore in the potassium-selective transport layer comprises valinomycin, gramicidin A, rhidomycin, rasaloside, maduramycin, monensin, naracin, nigericin, nonactin, nystatin, salinomycin, crown ether, cryptand, any derivative or conjugate thereof, or any combination thereof, for example, the potassium ionophore comprises valinomycin, or any derivative or conjugate thereof, according to claim 1 or 2.

4. The analyte sensor according to any one of claims 1 to 3, wherein the polymer of the potassium-selective transport layer comprises polyvinyl chloride (PVC), and the plasticizer comprises a PVC plasticizer.

5. The analyte sensor according to any one of claims 1 to 4, wherein the plasticizer comprises a phthalate compound, an adipate compound, an adipinate compound, a glutarate compound, a sebacate compound, a phosphate compound, a trimellitate compound, an epoxy compound, or a combination thereof, for example, the plasticizer comprises 2-nitrophenyloctyl ether (NPOE).

6. The analyte sensor according to any one of claims 1 to 5, wherein the electrolyte includes a hydrophobic electrolyte.

7. The electrolyte contains a salt comprising a quaternary ammonium cation having the formula R 1 R 2 R 3 R 4 N + and is the analyte sensor according to claim 6, wherein in the formula, R 1 to R 4 are each independently C 1 -C 4 alkoxy, C 2 -C 8 alkoxyalkoxy, C 3 -C 6 cycloalkyl, -OH, -NH 2 , -SH, -CO 2 (C 1 -C 6 ) alkyl and -OC(O)(C 1 -C 6 ) alkyl, and is optionally substituted by 1 to 3 groups selected therefrom, and C 1 -C 30 is selected from a straight-chain or branched alkyl or alkenyl group, for example, R 1 to R 4 are each independently selected from C 5 -C 15 alkyl groups optionally substituted as described above.

8. The analyte sensor according to claim 6 or 7, wherein the electrolyte comprises a salt containing a tetraoctylammonium cation.

9. The electrolyte is, formula BX 4 - or PX 6 - An analyte sensor according to any one of claims 6 to 8, comprising a salt containing the anion of, wherein each X independently comprises C 1 -C 4 Alkoxy, C 2 -C 8 Alkoxyalkoxy, C 3 -C 6 Cycloalkyl, -OH, -NH 2 -SH, -CO 2 (C 1 -C 6 ) Alkyl, F, Cl and -OC(O)(C 1 -C 6 ) C may be substituted with 1 to 3 groups selected from alkyl groups. 5 -C 15 An analyte sensor selected from aliphatic or aromatic hydrocarbyl groups.

10. Each X may be independently substituted as defined in claim 10, C 5 -C 15 aromatic group, C 5 -C 15 Alkyl alkyl group or C 5 -C 15 The analyte sensor according to claim 9, selected from alkenyl groups.

11. The analyte sensor according to any one of claims 6 to 10, wherein the electrolyte comprises a tetrakis(pentafluorophenyl) borate (TOATB) anion, for example, the electrolyte comprises tetraoctylammonium tetrakis(pentafluorophenyl) borate (TOATB).

12. The analyte sensor according to any one of claims 4 to 11, wherein the polymer and plasticizer in the potassium-selective transport layer are present in a weight ratio of 1:1 to 1:10, for example, 1:2 to 1:8, for example, 1:3 to 1:

5.

13. The analyte sensor according to any one of claims 1 to 12, wherein the potassium ionophore is present in the potassium-selective transport layer at a concentration of 0.1% to 30% by mass of the polymer present in the potassium-selective transport layer.

14. The analyte sensor according to any one of claims 4 to 13, wherein the electrolyte is present in the potassium-selective transport layer at a concentration of 5 mM to 50 mM, for example, 5 mM to 20 mM, or for example, 5 mM to 15 mM.

15. The analyte sensor according to any one of claims 1 to 14, wherein the electrolyte gel layer comprises an electrolyte hydrogel layer containing an aqueous electrolyte solution and a crosslinked hydrophilic polymer.

