Improved Solid-State Magnesium Ion-Selective Microelectrodes and Methods for Their Manufacture and Use

Abstract

To provide, e.g., a magnesium sensing membrane for use in a potentiometric ion-selective microelectrode that exhibits an increased lower detection limit.SOLUTION: A magnesium sensing membrane comprises: an ionophore having a tripodal stereochemical structure; a lipophilic borate present in an amount that provides a molar ratio of the lipophilic borate to the ionophore in a range from about 40 mol% to 67 mol%, the lipophilic borate being selected from potassium tetrakis(4-chlorophenyl)borate (KTpCIPB) and sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB); a specific lipophilic electrolyte present in an amount that provides a molar ratio of the lipophilic electrolyte to the ionophore of 0.5 mol% to 50 mol%: and a specific polymer matrix in which the ionophore, the lipophilic borate and the lipophilic electrolyte are disposed.SELECTED DRAWING: None

Description

Technical Field

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

[0002] Description of research or development by federal government funds Not applicable.

Background Art

[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, among others, magnesium, sodium, potassium, calcium, and chloride. 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 plays an important role, particularly for magnesium ion (Mg 2+ ) - selective electrodes. The level of borate present in the sensing membrane varies the selectivity coefficient of Mg 2+ for interfering cations such as Ca + , Na + , and K 2+ based on the cation charge number, complex stoichiometry with neutral ionophores, and response kinetics. Mg 2+Regarding ISE, a borate-to-ionophore molar ratio of 155 mol% has been considered the optimized formulation that results in 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 very important for obtaining a stable response of the microsensor for various reasons. First, the solid-state iMg microsensor is used when testing Mg in blood. In blood samples with low Mg concentrations (< about 0.2 mM), it is difficult for the sensor to differentiate responses among blood samples with various Mg concentrations. In other words, the Mg microsensor can be difficult when testing hypomagnesemia samples, especially when the Mg concentration is lower than 0.2 mM. For example, the bench-top iMg analyzer (Nova Stat Profile Critical Care Xpress (CCX), Nova Biomedical Corporation, Waltham, MA) is passive in testing blood samples with low Mg where Mg is < 0.2 mM. 2+ 2+ 2+ 2+ 2+ 2+ 2+

[0006] Second, Mg ionophores (such as ETH5506, ETH3832, ETH7025, etc.) have a relatively "weak" binding ability to the target ion compared to "strong" ionophores for other ions (e.g., ETH1001 for Ca, valinomycin-K, sodium ionophore X-Na, etc.). High membrane impedance reduces the sensitivity of the Mg sensor in samples with low Mg concentrations. In this way, the detection limit of Mg increases. 2+ + + 2+ 2 ​​​​​​​​​​​​​

[0007] Thirdly, low Mg 2+ The response of the microsensor in the sample is unstable with respect to "run accuracy" and "overall accuracy". This is caused by the high impedance of the microsensor, which has a much smaller size than conventional solid-state iMg sensors.

[0008] Fourthly, for iMg sensors using Cal reagent containing surfactant (Brij700), the film formulation (ionophore of ETH5506) is optimized with half the amount of lipophilic borate (KTpCIPB) compared to the ideal iMg formulation in surfactant-free Cal reagent. This enables the iMg sensor to operate accurately with respect to the influence of the surfactant on the iMg response. However, the reduction in the amount of borate present in the membrane results in a very high impedance (>gigaohm), especially for microsensors, and thereby the iMg microsensor becomes non-responsive to Mg present in the blood sample. 2+ non-responsive to the analyte.

[0009] Previously, the cation ISE membrane impedance was effectively reduced by adding anionic lipophilic borates such as KTpCIPB. Current ISE membrane formulations (Na, K, Ca, and pH) optimize the lipophilic anion salt content, which ensures fast response kinetics and rapid wet-up procedures. For iMg ISE, the lipophilic borate content functions to adjust the selectivity pattern for interfering cations (Ca 2+ , K + , and Na + with respect to Mg 2+ ). For iMg sensors used in blood tests, problems are faced when only one lipophilic borate is used in the iMg sensor: (a) too high a content of borate causes surfactant interference in the signal response; and (b) an iMg sensor with too low a content is subject to Ca, the main interfering cation. g sensor, when only one lipophilic borate is used in the iMg sensor: (a) too high a content of borate causes surfactant interference in the signal response; and (b) an iMg sensor with too low a content is subject to Ca, the main interfering cation. 2+Loses selectivity for. The optimal borate-to-ionophore ratio must be maintained for the iMg sensor present in the blood analyzer. However, even at such an optimal ratio, solid iMg microsensors still face difficulties in low Mg 2+ blood samples (<0.2 mM).

