Improved solid-state magnesium ion selective microelectrode and method of production and use thereof

Abstract

To provide a magnesium sensing membrane for use in a potentiometric ion selective microelectrode indicating an increased detection lower limit.SOLUTION: The magnesium sensing membrane includes: an ionophore having a tripodal stereochemical structure; a lipophilic borate salt present in an amount that provides a mol ratio of lipophilic borate salt to ionophore in a range from 50 mol% to 100 mol%, which is selected from potassium tetrakis (4-chlorophenyl) borate (KTpCIPB) and sodium tetrakis [3,5-bis(trifluoromethyl)phenyl] borate (NaTFPB); a specific lipophilic electrolyte that provides a mol ratio of lipophilic electrolyte to ionophore in a range from 0.5 mol% to 50 mol%, wherein the lipophilic electrolyte is present in an amount from 0.5 mass% to 1.5 mass%; and a polymer matrix in which the ionophore, lipophilic borate salt, and lipophilic electrolyte are disposed, wherein the polymer matrix includes a polymer and a plasticizer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference to related applications / Incorporation by Reference This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 888,643, filed August 19, 2019. The entire contents of the above-referenced patents / patent applications are expressly incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable. [Background technology]

[0003] background The use of ion-selective electrodes (ISEs) to determine the presence and amount of various analytes in biological samples has become a useful diagnostic technique. In fact, ISEs have been used to detect analytes such as magnesium, sodium, potassium, calcium, and chloride, among others. Some of these ISEs are often housed within clinical diagnostic instruments for the simultaneous analysis of multiple analytes.

[0004] The concentration of lipophilic borate present in the sensing membrane is important for the selective detection of magnesium ions (Mg 2+ The level of borate present in the sensing membrane is known to play an important role in Ca selective electrodes, based on the number of cationic charges, the complex stoichiometry with the neutral ionophore, and the response kinetics. 2+ , Na + , and K + against interfering cations such as Mg 2+ The selectivity coefficient of Mg is changed. 2+For ISEs, a borate-to-ionophore molar ratio of 155 mol% has been considered the optimized formulation yielding the best selectivity pattern. Commonly used lipophilic borates are potassium tetrakis(4-chlorophenyl)borate (KTpClPB) or sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB).

[0005] Membrane impedance is a key factor in determining low detection limits and solid-state Mg for a variety of reasons. 2+ It is very important to obtain a stable response of the microsensor. First, the solid-state iMg microsensor can measure blood Mg. 2+ Used to test low Mg 2+ The sensor detects various Mg concentrations in blood samples (<0.2 mM). 2+ It is difficult to differentiate the response between blood samples of different concentrations. In other words, the Mg microsensor is particularly useful when testing hypomagnesemic samples. 2+ It can be difficult when the concentration is below 0.2 mM. For example, the iMg analyzer (Nova Stat Profile Critical Care Xpress (CCX), Nova Biomedical Corporation, Waltham, MA) based on Mg 2+ Low Mg <0.2mM 2+ They are reluctant to test blood samples.

[0006] Second, Mg ionophores (such as ETH5506, ETH3832, and ETH7025) are "strong" ionophores for other ions (e.g., Ca). 2+ ETH1001, valinomycin-K + , sodium ionophore X-Na + It has a relatively "weak" binding capacity for target ions compared to other membranes. The high membrane impedance is due to the low Mg 2+ This reduces the sensitivity of the Mg sensor in samples with high Mg concentrations. 2 Under detection of The limit increases.

[0007] Third, low Mg 2+ The response of the microsensor in the sample is unstable in terms of "within run precision" and "total precision." This is caused by the high impedance of the microsensor, which has a much smaller size than conventional solid-state iMg sensors.

[0008] Fourth, for the iMg sensor using the Cal reagent containing surfactant (Brij700), the coating membrane formulation (ionophore ETH5506) is optimized with half the amount of lipophilic borate (KTpCIPB) compared to the ideal iMg formulation in the surfactant-free Cal reagent. This allows the iMg sensor to operate accurately against the effects of surfactants on the iMg response. However, the reduction in the amount of borate present in the membrane results in very high impedance (>gigaohms), especially for the microsensor, and this makes the iMg microsensor less sensitive to the Mg present in the blood sample. 2+ It becomes unresponsive to the analyte.

[0009] Previously, cation ISE membrane impedance was effectively reduced by adding anionic lipophilic borates such as KTpCIPB. The current ISE membrane formulation (Na, K, Ca, and pH) optimizes the lipophilic anion salt content, which ensures fast response kinetics and a rapid wet-up procedure. For iMg ISEs, the lipophilic borate content significantly influences the selectivity pattern for interfering cations (Ca). 2+ , K. + , and BiNa + Mg 2+ ) used in blood testing. As for the g sensor, problems are encountered when using only one lipophilic borate in the iMg sensor: (a) too high a content of borate results in surfactant interference on the signal response; and (b) too low a content of iMg sensor results in the main interfering cation, Ca. 2+The optimal borate-to-ionophore ratio must be maintained for the iMg sensor present in the hematology analyzer. However, even with such an optimal ratio, the solid-state iMg microsensor loses selectivity for low Mg. 2+ Difficulties are still encountered in blood samples (<0.2 mM).