16. The analyte sensor according to claim 15, wherein the crosslinked hydrophilic polymer comprises crosslinked polyvinyl alcohol, crosslinked polyethylene glycol, crosslinked acrylate polymer, crosslinked acrylamide polymer, crosslinked hyaluronic acid polymer, crosslinked chitosan, crosslinked heparin, crosslinked alginate, or crosslinked fibrin.

17. The analyte sensor according to claim 15 or 16, wherein a crosslinked hydrophilic polymer is formed by the reaction of a hydrophilic monomer and a crosslinking agent, and optionally, the hydrophilic monomer and the crosslinking agent are reacted in amounts of 90 mol% to 99 mol% of the hydrophilic monomer and 1 mol% to 10 mol% of the crosslinking agent, for example, 95 mol% to 99 mol% of the hydrophilic monomer and 1 mol% to 5 mol% of the crosslinking agent.

18. The analyte sensor according to any one of claims 15 to 17, wherein the crosslinked polymer comprises a crosslinked N,N-dimethylacrylamide polymer.

19. The analyte sensor according to claim 18, wherein the N,N-dimethylacrylamide polymer is crosslinked with ethylene glycol dimethacrylate.

20. The analyte sensor according to any one of claims 15 to 19, wherein the aqueous electrolyte solution includes an aqueous solution of a Group 1 metal salt, for example, an aqueous solution of a Group 1 metal halide salt, for example, an aqueous solution of lithium chloride.

21. The analyte sensor according to claim 20, wherein a Group 1 metal salt is present in an aqueous electrolyte solution at a concentration of 50 mM to 150 mM, for example, 75 mM to 125 mM.

22. The analyte sensor according to any one of claims 15 to 21, wherein the aqueous electrolyte solution further comprises a quaternary ammonium halide salt and / or a buffer, for example, the quaternary ammonium halide salt is present in the aqueous electrolyte solution at a concentration of 1 mM to 5 mM if a quaternary ammonium halide salt is present, or the buffer is present if a buffer is present.

23. An analyte sensor according to any one of claims 15 to 22, wherein an aqueous electrolyte solution is present in the electrolyte hydrogel layer in an amount of 60% to 95% by mass, and a crosslinked hydrophilic polymer is present in the electrolyte hydrogel layer in an amount of 5% to 40% by mass, for example, an aqueous electrolyte solution is present in the electrolyte hydrogel layer in an amount of 70% to 90% by mass, and a crosslinked hydrophilic polymer is present in the electrolyte hydrogel layer in an amount of 10% to 30% by mass.

24. The analyte sensor according to any one of claims 1 to 23, further comprising a support layer on which a first working electrode is disposed, wherein the support layer comprises, for example, a polymer, such as PET.

25. The analyte sensor according to any one of claims 1 to 24, wherein the portion of the first working electrode comprises silver, gold, osmium, iron, cobalt, nickel, copper, ruthenium, platinum, salts or complexes thereof, or any combination thereof.

26. The analyte sensor according to any one of claims 1 to 25, wherein a portion of the first working electrode contains a transition metal ion in the form of a salt or complex, and for example, the transition metal ion contains silver or osmium ions.

27. The analyte sensor according to any one of claims 1 to 26, wherein a portion of the first working electrode comprises silver and silver chloride (AgCl), and for example, the first working electrode comprises a silver / silver chloride electrode layer.

28. The analyte sensor according to claim 27, wherein the sensor includes a support layer on which a silver / silver chloride electrode layer is disposed.

29. The analyte sensor according to any one of claims 1 to 28, wherein the transition metal ion is in the form of a transition metal complex, and optionally the transition metal complex is grafted onto a polymer backbone in the form of a redox polymer.

30. The analyte sensor according to any one of claims 1 to 29, wherein the portion of the first working electrode includes an osmium(III) ion, an osmium(II) ion, or a combination thereof, for example, the osmium(II) ion, the osmium(III) ion, or both, in the form of an osmium ion complex, for example, the osmium ion complex is grafted onto a polymer backbone in the form of a redox polymer.

31. The analyte sensor according to claim 30, wherein the first working electrode includes a carbon electrode layer, and a portion of the outer surface of the first working electrode includes a layer of osmium ion complex or redox polymer disposed on a portion of the outer surface of the carbon electrode layer.