[0010] The lipophilic electrolyte known as ETH500 (tetradodecylammonium tetrakis(4-chlorophenyl)borate, Mw = 1148) has been previously used in conventional ISE macrosensors (i.e., for Na, K, Ca, pH, and Mg) to improve response kinetics (see, for example, Legin et al. (Non-Patent Document 1); Spichiger et al. (Non-Patent Document 2); Spichiger (Non-Patent Document 3); Eugster et al. (Non-Patent Document 4); and Eugster et al. (Non-Patent Document 5)). However, no studies have been reported on improving the detection limit of iMg solid-state sensors or the use of ETH500 in microsensors. Furthermore, the amount of ETH500 utilized in conventional ISEs is substantially high (i.e., higher than 50 mol%), which changes the dielectric permittivity of the membrane.

Prior Art Documents

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0012] Therefore, there is a need for a novel and improved magnesium-sensing membrane composition for potentiometric ion-selective microelectrodes that overcomes the disadvantages of the prior art. This disclosure relates to such membranes and microelectrodes containing them, as well as related compositions, kits, devices, and methods.

Brief Description of the Drawings

[0013] Brief Description of Some of the Drawings

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0014] Detailed Description Before explaining at least one embodiment of the concepts of the present invention in detail using exemplary drawings, experiments, results, and inspection methods, it is understood that the concepts of the present invention are not limited to the details of the construction and arrangement of the components described in the following description, or illustrated in the drawings, experiments, and / or results, in the context of its application. The concepts of the present invention are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are intended to be illustrative and not limiting. Also, it is understood that the expressions and terms used herein are for purposes of description and should not be construed as limiting.

[0015] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Enzyme reactions and purification techniques are performed according to the 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 specific references cited and discussed throughout this specification. The nomenclature utilized in connection with the analytical chemistry, synthetic organic chemistry, and the assays and techniques of pharmaceutical and medicinal chemistry described herein is well known and generally used in the art.

[0016] All patents, published patent applications, and non-patent publications referred to in the specification are indicative of the level of skill of those of ordinary skill in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referred to in part in this application are incorporated herein by reference in their entirety as if each individual patent or publication was specifically and individually indicated to be incorporated by reference To the same extent as shown when incorporated, they are hereby incorporated by reference in their entirety into this specification.

[0017] All of the compositions and / or methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of this disclosure have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made in the compositions and / or methods and in the steps or series of steps of the methods without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope, and concept of the inventive concept defined by the appended claims.

[0018] As used in accordance with this disclosure, the following terms are to be understood to have the following meanings unless otherwise indicated: The use of the terms "a" or "an" when used in connection with the term "comprising" in the claims and / or the specification may mean "one", but is also consistent with the meaning 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, a 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 "plural" refers to "two or more". The use of the term "or" in the claims is used to mean "and / or" unless it is clear that only alternatives are meant or the alternatives are mutually exclusive, but the disclosure supports definitions that refer to only alternatives and "and / or". Throughout this application, the term "about" is used to indicate that a value includes the inherent error variability of a device, the method used to determine that value, or the variability that exists between test subjects. For example, but not by way of limitation, when the term "about" is utilized, the specified value can vary by ±20% or ±10% or ±5% or ±1% or ±0.1% from the specified value, such variations being appropriate for carrying out the disclosed method and being understood by one of ordinary skill in the art. The use of the term "at least one" is understood to include not only one but any amount greater than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can be extended up to 100 or 100 or more by the term to which it is attached; further, an amount of 100 / 1000 should not be considered limiting as greater limits can also produce satisfactory results. Further, the term "at least one of X, Y, and Z" will be understood to include only X, only Y, and only Z, and also any combination of X, Y, and Z. The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is for the sole purpose of differentiating between two or more items and is not intended to imply, for example, the order or sequence or importance of one item with respect to another or the order in which to add items.