[0010] A lipophilic electrolyte known as ETH500 (tetrakis(4-chlorophenyl)borate tetradodecylammonium salt, Mw=1148) has previously been used in conventional ISE macrosensors (i.e., Na, K, Ca, pH, and Mg) to improve response kinetics (see, e.g., Legin et al. (2002); Spichiger et al. (2002); Spichiger et al. (2003); Eugster et al. (2004); and Eugster et al. (2005)). However, no studies have been reported on improving the detection limit of iMg solid-state sensors or on the use of ETH500 in microsensors. Furthermore, the amount of ETH500 utilized in conventional ISEs is substantially high (i.e., greater than 50 mol%), which alters the dielectric permittivity of the film. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Legin et al. Electrochimica Acta (2004) 49:5203-5207 [Non-patent document 2] Spichiger et al. J Anal Chem (1991) 341:727-731 [Non-patent document 3] Spichiger, Electroanalysis (1993) 5:739-745 [Non-patent document 4] Eugster et al. Clin Chem (1993) 39:855-859 [Non-Patent Document 5] Eugster et al. Anal Chem (1993) 65:689-695 Summary of the Invention [Problem to be solved by the invention]

[0012] Therefore, new and improved magnesium-sensing membrane compositions for potentiometric ion-selective microelectrodes that overcome the disadvantages of the prior art are desirable. The present disclosure relates to such membranes and microelectrodes containing them, as well as compositions, kits, devices, and methods related thereto. [Brief explanation of the drawings]

[0013] A brief description of some of the drawings [Figure 1] FIG. 1 graphically illustrates the effect of adding a lipophilic electrolyte (ETH500) on membrane impedance in an iMg microsensor membrane. [Figure 2] Figure 2 graphically illustrates the response recovery of an iMg microsensor containing 0.0 wt% ETH500 in a solution system of 0.5 mM Mg2+ to 0.1 mM Mg2+. [Figure 3] Figure 3 graphically illustrates the response recovery of an iMg microsensor containing 0.5 wt% ETH500 in a solution system of 0.5 mM Mg2+ to 0.1 mM Mg2+. [Figure 4] Figure 4 graphically illustrates the response recovery of an iMg microsensor containing 1.5 wt% ETH500 in a solution system of 0.5 mM Mg2+ to 0.1 mM Mg2+. [Figure 5] FIG. 5 graphically illustrates the AQC stability of iMg microsensors containing 0.0 wt % ETH500. [Figure 6] FIG. 6 graphically illustrates the AQC stability of iMg microsensors containing 0.5 wt % ETH500. [Figure 7] FIG. 7 graphically illustrates the AQC stability of iMg microsensors containing 1.5 wt % ETH500. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description Before describing in detail at least one embodiment of the inventive concept using illustrative figures, experiments, results, and testing methods, it is to be understood that the inventive concept is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the figures, experiments, and / or results. The inventive concept is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be accorded the broadest possible scope and meaning; and the embodiments are intended to be illustrative, not all-inclusive. It is also to be understood that the phraseology and terminology used herein is for the purpose of description and should not be construed as limiting.

[0015] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more detailed references cited and discussed throughout the specification. Nomenclature utilized in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry laboratory methods and techniques described herein is that well known and commonly used in the art.

[0016] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the levels of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any part of this application are to be construed as though each individual patent or publication were specifically and individually indicated by reference. To the same extent as if expressly indicated as incorporated herein by reference in their entireties.

[0017] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied in the compositions and / or methods and in the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept as defined by the appended claims.

[0018] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The use of the words "a" or "an" when used in the claims and / or specification in connection with the term "comprising" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" can refer to one or more, two or more, three or more, four or more, or a greater number of compounds. The term "plurality" refers to "two or more." The use of the term "or" in the claims is used to mean "and / or" unless clearly indicated to refer to alternatives only or the alternatives are mutually exclusive; however, the disclosure supports a definition that refers to alternatives only and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects. For example, but not as a limitation, when the term "about" is used, the specified value may vary by ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, with such variations being appropriate for practicing the disclosed methods and understood by those of ordinary skill in the art. Use of the term "at least one" will be understood to include not only 1 but any amount greater than 1, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may be expanded up to 100 or even 100 or more, depending on the term to which it is attached; further, an amount of 100 / 1000 should not be considered limiting, as larger limits may also produce satisfactory results. Furthermore, the term "at least one of X, Y, and Z" will be understood to include X only, Y only, and Z only, as well as any combination of X, Y, and Z. The use of ordinal terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for purposes of differentiating between two or more items and is not intended to imply, for example, an order or sequence or importance or ranking of one item relative to another item.