32. The analyte sensor according to claim 31, wherein the sensor includes a support layer on which a carbon electrode layer is disposed.

33. (i) The potassium-selective transport layer comprises PVC, valinomycin, tetraoctylammonium tetrakis(pentafluorophenyl) borate (TOATB), and 2-nitrophenyl octyl ether (NPOE), and (ii) the electrolyte gel layer comprises an electrolyte hydrogel layer comprising an aqueous electrolyte solution of a group 1 metal halide salt and a crosslinked N,N-dimethylacrylamide, according to any one of claims 1 to 32.

34. The analyte sensor according to any one of claims 1 to 33, wherein the potassium-selective transport layer has a thickness of 1 to 200 μm, the electrolyte gel layer has a thickness of 1 to 50 μm, and / or the first working electrode layer has a thickness of 1 nm to 20 μm.

35. The analyte sensor according to any one of claims 1 to 34, further comprising a layer of dielectric material disposed on a second portion of the outer surface of a first working electrode in which no potassium-responsive active region is located.

36. The analyte sensor according to any one of claims 1 to 35, wherein the first working electrode and the potassium-responsive active region are completely or partially encapsulated by a mass transport restriction membrane, for example, the first working electrode and the potassium-responsive active region are completely encapsulated by a mass transport restriction membrane.

37. The analyte sensor according to claim 36, wherein the substance transport restriction membrane comprises a polymer membrane, for example, the polymer membrane comprises a copolymer of polyvinylpyridine, polyvinylimidazole, vinylpyridine, and styrene, or a combination thereof.

38. The analyte sensor according to claim 37, wherein the polymer of the polymer film is crosslinked, and optionally, the polymer of the polymer film is crosslinked by reaction with polyethylene glycol tetraglycidyl ether.

39. The analyte sensor according to any one of claims 1 to 38, further comprising one or more additional working electrodes and / or one or more additional active regions, wherein optionally one or more additional working electrodes or one or more additional active regions are as defined in any one of claims 1 to 38.

40. The analyte sensor according to any one of claims 1 to 39, wherein the first working electrode further comprises one or more additional active regions, optionally, one or more additional active regions as defined in any one of claims 1 to 39.

41. The analyte sensor according to claim 39 or 40, wherein one or more additional active regions are configured to respond to potassium, or to one or more additional analytes.

42. The analyte sensor according to any one of claims 1 to 41, wherein the sensor further comprises a counter electrode, a reference electrode, or both.

43. The analyte sensor according to any one of claims 1 to 42, wherein the sensor is an in vivo analyte sensor, and for example, the sensor includes a sensor tail having a first working electrode and, if present, any other arbitrary working electrodes.

44. The analyte sensor according to claim 43, wherein the sensor has a length of 5 mm to 10 mm, a width of 0.3 mm to 0.5 mm, and a thickness of 0.2 mm to 0.4 mm.

45. The analyte sensor according to any one of claims 1 to 44, wherein the sensor is suitable for detecting the concentration of potassium in a body fluid, and the body fluid includes, for example, skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid.

46. The analyte sensor according to any one of claims 43 to 45, further comprising a sensor housing, wherein an adhesive layer in contact with the sensor housing is adapted to adhere to the tissue surface of the sensor housing, and a sensor tail protrudes from the sensor housing through the adhesive layer.

47. A method for detecting potassium ions in a fluid, (a) A step of preparing an analyte sensor including a first working electrode and a potassium-responsive active region disposed on at least a portion of the outer surface of the first working electrode, wherein the portion of the outer surface of the first working electrode contains a transition metal ion in the form of a salt or complex, and may optionally contain a transition metal, and the potassium-responsive active region is (i) A potassium-selective transport layer that forms the outer surface of a potassium-responsive active region, comprising a polymer, a potassium ionophore, a plasticizer, and an electrolyte, and (ii) an electrolyte gel layer disposed between the potassium-selective transport layer and the portion of the outer surface of the first working electrode. Steps including, (b) The step of applying a potential to the first working electrode, (c) A step of obtaining a signal indicating oxidation and / or reduction of a transition metal ion in salt or complex form, and optionally the transition metal of the first working electrode, wherein the signal indicates the concentration of potassium ions in the fluid. (d) A step of determining the concentration of potassium ions in the fluid from the signal obtained in step (c) and Methods that include...