[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 comprising, e.g., "have" and "has"), "including" (and any form of comprising, 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 preceding 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, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. As will be appreciated by those of skill in the art, typically there is no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0021] As used herein, the term "substantially" means that substantially the described event or circumstance occurs completely or that substantially the described event or circumstance occurs to a significant extent. For example, the term "substantially" means that substantially the described event or circumstance occurs 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 "coupled to" includes both direct coupling of two parts to each other and indirect coupling of two parts to each other. Non-limiting examples of coupling include covalent attachment of a moiety to another moiety either directly or via a spacer group, non-covalent attachment of a moiety to another moiety by direct or moiety-attached specific binding pair members, for example incorporating a moiety into another moiety by dissolving one moiety in the other or by synthesis, and coating one moiety onto another moiety.

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

[0024] The terms "analog" and "derivative" are used interchangeably herein and refer to substances that contain the same basic carbon skeleton and carbon functional groups as a given compound in their structure, but may also contain one or more of their substitutions. As used herein, the term "substitution" is understood to refer to substitution at the residue R of at least one substituent on the compound. In certain non-limiting embodiments, R may include a halide selected from H, hydroxy, thiol, fluoride, chloride, bromide or iodide, and one selected from the following C1-C4 compounds: optionally substituted, linear, branched or cyclic alkyl, and linear, branched or cyclic alkenyl, where any substituent is one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocyclic alkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, each of which is optionally substituted, where any substituent is selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocyclic alkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxy, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxy and -C(O))-alkyl.

[0025] As used herein, the term "sample" refers to any It is understood to include various types of biological samples. In certain embodiments, the sample can be any fluid sample and / or a sample that can be fluid (e.g., a biological sample mixed with a fluid substance). Examples of biological samples that can be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, cerebrospinal fluid (CSF), surgical drain fluid, skin, interstitial fluid, tears, mucus, urine, swabs, combinations, and the like. Although the present disclosure is directed to biological samples, it will be understood by those skilled in the art that the concepts disclosed herein can be applied to any sample in which the concentration of magnesium can be determined, and thus, 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 installation of the sensor in the fluid analyzer until a stable signal is obtained from the calibration reagent.

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

[0028] As used herein, a circuit can be analog and / or digital components, or one or more appropriately programmed processors (e.g., a microprocessor) along with associated hardware and software, or a logic circuit incorporated into hardware. Also, a "component" can perform one or more functions. The term "component" can 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] When executed by one or more components, software may include one or more computer-readable instructions that cause the components to perform a particular function. Of course, 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 electrically based, optically based, and / or the like.

[0030] Of course, as used herein, the term "user" is not limited to humans and may include, for example, computers, servers, websites, processors, network interfaces, humans, 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 Nernstian or Nicolsky-Eisenman slopes, offsets, and / or selectivity coefficients determined in an initial three-point calibration (i.e., "full calibration").

[0032] As used herein, the term "calibration logic" refers to the 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 the program logic of a control system used by a processor to interpret data from a magnesium ion-selective electrode for an initial three-point calibration (i.e., "full calibration").

[0033] As used herein, the term "recalibration information" may refer to one or more of the slopes, offsets, and / or selectivity coefficients determined using a Nernstian or Nicolsky-Eisenman equation using information derived from any post-three-point calibration, two-point calibration, or one-point calibration after an 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 the program logic of a control system used by a processor to interpret data from a magnesium ion-selective electrode for additional three-point calibrations, one-point calibrations, and two-point calibrations (as further defined herein) after an initial three-point calibration.

[0035] Turning now to the concepts of the presently disclosed and / or claimed inventions, a novel and improved magnesium-sensing membrane for a microelectrode is provided, which exhibits improved stability over existing magnesium-sensing membranes. This novel magnesium-sensing membrane can be used in the development of novel 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 tripod-shaped 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 lower than about 50 mol% 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%, 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%, about 0.5 mol%, and the like. Further, the lipophilic electrolyte:ionophore ratio can be within the range between any two of the values listed above; for example (by way of illustration and not limitation), the lipophilic electrolyte:ionophore ratio can be 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 in a similar range.