[0019] As used in this specification and the claims, the terms "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include") or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or any combination thereof" refers to any combination of A, B, C, or any combination thereof. A "combination of" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular situation, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repeats of one or more items or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will appreciate that typically there is no limit to the number of items or terms in any combination unless otherwise apparent from the context.

[0021] As used herein, the term "substantially" means that substantially the described events or circumstances occur entirely, or that substantially the described events or circumstances occur to a significant extent. For example, the term "substantially" means that substantially the described events or circumstances occur at least 90% of the time, or at least 95% of the time, or at least 98% of the time.

[0022] As used herein, the phrase "conjugated to" includes both direct binding of two moieties to each other as well as indirect binding of two moieties to each other. Non-limiting examples of binding include covalent binding of one moiety to another moiety by direct bonding or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by a specific binding pair member bound to the moiety, incorporation of one moiety into another moiety, for example, by dissolving one moiety in another moiety or by synthesis, and coating one moiety to another moiety.

[0023] As used herein, the term "purified" means that at least one order of magnitude of purification is achieved as compared to the starting material or naturally occurring material, for example, but not limited to, two, three, four, or five orders of magnitude of purification of the starting material or naturally occurring material. Thus, as used herein, the term "purified" does not mean that a material is necessarily 100% purified, and thus the term does not exclude the presence of other materials that are present in the purified composition.

[0024] The terms "analog" and "derivative" are used interchangeably herein and refer to a substance that contains in its structure the same basic carbon skeleton and carbon functional groups as a given compound, but may also contain one or more substitutions thereof. As used herein, the term "substituted" is understood to refer to the replacement of at least one substituent on the compound with a residue R. In certain non-limiting embodiments, R can include a halide selected from H, hydroxy, thiol, fluoride, chloride, bromide, or iodide, a C1-C4 compound selected from the following: optionally substituted straight-chain, branched, or cyclic alkyl, and straight-chain, branched, or cyclic alkenyl, where the optional substituents are one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocyclo. and alkyl, each of which is optionally substituted, wherein the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxy, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), carboxy, and -C(O)-alkyl.

[0025] As used herein, the term "sample" refers to any sample that may be utilized in accordance with the present disclosure. It is understood that the term "biological sample" encompasses any type of biological sample. In certain embodiments, the sample may be any fluid sample and / or a sample capable of being fluid (e.g., a biological sample mixed with a fluid substance). Examples of biological samples that may be utilized include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), saliva, sputum, cerebrospinal fluid (CSF), surgical drainage fluid, skin, interstitial fluid, tears, mucus, urine, swabs, combinations, etc. While the present disclosure is directed to biological samples, it will be understood by those skilled in the art that the concepts disclosed herein may be applied to any sample in which the concentration of magnesium can be determined, and as such, it should be noted that the scope of the present disclosure is not limited to biological samples.

[0026] As used herein, the term "wet-up" will be understood to refer to the hydration process from the placement of the sensor in the fluid analyzer to the point where a stable signal is obtained from the calibration reagent.

[0027] The term "recovery," as used herein, alone or in combination with other terms (e.g., without limitation, "quality control recovery," "recovery phase," and "recovery uptake"), is understood to mean the yield of an analytical process compared to an assigned or reference value.

[0028] As used herein, a circuit may be analog and / or digital components, or one or more appropriately programmed processors (e.g., microprocessors) and associated hardware and software, or logic circuitry embedded in hardware. Also, a "component" may perform one or more functions. The term "component" may include hardware, such as a processor (e.g., a microprocessor), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a combination of hardware and software, and / or the like.

[0029] Software may include one or more computer-readable instructions that, when executed by one or more components, cause the components to perform a particular function. It should be understood that the algorithms described herein may be stored on one or more non-transitory memories. Exemplary non-transitory memories may include random access memory, read-only memory, flash memory, and / or the like. Such non-transitory memories may be electrical-based, optical-based, and / or the like.

[0030] It should further be understood that the term "user" as used herein is not limited to humans and can include, for example, computers, servers, websites, processors, network interfaces, people, user terminals, virtual computers, combinations thereof, and the like.

[0031] As used herein, the term "calibration information" may refer to one or more of the slope, offset, and / or selectivity coefficient of the Nernstian or Nicolsky-Eisenman equation determined in an initial three-point calibration (i.e., a "full calibration").

[0032] As used herein, the term "calibration logic" refers to program logic used by a processor in a control system to interpret data measured by one or more ion-selective electrodes. In particular, the term "calibration logic" relates to program logic in the control system used by the processor to interpret data from a magnesium ion-selective electrode for an initial three-point calibration (i.e., a "full calibration").

[0033] As used herein, the term "recalibration information" may refer to one or more of the slope, offset, and / or selectivity coefficient determined using the Nernstian or Nicolsky-Eisenman equations using information derived from any subsequent three-point, two-point, or one-point calibrations after the initial three-point calibration.