48. The method according to claim 47, wherein the sensor is as defined in any one of claims 1 to 47.

49. The method according to claim 47 or 48, wherein the fluid includes bodily fluids.

50. The method according to claim 49, wherein the body fluid includes skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid.

51. The method according to any one of claims 47 to 50, wherein the sensor is located in vivo, and the method includes the step of determining the concentration of potassium ions in a body fluid in vivo.

52. The method according to any one of claims 47 to 51, wherein the first working electrode comprises silver and silver chloride (AgCl), and the signal in step (c) indicates the reduction of silver chloride (AgCl) to silver and / or the oxidation of silver to silver chloride (AgCl).

53. The method according to any one of claims 47 to 51, wherein the first working electrode comprises an osmium(II) complex, an osmium(III) complex, or both, and the signal of step (c) indicates the reduction of osmium(III) to osmium(II) and / or the oxidation of osmium(II) to osmium(III).

54. The method according to any one of claims 47 to 53, wherein the detection method comprises a voltammetry method, step (b) comprising applying a fluctuating potential to a first working electrode, and the signal of step (c) comprising measuring a peak current.

55. The method according to any one of claims 47 to 53, wherein the detection method comprises an amperometric method, step (b) comprising applying a constant potential to a first working electrode, and step (c) measuring the obtained current.

56. The method according to any one of claims 47 to 53, wherein the detection method comprises a chronoamperometric method, the step of applying a potential that alternately repeats two different values, and the step of measuring the resulting current.

57. Use of an analyte sensor according to any one of claims 1 to 46 for detecting the concentration of potassium ions in a fluid.

58. The use according to claim 57, wherein the fluid is a bodily fluid.

59. The use according to claim 57 or 58, wherein the bodily fluid and analyte sensors are in vivo.

60. The use according to any one of claims 57 to 59, wherein the body fluid includes skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid.

61. A method for detecting potassium ions in a fluid, (a) A step of preparing an analyte sensor comprising a first working electrode and a potassium-responsive active region disposed on at least a portion of the outer surface of the first working electrode, wherein the portion of the outer surface of the first working electrode comprises a transition metal ion in the form of a salt or complex, and may also comprise a transition metal, and the potassium-responsive active region comprises a potassium-selective transport layer forming the outer surface of the potassium-responsive active region, the potassium-selective transport layer comprising a polymer, a potassium ionophore, a plasticizer, and an electrolyte. (b) The step of applying a potential to the first working electrode, (c) A step of obtaining a signal indicating the oxidation and / or reduction of a transition metal ion in the form of a salt or complex, and optionally, the transition metal of the first working electrode, wherein the signal indicates the concentration of potassium ions in the fluid. (d) A step of determining the concentration of potassium ions in the fluid from the signal obtained in step (c) and Includes, Steps (b) and (c) include obtaining a signal indicating the concentration of potassium ions in a fluid using chronoamperometry, wherein the chronoamperometry includes the sequential application of first and second potentials, the first potential being applied for a duration of 10 milliseconds to 60 seconds, the second potential being applied for a duration of 10 milliseconds to 60 seconds, and the first potential being different from the second potential. method.

62. The method according to claim 61, wherein the chronoamperometry includes the continuous application of alternating positive and negative potentials, and the potentials are applied for a duration of 10 milliseconds to 60 seconds.

63. The method according to claim 61 or 62, wherein the chronoamperometry includes the continuous application of alternating positive and negative potentials, the potentials being applied for a duration of 500 milliseconds to 60 seconds.

64. The method according to any one of claims 61 to 63, wherein the chronoamperometry includes the continuous application of alternating positive and negative potentials, the potentials being applied for a duration of 500 milliseconds to 20 seconds.

65. The method according to claim 63 or 64, wherein the duration of the positive potential is different from the duration of the negative potential.

66. The method according to claim 65, wherein a positive potential is applied for a longer duration than a negative potential.