[0038] Any ionophore having a tripod-shaped stereochemical structure that is known or not known but contemplated within the art and that can function in accordance with the present disclosure is within the scope of the present disclosure. In one embodiment, the ionophore can have at least one maleimide functional group. Non-limiting examples of ionophores that can be utilized in accordance with the present disclosure include Formulas I-IV:

Chemical formula

[0039] In formula IV, n is in the range of about 6 to about 8. The ionophores represented by any of the structures of formulas I to III are known in the art by the product designations 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 German version of the Swiss Federal Institute of Technology (Eidgenössische Technische Hochschule). is shown.

[0040] In certain embodiments (but not limited to), the ionophore is represented by the structure of formula I (i.e., ETH5506).

[0041] Any lipophilic borate that is known or contemplated within the art and that can function as described herein can be utilized in accordance with the present disclosure. Non-limiting examples of lipophilic borates that can 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 can be present in any concentration that allows the membrane to function in accordance with the present disclosure. For example (by way of illustration and not limitation), the lipophilic borate can be present in an amount that results in 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%, 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%, and further in a range formed by any two of the above values (or any value between two of the above values), for example (but not limited to) in the range of about 40 mol% to about 100 mol%, in the range of about 50 mol% to about 100 mol%, etc.

[0043] Any lipophilic electrolyte, whether known or not, contemplated within the art and capable of functioning as described herein can be utilized in accordance with the present disclosure. A non-limiting example of a lipophilic electrolyte that can be utilized herein is of Formula V:

Chemical formula

[0044] Any polymer that is known or contemplated within the art and can function as described herein may be utilized as part of the polymer matrix in accordance with the present disclosure. Non-limiting examples of polymers that may be utilized herein include poly(vinyl chloride), polyurethane, and combinations thereof.

[0045] Any plasticizer that is known or contemplated within the art and can function 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 the following Formulas VI - VIII:

Chemical Formula

[0046] The membranes can be provided in any dimensions that enable the potentiometric ion-selective microelectrodes formed therefrom to function in accordance with the present disclosure. In certain non-limiting embodiments, the membranes for the potentiometric ion-selective microelectrodes typically have a diameter of less than 0.5 cm and a thickness of less than about 100 μm. Further, the membranes for the microelectrodes may or may not have an internal electrolyte solution, or may have an internal solid-state electrolyte solution up to a nanoliter volume (with a thickness of less than about 3 μm). In comparison, the membranes for the microelectrodes would have a diameter greater than about 1.0 cm and a thickness in the range of about 100 μm to about 150 μm with an internal electrolyte aqueous solution of more 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 above herein or otherwise contemplated. The potentiometric ion-selective electrode has a significantly better lower limit of detection than any current iMg sensor (the lower limit of detection of which is greater than 0.2 mM). For example (but not by way of limitation), the potentiometric ion-selective microelectrode may have a lower limit of detection that is lower than or equal to about 0.1 mM Mg 2+ and 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 the method, any of the potentiometric ion-selective electrodes described or otherwise contemplated are 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 containing 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 may be utilized in accordance with the present disclosure. Non-limiting examples of poly(ethylene oxide) surfactants that may be utilized in accordance with the present disclosure are of Formulas IX-XI:

Chemical formula

[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 sold 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 symbol designation Brij-35. A non-limiting example of a surfactant represented by the structure of Formula XI (wherein n is about 10 0) is the nonionic surfactant polyoxyethylene (100) stearyl ether, which is known in the art by the product symbol designation Brij-700 (CAS number 9005-00-9). A specific non-limiting example of a surfactant represented by the structure of Formula XI is disclosed in U.S. Patent No. 8,496,900, issued to Zhang et al. on July 30, 2013.

[0052] Yet another embodiment of the present disclosure includes a kit containing any one or more of the membranes, microelectrodes, and / or reagents described or contemplated herein, whether or not described. By way of non-limiting example, the kit can include any of the magnesium-sensing membranes described herein and / or any of the potentiometric ion-selective electrodes containing such membranes. Further, the kit can further include one or more reagents including a surfactant described or contemplated herein, whether or not described. Alternatively (and / or in addition thereto), the reagent(s) can be one or more calibration reagents, one or more cleaning reagents, or one or more quality control reagents, or any combination of the foregoing.