[0034] As used herein, the term "recalibration logic" also refers to program logic used by a processor within a control system to interpret data measured by one or more ion-selective electrodes. In particular, the term "recalibration logic" relates to program logic of a control system used by a processor to interpret data from a magnesium ion-selective electrode for additional three-point, one-point, and two-point calibrations (as further defined herein) after an initial three-point calibration.

[0035] In light of the presently disclosed and / or claimed inventive concepts, a new and improved magnesium-sensing membrane for microelectrodes is provided, which exhibits improved stability over existing magnesium-sensing membranes. This new magnesium-sensing membrane can be used in the development of new potentiometric ion-selective microelectrodes suitable for central laboratory and / or POC applications.

[0036] Certain embodiments of the present disclosure relate to a magnesium-sensing membrane for a potentiometric ion-selective microelectrode for detecting ionized magnesium in a biological sample. The magnesium-sensing membrane may be a conventional membrane, i.e., a solid-state planar membrane. The magnesium-sensing membrane includes an ionophore having a tripodal stereochemical structure, a lipophilic borate, a lipophilic electrolyte, and a polymer matrix in which the ionophore, lipophilic borate, and lipophilic electrolyte are disposed. The polymer matrix includes a polymer and a plasticizer.

[0037] The lipophilic electrolyte is present in an amount that results in a molar ratio of lipophilic electrolyte to ionophore that is less than or equal to about 50 mol%. Non-limiting examples of lipophilic electrolyte:ionophore ratios that can be utilized include about 49 mol%, about 48 mol%, about 47 mol%, about 46 mol%, about 45 mol%, about 44 mol%, about 43 mol%, about 42 mol%, about 41 mol%, about 40 mol%, about 39 mol%, about 38 mol%, about 37 mol%, about 36 mol%, about 35 mol%, about 34 mol%, about 33 mol%, about 32 mol%, about 31 mol%, about 30 mol%, about 29 mol%, about 28 mol%, about 27 mol%, and the like. Examples include about 26 mol%, about 25 mol%, about 24 mol%, about 23 mol%, about 22 mol%, about 21 mol%, about 20 mol%, about 19 mol%, about 18 mol%, about 17 mol%, about 16 mol%, about 15 mol%, about 14 mol%, about 13 mol%, about 12 mol%, about 11 mol%, about 10 mol%, about 9 mol%, about 8 mol%, about 7 mol%, about 6 mol%, about 5 mol%, about 4 mol%, about 3 mol%, about 2 mol%, about 1 mol%, and about 0.5 mol%. Additionally, the lipophilic electrolyte:ionophore ratio can be within a range between any two of the values listed above; for example (and not by way of limitation), the lipophilic electrolyte:ionophore ratio is in the range of about 0.5 mol% to about 49 mol%, or in the range of about 5 mol% to about 48 mol%, or in the range of about 15 mol% to about 45 mol%, or similar ranges.

[0038] Any ionophore having a tripodal stereochemistry, known or not, contemplated within the art and capable of functioning in accordance with the present disclosure is within the scope of the present disclosure. In one embodiment, the ionophore may have at least one malonimide functional group. Non-limiting examples of ionophores that may be utilized in accordance with the present disclosure include those represented by Formulae I-IV: [ka] Examples of ionophores include those represented by any of the following structures:

[0039] In Formula IV, n is in the range of about 6 to about 8. Ionophores represented by any of the structures of Formulas I-III are known in the art by the product designation ETH5506, ETH5504, and ETH3832, respectively. When n is 6 in Formula IV, the ionophore is known by the product designation ETH5282; when n is 8 in Formula IV, the ionophore is known by the product designation ETH7025. "ETH" refers to the Swiss Federal Institute of Standards and Technology (FSU). This shows the German version of the Swiss Federal Institute of Technology (Eidgenoesissche Technische Hochschule).

[0040] In certain (non-limiting) embodiments, the ionophore is represented by the structure of Formula I (ie, ETH5506).

[0041] Any lipophilic borate salt, known or not, contemplated within the art and capable of functioning as described herein may be utilized in accordance with the present disclosure. Non-limiting examples of lipophilic borate salts that may be utilized herein include potassium tetrakis(4-chlorophenyl)borate (KTpClPB) and sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB).

[0042] Furthermore, the lipophilic borate may be present in any concentration that enables the membrane to function in accordance with the present disclosure. For example (and not by way of limitation), the lipophilic borate may have a molar ratio of lipophilic borate to ionophore of about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, about 70 mol%, about 71 mol%, about 72 mol%, about 73 mol%, about 74 mol 1%, about 75 mol%, about 76 mol%, about 77 mol%, about 78 mol%, about 79 mol%, about 80 mol%, about 81 mol%, about 82 mol%, about 83 mol%, about 84 mol%, about 85 mol%, about 86 mol%, about 87 mol%, about 88 mol%, about 89 mol%, about 90 mol%, about 91 mol%, about 92 mol%, about 93 mol%, about 94 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, about 99 mol%, or about 100 mol%, as well as a range formed by any two of the above values (or any value between two of the above values), such as (but not limited to) a range of about 40 mol% to about 100 mol%, a range of about 50 mol% to about 100 mol%, etc.