67. The method according to claim 65 or 66, wherein the duration of the positive potential is 2 to 5 seconds, and the duration of the negative potential is 1 to 2 seconds.

68. The method according to any one of claims 61 to 67, wherein the chronoamperometry includes pulse amperometry.

69. The method according to any one of claims 61 to 68, wherein the chronoamperometry includes the continuous application of alternating positive and negative potentials, and the negative or positive potential includes potentials of 50 mV to 500 mV, for example 100 mV to 300 mV, for example 150 mV to 250 mV.

70. The method according to any one of claims 61 to 69, wherein the fluid includes bodily fluids.

71. The method according to claim 70, wherein the body fluid includes skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid.

72. The method according to any one of claims 61 to 71, comprising the step of determining the concentration of potassium ions in an in vivo body fluid, wherein the sensor is located in vivo.

73. The method according to any one of claims 61 to 72, wherein the polymer of the potassium-selective transport layer comprises polyvinyl chloride (PVC).

74. The method according to any one of claims 61 to 73, wherein the potassium ionophore in the potassium-selective transport layer comprises valinomycin, gramicidin A, rhidomycin, rasaloside, maduramycin, monensin, naracin, nigericin, nonactin, nystatin, salinomycin, crown ether, cryptand, any derivative or conjugate thereof, or any combination thereof, for example, the potassium ionophore comprises valinomycin or any derivative or conjugate thereof.

75. The method according to any one of claims 61 to 74, wherein the polymer of the potassium-selective transport layer comprises polyvinyl chloride (PVC) and the plasticizer comprises a PVC plasticizer.

76. The method according to any one of claims 61 to 75, wherein the plasticizer comprises a phthalate compound, an adipate compound, an adipinate compound, a glutarate compound, a sebacate compound, a phosphate compound, a trimellitate compound, an epoxy compound, or a combination thereof, for example, the plasticizer comprises 2-nitrophenyloctyl ether (NPOE).

77. The method according to any one of claims 61 to 76, wherein the electrolyte includes a hydrophobic electrolyte.

78. The electrolyte is, formula R 1 R 2 R 3 R 4 N + The method according to claim 77, comprising a salt containing a quaternary ammonium cation having R, wherein R 1 From R 4 These are each independently C1-C4 alkoxy, C2-C8 alkoxyalkoxy, C3-C6 cycloalkyl, -OH, and -NH. 2 -SH, -CO 2 Selected from C1-C30 linear or branched alkyl or alkenyl groups, which may be substituted with 1 to 3 groups selected from (C1-C6) alkyl and -OC(O)(C1-C6) alkyl, for example, R 1 From R 4 The method wherein each of the C5-C15 alkyl groups is independently selected from those which may be substituted as described above.

79. The method according to claim 77 or 78, wherein the electrolyte comprises a salt containing a tetraoctylammonium cation.

80. The electrolyte is, formula BX 4 - or PX 6 - A method according to any one of claims 77 to 79, comprising a salt containing the anion of, where each X independently is C1-C4 alkoxy, C2-C8 alkoxyalkoxy, C3-C6 cycloalkyl, -OH, -NH2, -SH, -CO 2 The method is selected from a C5-C15 aliphatic or aromatic hydrocarbyl group which may be substituted with one to three groups selected from (C1-C6)alkyl, F, Cl, and -OC(O)(C1-C6)alkyl.

81. The method according to claim 80, wherein each X is independently selected from a C5-C15 aromatic group, a C5-C15 alkyl group, or a C5-C15 alkenyl group, which may be substituted as defined in claim 77.

82. The method according to any one of claims 77 to 81, wherein the electrolyte comprises a tetrakis(pentafluorophenyl) borate (TOATB) anion, for example, the electrolyte comprises tetraoctylammonium tetrakis(pentafluorophenyl) borate (TOATB).

83. The method according to any one of claims 61 to 82, wherein the polymer and plasticizer in the potassium-selective transport layer are present in a weight ratio of 1:1 to 1:10, for example 1:2 to 1:8, for example 1:3 to 1:

5.