[0053] Furthermore, the kit may further contain other reagents for performing any of the specific methods described or contemplated herein, whether or not described. The nature of these additional reagents depends on the particular assay format, and their identification is well within the skill of the art.

[0054] The components / reagents can be placed in separate containers / compartments of the kit, respectively, or the various components / reagents can be combined in one or more containers / compartments of the kit depending on the competing nature of the components / reagents and / or the stability of the components / reagents. The kit may further include other separately packaged reagents for performing the assay. The relative amounts of the various components / reagents in the kit can vary widely to yield concentrations of the components / reagents that substantially optimize the reactions that need to occur during the assay method and, further, to substantially optimize the stability / sensitivity of the assay. Positive and / or negative controls can be included in the kit. The kit can further include a set of written instructions that explain how to use the kit. For example, by way of non-limitation, the kit can further include instructions for rinsing, calibrating, and / or operating a potentiometric ion-selective electrode. Kits of this nature can be used in any of the methods described or contemplated herein.

Examples

[0055] Example Examples are provided hereinafter in this specification. However, it should be understood that the present disclosure is not limited in its application to the specific experiments, results, and test procedures disclosed hereinafter in this specification. Rather, the examples are provided merely as one of various embodiments and are intended to be neither comprehensive nor exemplary.

[0056] In this example, a solid-state microsensor for iMg using ETH5506 as an ionophore was fabricated, where the coating was doped with the lipophilic electrolyte ETH500 (tetradodecylammonium tetrakis(4-chlorophenyl)borate, Mw = 1148, a lipophilic additive) in the range of 15 mol% to 45 mol% of the ionophore ETH5506. The dielectric characteristics (dielectric properties) of the bulk sensing membrane increased and the impedance decreased; thus, the major interfering cation Ca 2+The selectivity for [substance] was improved, and the detection limit decreased 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 by adding ETH500.

[0057] When ETH500 is added in excess of 50 mol%, the dielectric constant of the iMg sensing membrane tends to be affected by the ion exchange process rather than the complex formation process between the ionophore and the target ion. As a result, the monovalent cation interference becomes more significant (e.g., Na + interference).

[0058] LOQ Mg 2+ ETH500 in the iMg microsensor for improving The iMg microsensor was fabricated as follows. The coating film was fabricated using an ETH5506-based iMg coating film cocktail. The film thickness was 70 - 100 micrometers, and the microsensor diameter was 200 micrometers. The internal electrolyte solution was 40 mM MgCI in a methocel-based formulation, and the dried IE thickness was 2 and the dried IE thickness was <3 micrometers.

[0059] Table 1 and Figure 1 clearly demonstrated that the addition of ETH500 to the iMg microsensor membrane significantly decreased the membrane impedance from >250 M ohms to <20 M ohms.

[0060]

Table 1

[0061] Figures 2 - 4 and Table 2 show the response recovery of the iMg microsensor using 0.0 wt%, 0.5 wt%, and 1.5 wt% of ETH500 in a solution system from 0.5 mM Mg 2+ to 0.1 mM Mg 2+ .

[0062]

Table 2

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

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

[0065] Discussion This disclosure is the first to introduce ETH500 in an iMg microsensor for a blood analyzer, where the iMg microsensor contains an optimal content of a lipophilic electrolyte (ETH500) in addition to a lipophilic borate (e.g., but not limited to, KTpCIPB) to overcome surfactants that affect the iMg response.

[0066] Using a solid state iMg microsensor constructed as described hereinabove, low Mg at levels of ≦0.1 mM 2+ concentrations were tested, which is significantly better than conventional iMg sensors over a four-week period (iMg ISE in the research and development phase; Siemens Healthcare Diagnostics, Inc., Tarrytown, NY), and iMg sensors (>0.2 mM) based on NOVA CCX. Furthermore, the iMg microsensor described herein showed more stable recovery with the addition of ETH500 thereby.

[0067] ETH500 has been previously reported in studies of conventional ISEs (i.e., Na, K, Ca, pH, and Mg) to improve response kinetics, but there are no reported studies 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 has been used as a preferred selective ionophore for Mg 2+ However, this has a relatively weak binding affinity for its target cation Mg 2+ compared to other cation ionophore-cation pairs (ETH1001-pair-Ca + NaX-pair-Na + valinomycin-pair-K 2+ ). Compared to conventional microsensors, the iMg microsensor size results in an increase in membrane impedance (from M ohms to G ohms). As shown herein, adding ETH500 as a lipophilic ion pair changes the dielectric constant of the membrane.