[0043] Any lipophilic electrolyte, known or not known, contemplated within the art and capable of functioning as described herein may be utilized in accordance with the present disclosure. One non-limiting example of a lipophilic electrolyte that may be utilized herein is represented by Formula V: [ka] The compound may have a structure represented by:

[0044] known or not known, are contemplated within the art and are described herein. Any polymer that can function as part of the polymer matrix in accordance with the present disclosure can be utilized as part of the polymer matrix in accordance with the present disclosure. Non-limiting examples of polymers that can be utilized herein include poly(vinyl chloride), polyurethane, and combinations thereof.

[0045] Any plasticizer, known or unknown, contemplated within the art and capable of functioning as described herein, may be utilized as part of the polymer matrix in accordance with the present disclosure. Non-limiting examples of plasticizers that may be utilized herein include those represented by the following formulae VI-VIII: [ka] Examples include those represented by the following:

[0046] Membranes can be provided in any dimensions that enable potentiometric ion-selective microelectrodes formed therefrom to function according to the present disclosure. In certain non-limiting embodiments, membranes for potentiometric ion-selective microelectrodes typically have a diameter of less than 0.5 cm and a thickness of less than about 100 μm. Furthermore, membranes for microelectrodes may have no internal electrolyte solution or may have up to nanoliter volumes of internal solid-state electrolyte solution (thickness of less than about 3 μm). In comparison, membranes for microelectrodes will have a diameter of greater than about 1.0 cm and a thickness in the range of about 100 μm to about 150 μm, with an internal electrolyte salt solution of greater than about 1 mL.

[0047] Another embodiment of the present disclosure relates to a potentiometric ion-selective electrode for detecting ionized magnesium in a biological sample. The potentiometric ion-selective electrode includes any of the magnesium-sensing membranes described or otherwise contemplated herein above. The potentiometric ion-selective electrode has a significantly better detection limit than any current iMg sensor (which has a detection limit greater than 0.2 mM). For example (but not by way of limitation), the potentiometric ion-selective microelectrode detects ionized magnesium below or equal to about 0.1 mM. 2+ It may have a lower limit of detection.

[0048] Another embodiment of the present disclosure relates to a method for measuring the level of magnesium ions present in a biological sample, in which any potentiometric ion selective electrode described or otherwise contemplated is contacted with the biological sample, and the level of magnesium ions present in the biological sample is measured using the potentiometric ion selective electrode.

[0049] The method may further include contacting the potentiometric ion-selective electrode with a reagent comprising a poly(ethylene oxide) surfactant. The poly(ethylene oxide) surfactant may be utilized at any concentration that enables the surfactant and the potentiometric ion-selective electrode to function in accordance with the present disclosure. A non-limiting example of a poly(ethylene oxide) surfactant concentration within the scope of the present disclosure is less than about 100 mg / L.

[0050] Any poly(ethylene oxide) surfactant known or otherwise contemplated within the art and capable of functioning as described herein can be utilized in accordance with the present disclosure. Non-limiting examples of poly(ethylene oxide) surfactants that can be utilized in accordance with the present disclosure include those represented by Formulae IX-XI: [ka] It is represented by the structure:

[0051] In Formula IX, n is in the range of about 9 to about 10; in Formula XI, n is about 100. A non-limiting example of a surfactant represented by the structure of Formula IX (e.g., t-octylphenoxypolyethoxyethanol) is available under the trade name TRITON TM X-100 (Sigma-Aldrich, St. Louis, MO). A non-limiting example of a surfactant represented by the structure of formula X (e.g., polyethylene 23 lauryl ether) is known in the art by the product designation Brij-35. Formula XI (where n is about 10 A non-limiting example of a surfactant represented by the structure of Formula XI (wherein X is 0) is polyoxyethylene (100) stearyl ether nonionic surfactant, which is known in the art by the product designation Brij-700 (CAS No. 9005-00-9). A specific non-limiting example of a surfactant represented by the structure of Formula XI is disclosed in U.S. Pat. No. 8,496,900 (issued July 30, 2013 to Zhang et al.).

[0052] Yet another embodiment of the present disclosure includes a kit containing any one or more of the membranes, microelectrodes, and / or reagents described herein or otherwise contemplated. For example, but not by way of limitation, the kit may include any of the magnesium-sensing membranes described herein and / or any of the potentiometric ion-selective electrodes containing such membranes. Furthermore, the kit may further include one or more reagents, including surfactants, described herein or otherwise contemplated. Alternatively (and / or in addition), the reagents may be one or more calibration reagents, one or more cleaning reagents, or one or more quality control reagents, or any combination of the above.