84. The method according to any one of claims 61 to 83, wherein the potassium ionophore is present in the potassium-selective transport layer at a concentration of 0.1% to 30% by mass of the polymer present in the potassium-selective transport layer.

85. The method according to any one of claims 61 to 84, wherein the electrolyte is present in the potassium-selective transport layer at a concentration of 5 mM to 50 mM, for example, 5 mM to 20 mM, or for example, 5 mM to 15 mM.

86. The method according to any one of claims 61 to 85, wherein the analyte sensor further comprises an electrolyte gel layer disposed between a potassium-selective transport layer and the portion of the outer surface of the first working electrode.

87. The method according to claim 86, wherein the electrolyte gel layer comprises an electrolyte hydrogel layer containing an aqueous electrolyte solution and a crosslinked hydrophilic polymer.

88. The method according to claim 87, wherein the crosslinked hydrophilic polymer comprises crosslinked polyvinyl alcohol, crosslinked polyethylene glycol, crosslinked acrylate polymer, crosslinked acrylamide polymer, crosslinked hyaluronic acid polymer, crosslinked chitosan, crosslinked heparin, crosslinked alginate, or crosslinked fibrin.

89. The method according to claim 87 or 88, wherein a crosslinked hydrophilic polymer is formed by the reaction of a hydrophilic monomer and a crosslinking agent, and optionally, the hydrophilic monomer and the crosslinking agent are reacted in amounts of 90 mol% to 99 mol% of the hydrophilic monomer and 1 mol% to 10 mol% of the crosslinking agent, for example, 95 mol% to 99 mol% of the hydrophilic monomer and 1 mol% to 5 mol% of the crosslinking agent.

90. The method according to any one of claims 87 to 89, wherein the crosslinked polymer comprises a crosslinked N,N-dimethylacrylamide polymer.

91. The method according to claim 90, wherein the N,N-dimethylacrylamide polymer is crosslinked with ethylene glycol dimethacrylate.

92. The method according to any one of claims 87 to 91, wherein the aqueous electrolyte solution includes an aqueous solution of a Group 1 metal salt, for example, an aqueous solution of a Group 1 metal halide salt, for example, an aqueous solution of lithium chloride.

93. The method according to claim 92, wherein the Group 1 metal salt is present in the aqueous electrolyte solution at a concentration of 50 mM to 150 mM, for example, 75 mM to 125 mM.

94. The method according to any one of claims 87 to 93, wherein the aqueous electrolyte solution further comprises a quaternary ammonium halide salt and / or a buffer, for example, the quaternary ammonium halide salt is present in the aqueous electrolyte solution at a concentration of 1 mM to 5 mM if a quaternary ammonium halide salt is present, or the buffer is present if a buffer is present.

95. The method according to any one of claims 87 to 94, wherein an aqueous electrolyte solution is present in the electrolyte hydrogel layer in an amount of 60% to 95% by mass, and a crosslinked hydrophilic polymer is present in the electrolyte hydrogel layer in an amount of 5% to 40% by mass, for example, the aqueous electrolyte solution is present in the electrolyte hydrogel layer in an amount of 70% to 90% by mass, and the crosslinked hydrophilic polymer is present in the electrolyte hydrogel layer in an amount of 10% to 30% by mass.

96. The method according to any one of claims 61 to 95, wherein the sensor further comprises a support layer on which a first working electrode is disposed, and the support layer comprises, for example, a polymer, such as PET.

97. The method according to any one of claims 61 to 96, wherein the portion of the first working electrode comprises silver, gold, osmium, iron, cobalt, nickel, copper, ruthenium, platinum, salts or complexes thereof, or any combination thereof.

98. The method according to any one of claims 61 to 97, wherein the portion of the first working electrode contains a transition metal ion in the form of a salt or complex, and for example, the transition metal ion contains silver or osmium ions.

99. The method according to any one of claims 61 to 98, wherein the portion of the first working electrode comprises silver and silver chloride (AgCl), and for example, the first working electrode comprises a silver / silver chloride electrode layer.

100. The method according to claim 99, wherein the sensor includes a support layer on which a silver / silver chloride electrode layer is disposed.