[0069] According to the Born's formula listed below:

Equation

[0070] Thus, the results of this example demonstrate that an iMg microsensor doped with 0.5% - 1.5% ETH500 can clearly distinguish the response signals between 0.2 mM and 0.1 mM Mg 2+ in the sample, and this improvement in the detection limit compared to the prior art is a definite advantage of the iMg microsensor of the present disclosure.

[0071] In the conventional iMg membrane (macrosensor), the borate counter-ionophore ratio is for Ca 2+ versus Mg 2+It is very important to ensure selectivity; 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), the currently preferred borate counter-ionophore ratio is 150 mol%. When such an iMg sensor comes into contact with a surfactant in a reagent, the response signal is distorted due to strong surfactant interference (Malinowska et al. (Analytica Chimica Acta (1999) 382:265-275)). An iMg sensor formulation disclosed in recent years (U.S. Patent No. 10,241,071, issued to Zhang et al. on March 26, 2019) can successfully overcome surfactant interference when iMg is used in a blood analyzer; this iMg sensor formulation has a borate counter-ionophore ratio ranging from 50 mol% to 100 mol%. Such a decrease in the borate content in a conventional iMg membrane decreases the membrane permittivity and, therefore, increases the lower limit of detection (LLOD). Adding ETH500 to the iMg macro-sensor disclosed herein compensates for the membrane permittivity loss due to borate reduction and 2+ improves the iMg micro-sensor sensitivity in low Mg samples.

[0072] In summary, the present disclosure is the first to introduce 0.5-1.5 wt% (15 mol%-45 mol% relative to the ionophore) ETH500 into an iMg micro-sensor for a blood Mg analyzer containing a calibration reagent containing a surfactant. The iMg formulation of a conventional sensor (i.e., the optimal 50 mol%-100 mol% borate-to-ionophore ratio of the ‘071 patent) was used as the basic formulation. The micro-sensor disclosed herein improves the iMg micro-sensor detection limit to 0.1 mM Mg 2+ which is substantially the basis of conventional iMg sensors and the NOVA CCX iMg sensor system (which has the lowest Mg 2+ detection limit). 2+It is substantially superior to (only able to detect at a concentration of 0.2 mM). This is a very significant improvement of the iMg sensor when testing hypomagnesemia samples with high precision and accuracy.

[0073] Furthermore, the iMg microsensor of the present disclosure results in a more stable sensor response due to the impedance reduction of the microsensor. Also, the overall precision of the recovery is significantly improved.

[0074] Accordingly, in accordance with the present disclosure, compositions, kits, and devices, as well as methods for manufacturing and using them, are provided, which fully meet the objectives and advantages described above in this specification. The present disclosure has been described in conjunction with the specific drawings, experiments, results, and language described above in this specification, but it is obvious 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 the present disclosure.

Claims

1. A magnesium-sensing membrane for a potentiometric ion-selective microelectrode for detecting ionized magnesium in a biological sample, wherein the magnesium-sensing membrane comprises: An ionophore having a tripod-shaped stereochemical structure; A lipophilic borate present in an amount that provides a molar ratio of lipophilic borate to ionophore within the range of 40 mol% to 58 mol%, selected from sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB); A lipophilic electrolyte present in an amount that provides a molar ratio of lipophilic electrolyte to ionophore from 0.5 mol% to 40 mol% [wherein the lipophilic electrolyte is represented by Formula V: 【Chemical 1】 ; and A polymer matrix in which the ionophore, lipophilic borate, and lipophilic electrolyte are disposed [wherein the polymer matrix contains a polymer and a plasticizer] comprising, The ionophore is selected from ionophores represented by Structures I-IV: 【Chemical Formula 2】 wherein in Formula IV, n ranges from 6 to 8, the above magnesium-sensing membrane.

2. The magnesium-sensing membrane according to Claim 1, wherein the molar ratio of lipophilic electrolyte to ionophore is in the range of 15 mol% to 40 mol%.