[0053] In addition, the kits may further contain other reagents for carrying out any of the particular methods described herein or otherwise contemplated. The nature of these additional reagents will depend on the particular assay format, and their identification is well within the skill of one in the art.

[0054] The components / reagents may each be placed in separate containers / compartments of the kit, or the various components / reagents may be combined in one or more containers / compartments of the kit, depending on the competing properties of the components / reagents and / or their stability. The kit may further include other separately packaged reagents for performing the assay. The relative amounts of the various components / reagents in the kit may vary widely to produce component / reagent concentrations that substantially optimize the reactions required to occur during the assay method and further to substantially optimize the stability / sensitivity of the assay. Positive and / or negative controls may be included in the kit. The kit may further include a set of written instructions describing how to use the kit. For example, but not by way of limitation, the kit may further include instructions for rinsing, calibrating, and / or operating a potentiometric ion-selective electrode. Kits of this nature may be used in any of the methods described or otherwise contemplated herein. [Example]

[0055] Example Examples are provided herein below. However, it should be understood that the present disclosure is not limited to the specific experiments, results, and testing procedures disclosed herein below in its application. Rather, the examples are provided merely as one of various embodiments and are intended to be illustrative rather than comprehensive.

[0056] In this example, a solid-state microsensor for iMg was fabricated using ETH5506 as the ionophore, where the coating film was doped with the lipophilic electrolyte ETH500 (tetrakis(4-chlorophenyl)borate tetradodecylammonium salt, a lipophilic additive with Mw = 1148) in the range of 15 mol% to 45 mol% of the ionophore ETH5506. The permittivity characteristics of the bulk sensing film increased and the impedance decreased; therefore, the main interfering cation Ca was removed. 2+The selectivity for iMg was improved, lowering the detection limit to <0.1 mM; this detection limit is significantly better than that of the underlying Nova CCX iMg sensor (>0.2 mM). Furthermore, the iMg microsensor described herein showed more stable recovery with the addition of ETH500.

[0057] When ETH500 was added above 50 mol%, the dielectric constant of the iMg sensing membrane tended to be affected by the ion exchange process rather than the complexation process between the ionophore and the target ion, resulting in more monovalent cation interference (e.g., Na + interference).

[0058] LOQ mg 2+ iMg Microsensor ETH500 to improve The iMg microsensor was fabricated as follows. The coating membrane was fabricated using an ETH5506-based iMg coating membrane cocktail. The membrane thickness was 70-100 micrometers, and the microsensor diameter was 200 micrometers. The internal electrolyte solution was 40 mM MgCl2 in a methocel-based formulation, and the dried IE thickness was It was <3 micrometers.

[0059] Table 1 and Figure 1 clearly demonstrate that the addition of ETH500 to the iMg microsensor membrane significantly reduced the membrane impedance from >250 MOhms to <20 MOhms.

[0060] [Table 1]

[0061] 2 to 4 and Table 2 show the results for 0.5 mM Mg 2+ to 0.1mM Mg 2+ 1 shows the response recovery of the iMg microsensor using 0.0 wt %, 0.5 wt %, and 1.5 wt % ETH500 in the solution system.

[0062] [Table 2]

[0063] Response stability of iMg microsensor in three levels of automated QC solution Three sensors (ETH500: 0%, 0.5%, and 1.5%) were assembled in the same sensor module (RP Coox module). 2+ The concentrations were as follows: AQC1, 0.9 mM Mg 2+ ;AQC2, 0.6mM Mg 2+ and AQC3, 0.3 mM Mg 2+ Data system number: SN31467. The calibration reagents utilized were developed for use with the RAPIDPoint 500 (RP500) blood gas analyzer (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY).

[0064] AQC performance of iMg microsensors with various ETH500 mass%: The iMg microsensor without ETH500 (Fig. 5) had lower stability when compared to the iMg microsensors with two different concentrations of ETH500 (Figs. 6 and 7). Furthermore, the iMg microsensor without ETH500 (Fig. 5) showed a lower AQC stability than the iMg microsensors with low Mg 2+ The iMg microsensor showed a positively biased recovery for the AQC (AQC3). When ETH500 was added (Figs. 6 and 7), the iMg microsensor showed a positively biased recovery for the AQC (AQC3). 2+ AQC (AQC3) showed a significantly reduced bias.

[0065] Consideration The present disclosure is the first to introduce ETH500 in an iMg microsensor for hematology analyzers, where the iMg microsensor contains an optimal content of lipophilic electrolyte (ETH500) in addition to lipophilic borate (e.g., but not limited to, KTpCIPB) to overcome surfactant affecting iMg response.

[0066] Using a solid-state iMg microsensor constructed as described herein above, low Mg levels at ≦0.1 mM were detected. 2+ The concentrations tested were significantly better than those of a conventional iMg sensor (iMg ISE; Siemens Healthcare Diagnostics, Inc., Tarrytown, NY) over a 4-week period, as well as the iMg sensor based on NOVA CCX (>0.2 mM). Furthermore, the iMg microsensor described herein exhibited a significant improvement over the addition of ETH500. This resulted in a more stable recovery.