101. The method according to any one of claims 61 to 100, wherein the transition metal ion is in the form of a transition metal complex, and optionally the transition metal complex is grafted onto a polymer backbone in the form of a redox polymer.

102. The method according to any one of claims 61 to 101, wherein the portion of the first working electrode comprises an osmium(III) ion, an osmium(II) ion, or a combination thereof, for example, the osmium(II) ion, the osmium(III) ion, or both thereof, in the form of an osmium ion complex, for example, the osmium ion complex is grafted onto a polymer backbone in the form of a redox polymer.

103. The method according to claim 102, wherein the first working electrode includes a carbon electrode layer, and a portion of the outer surface of the first working electrode includes a layer of osmium ion complex or redox polymer disposed on the portion of the outer surface of the carbon electrode layer.

104. The method according to claim 103, wherein the sensor includes a support layer on which a carbon electrode layer is disposed.

105. The method according to any one of claims 61 to 104, wherein the potassium-selective transport layer comprises PVC, valinomycin, tetraoctylammonium tetrakis(pentafluorophenyl) borate (TOATB), and 2-nitrophenyloctyl ether (NPOE).

106. The method according to any one of claims 61 to 105, wherein the potassium-selective transport layer has a thickness of 1 to 200 μm, the electrolyte gel layer, if present, has a thickness of 1 to 50 μm, and / or the first working electrode layer has a thickness of 1 nm to 20 μm.

107. The method according to any one of claims 61 to 106, wherein the sensor further comprises a layer of dielectric material disposed on a second portion of the outer surface of a first working electrode in which no potassium-responsive active region is located.

108. The method according to any one of claims 61 to 107, wherein the first working electrode and the potassium-responsive active region are completely or partially encapsulated by a mass transport restriction membrane, for example, the first working electrode and the potassium-responsive active region are completely encapsulated by a mass transport restriction membrane.

109. The method according to claim 108, wherein the substance transport restriction membrane comprises a polymer membrane, for example, the polymer membrane comprises a copolymer of polyvinylpyridine, polyvinylimidazole, vinylpyridine and styrene, or a combination thereof.

110. The method according to claim 109, wherein the polymer of the polymer film is crosslinked, and optionally the polymer of the polymer film is crosslinked by reaction with polyethylene glycol tetraglycidyl ether.

111. The method according to any one of claims 61 to 110, wherein the sensor further comprises one or more additional working electrodes and / or one or more additional active regions, wherein optionally one or more additional working electrodes or one or more additional active regions are as defined in any one of claims 1 to 110.

112. The method according to any one of claims 61 to 111, wherein the first working electrode further comprises one or more additional active regions, optionally, one or more additional active regions as defined in any one of claims 1 to 111.

113. The method according to claim 111 or 112, wherein one or more additional active regions are configured to respond to potassium, or to one or more additional analytes.

114. The method according to any one of claims 61 to 113, wherein the sensor further includes a counter electrode, a reference electrode, or both.

115. The method according to any one of claims 61 to 114, wherein the sensor is an in vivo analyte sensor, and for example, the sensor includes a sensor tail comprising a first working electrode and any other working electrodes, if present.

116. The method according to claim 115, wherein the sensor has a length of 5 mm to 10 mm, a width of 0.3 mm to 0.5 mm, and a thickness of 0.2 mm to 0.4 mm.

117. The method according to claim 115 or 116, wherein the sensor further includes a sensor housing, and an adhesive layer in contact with the sensor housing is adapted to adhere to the tissue surface of the sensor housing, and a sensor tail protrudes from the sensor housing through the adhesive layer.

118. The method according to any one of claims 61 to 117, wherein the first working electrode comprises silver and silver chloride (AgCl), and the signal in step (c) indicates the reduction of silver chloride (AgCl) to silver and / or the oxidation of silver to silver chloride (AgCl).

119. The method according to any one of claims 61 to 118, wherein the first working electrode comprises an osmium(II) complex, an osmium(III) complex, or both, and the signal of step (c) indicates the reduction of osmium(III) to osmium(II) and / or the oxidation of osmium(II) to osmium(III).

120. An analyte sensor configured to perform the method described in any one of claims 61 to 119.

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