3. The magnesium-sensing membrane according to Claim 1, wherein the polymer is selected from the group consisting of poly(vinyl chloride), polyurethane, and combinations thereof.

4. The magnesium-sensing membrane according to Claim 1, wherein the membrane has a diameter of less than 0.5 cm and a thickness of less than 100 μm.

5. A potentiometric ion-selective microelectrode for detecting ionized magnesium in a biological sample, wherein the potentiometric ion-selective microelectrode comprises: An ionophore having a tripod-shaped stereochemical structure; A lipophilic borate present in an amount that provides a molar ratio of lipophilic borate to ionophore within the range of 40 mol% to 58 mol%, selected from sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB); A lipophilic electrolyte present in an amount that provides a molar ratio of lipophilic electrolyte to ionophore from 0.5 mol% to 40 mol% [wherein the lipophilic electrolyte is represented by Formula V: ; and [Chemical Formula 3] A polymer matrix in which the ionophore, lipophilic borate, and lipophilic electrolyte are disposed [wherein the polymer matrix contains a polymer and a plasticizer] ​ comprising the magnesium-sensing membrane according to claim 1; and Here, the potential difference measuring ion-selective microelectrode has a detection limit of less than 0.1 mM or equal to 0.1 mM of Mg ２＋ and the ionophore is selected from ionophores represented by formulas I-IV: 【Chemical Formula 4】 selected from ionophores represented by the structure of, wherein in formula IV, n is in the range of 6 to 8, the above-mentioned potential difference measuring ion-selective microelectrode. **Claim 6** The potential difference measuring ion-selective microelectrode according to claim 5, wherein the molar ratio of the lipophilic electrolyte to the ionophore is in the range of 15 mol% to 40 mol%. **Claim 7** The potential difference measuring ion-selective microelectrode according to claim 5, wherein the polymer is selected from the group consisting of poly(vinyl chloride), polyurethane, and combinations thereof. **Claim 8** The potential difference measuring ion-selective microelectrode according to claim 5, wherein the membrane has a diameter of less than 0.5 cm and a thickness of less than 100 μm. **Claim 9** A method for measuring the level of magnesium ions present in a biological sample, the method comprising: contacting a potential difference measuring ion-selective microelectrode with a biological sample; and measuring the level of magnesium ions in the biological sample using the potential difference measuring ion-selective microelectrode comprising, wherein the potential difference measuring ion-selective microelectrode detects ionized magnesium in the biological sample, and the potential difference measuring ion-selective microelectrode has an ionophore with a tripod-shaped stereochemical structure; a lipophilic borate present in an amount providing a molar ratio of the lipophilic borate to the ionophore in the range of 40 mol% to 58 mol%, selected from sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB); a lipophilic electrolyte present in an amount providing a molar ratio of the lipophilic electrolyte to the ionophore in the range of 0.5 mol% to 40 mol% [wherein the lipophilic electrolyte is represented by formula V: [Chemical Formula 5] ; and a polymer matrix in which the ionophore, lipophilic borate, and lipophilic electrolyte are disposed [wherein the polymer matrix contains a polymer and a plasticizer] comprising a magnesium-sensing membrane, the ionophore is selected from ionophores represented by formulas I-IV: 【Chemical Formula 6】 selected from ionophores represented by the structure of, wherein in formula IV, n is in the range of 6 to 8, the above method. **Claim 10** The method according to claim 9, wherein the molar ratio of the lipophilic electrolyte to the ionophore is in the range of 15 mol% to 40 mol%. **Claim 11** The polymer is selected from the group consisting of poly(vinyl chloride), polyurethane, and combinations thereof The method according to claim 9, selected from **Claim 12** The method according to claim 9, wherein the membrane has a diameter of less than 0.5 cm and a thickness of less than 100 μm. **Claim 13** The method according to claim 9, further comprising the step of contacting a potentiometric ion-selective electrode with a reagent containing a surfactant. **Claim 14** The concentration of the surfactant is less than 100 mg / L, and the surfactant is a poly(ethylene oxide) surfactant represented by one of the structures of Formula IX, X, or XI: 【Chemical Formula 7】 wherein in Formula IX, n ranges from 9 to 10; and in Formula XI, n is 100, The method according to claim 13. ​

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