[0067] Although ETH500 has previously been reported in studies of conventional ISEs (i.e., Na, K, Ca, pH, and Mg) to improve response kinetics, no studies have been reported on improving the detection limit of iMg solid-state sensors. Furthermore, the amount of ETH500 utilized in conventional ISEs is substantially higher than the amount utilized in the presently disclosed iMg microsensors.

[0068] ETH5506 is Mg 2+ However, this has not been shown to be the preferred selective ionophore for other cation ionophore-cation pairs (ETH1001 vs. Ca). 2+ , NaX-vs-Na + , valinomycin-vs-K + ) than its target cation Mg 2+ Compared to conventional microsensors, the iMg microsensor size results in an increased membrane impedance (from MOhms to MOhms). As shown herein, adding ETH500 as a lipophilic ion pair changes the dielectric constant of the membrane.

[0069] According to the Born equation given below:

number

[0070] Therefore, the results of this example show that the iMg microsensor doped with 0.5% to 1.5% ETH500 exhibited the same sensitivity as the 0.2 mM and 0.1 mM Mg 2+ This demonstrates that the response signals between iMg and iMg can be clearly distinguished, and this improvement in detection limit compared to the prior art is a definite advantage of the iMg microsensor of the present disclosure.

[0071] In conventional iMg membranes (macrosensors), the borate-to-ionophore ratio is Ca 2+ Mg 2+It is very important to ensure selectivity; the currently preferred borate-to-ionophore ratio is 150 mol%, as can be seen in O'Donnell et al. (Analytica Chimica Acta (1993) 281:129-134) and Zhang et al. (Analytical Sciences (2000) 16:11-18). When such an iMg sensor comes into contact with a surfactant in the reagent, the response signal is distorted due to strong surfactant interference (Malinowska et al. (Analytica Chimica Acta (1999) 382:265-275)). A recently disclosed iMg sensor formulation (U.S. Patent No. 10,241,071, issued March 26, 2019 to Zhang et al.) can successfully overcome surfactant interference when iMg is used in a hematology analyzer; this iMg sensor formulation has a borate-to-ionophore ratio ranging from 50 mol% to 100 mol%. This reduction in borate content in conventional iMg films reduces the film dielectric constant and therefore increases the lower limit of detection (LLOD). Adding ETH500 to the iMg macrosensor disclosed herein compensates for the film dielectric constant loss due to borate reduction and allows for low Mg 2+ Improve the sensitivity of the iMg microsensor in the sample.

[0072] In summary, the present disclosure provides a method for measuring blood Mg concentrations by adding 0.5 to 1.5% by mass (15 mol% to 45 mol% relative to the ionophore) of ETH500 to a calibration reagent containing a surfactant. 2+ This is the first time that an iMg microsensor for analytical instruments has been implemented. The iMg formulation of a conventional sensor (i.e., the optimal 50 mol% to 100 mol% borate-to-ionophore ratio of the '071 patent) was used as the base formulation. The microsensor disclosed herein achieves an iMg microsensor detection limit of 0.1 mM Mg. 2+ This is essentially the system that builds on the legacy iMg sensor and the NOVA CCX iMg sensor (which has the lowest Mg 2+This is substantially better than the iMg sensor (which can only detect a concentration of 0.2 mM). This is a very significant improvement of the iMg sensor in testing hypomagnesemic samples with high precision and accuracy.

[0073] Furthermore, the iMg microsensor of the present disclosure provides a more stable sensor response due to the reduced impedance of the microsensor. Additionally, the overall precision of recovery is significantly improved.

[0074] Thus, in accordance with the present disclosure, compositions, kits, and devices, as well as methods for making and using the same, are provided that fully satisfy the objects and advantages set forth hereinabove. While this disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that are within the spirit and broad scope of this disclosure.

Claims

1. 1. A magnesium-sensing membrane for a potentiometric ion-selective microelectrode for detecting ionized magnesium in a biological sample, the magnesium-sensing membrane comprising: Ionophores with tripodal stereochemistry; a lipophilic borate present in an amount resulting in a molar ratio of lipophilic borate to ionophore in the range of about 50 mol% to about 100 mol%; a lipophilic electrolyte present in an amount resulting in a molar ratio of lipophilic electrolyte to ionophore of less than or equal to about 50 mol %, wherein the lipophilic electrolyte is a compound represented by Formula V: 【Chemical 1】 and a polymer matrix in which an ionophore, a lipophilic borate, and a lipophilic electrolyte are disposed, wherein the polymer matrix comprises a polymer and a plasticizer; The magnesium-sensing membrane.

2. 2. The magnesium-sensing membrane of claim 1, wherein the molar ratio of lipophilic electrolyte to ionophore is in the range of about 15 mol % to about 45 mol %.

3. Ionophores are represented by Formulas I-IV: 【Chemistry 2】 is selected from ionophores represented by the structure 2. The magnesium-sensing membrane of claim 1, wherein in formula IV, n is in the range of about 6 to about 8.

4. The polymer may be selected from the group consisting of poly(vinyl chloride), polyurethane, and combinations thereof.

2. The magnesium-sensing membrane of claim 1, wherein the magnesium-sensing membrane is selected from:

5. 10. The magnesium-sensing membrane of claim 1, wherein the membrane has a diameter of less than about 0.5 cm and a thickness of less than about 100 μm.

6. 2. The magnesium-sensing membrane of claim 1, wherein the lipophilic borate is potassium tetrakis(4-chlorophenyl)borate (KTpCIPB).

7. 1. A potentiometric ion-selective microelectrode for detecting ionized magnesium in a biological sample, wherein the potentiometric ion-selective microelectrode comprises: Ionophores with tripodal stereochemistry; a lipophilic borate present in an amount resulting in a molar ratio of lipophilic borate to ionophore in the range of about 50 mol% to about 100 mol%; a lipophilic electrolyte present in an amount resulting in a molar ratio of lipophilic electrolyte to ionophore of less than or equal to about 50 mol %, wherein the lipophilic electrolyte is a compound represented by Formula V: 【Chemistry 3】 and a polymer matrix in which an ionophore, a lipophilic borate, and a lipophilic electrolyte are disposed, wherein the polymer matrix comprises a polymer and a plasticizer; a magnesium-sensing membrane comprising: wherein the potentiometric ion-selective microelectrode has a Mg content less than or equal to about 0.1 mM. 2+ The potentiometric ion-selective microelectrode as defined above, having a lower limit of detection.

8. 8. The potentiometric ion-selective microelectrode of claim 7, wherein the molar ratio of lipophilic electrolyte to ionophore is in the range of about 15 mol % to about 45 mol %.

9. Ionophores are represented by Formulas I-IV: 【Chemistry 4】 is selected from ionophores represented by the structure wherein in Formula IV, n is in the range of from about 6 to about 8.

8. The potentiometric ion-selective microelectrode of claim 7.

10. 8. The potentiometric ion-selective microelectrode of claim 7, wherein the polymer is selected from the group consisting of poly(vinyl chloride), polyurethane, and combinations thereof.

11. 8. The potentiometric ion-selective microelectrode of claim 7, wherein the membrane has a diameter of less than about 0.5 cm and a thickness of less than about 100 μm.

12. 8. The potentiometric ion-selective microelectrode of claim 7, wherein the lipophilic borate is potassium tetrakis(4-chlorophenyl)borate (KTpCIPB).

13. 1. A method for determining the level of magnesium ions present in a biological sample, the method comprising: contacting a potentiometric ion-selective microelectrode with a biological sample, wherein the potentiometric ion-selective microelectrode detects ionized magnesium in the biological sample, and wherein the potentiometric ion-selective microelectrode Ionophores with tripodal stereochemistry; a lipophilic borate present in an amount resulting in a molar ratio of lipophilic borate to ionophore in the range of about 50 mol% to about 100 mol%; a lipophilic electrolyte present in an amount resulting in a molar ratio of lipophilic electrolyte to ionophore of less than or equal to about 50 mol %, wherein the lipophilic electrolyte is a compound represented by Formula V: 【Chemistry 5】 and a polymer matrix in which an ionophore, a lipophilic borate, and a lipophilic electrolyte are disposed, wherein the polymer matrix comprises a polymer and a plasticizer; and Measuring the level of magnesium ions in a biological sample using a potentiometric ion-selective microelectrode The above method, comprising:

14. 14. The method of claim 13, wherein the molar ratio of lipophilic electrolyte to ionophore is in the range of about 15 mol % to about 45 mol %.

15. Ionophores are represented by Formulas I-IV: 【Chemistry 6】 is selected from ionophores represented by the structure 14. The method of claim 13, wherein in Formula IV, n is in the range of about 6 to about 8.

16. 14. The method of claim 13, wherein the polymer is selected from the group consisting of poly(vinyl chloride), polyurethane, and combinations thereof.

17. 14. The method of claim 13, wherein the membrane has a diameter of less than about 0.5 cm and a thickness of less than about 100 μm. Law.

18. 14. The method of claim 13, wherein the lipophilic borate is potassium tetrakis(4-chlorophenyl)borate (KTpCIPB).

19. 14. The method of claim 13, further comprising contacting the potentiometric ion selective electrode with a reagent comprising a surfactant.

20. The concentration of the surfactant is less than about 100 mg / L, and the surfactant is represented by Formula IX, X, or XI: 【Chemistry 7】 and wherein the surfactant is a poly(ethylene oxide) surfactant represented by one of the structures: wherein in Formula IX, n is in the range of about 9 to about 10; and In Formula XI, n is about 100.

20. The method of claim 19.

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