WO2015127354A1

WO2015127354A1 - Potentiometric sensor, kit and method of use

Info

Publication number: WO2015127354A1
Application number: PCT/US2015/017075
Authority: WO
Inventors: Todd ANDRADE
Original assignee: Siemens Healthcare Diagnostics Inc.
Priority date: 2014-02-24
Filing date: 2015-02-23
Publication date: 2015-08-27

Abstract

A potentiometric sensor for determining a concentration of magnesium within a sample includes a reference electrode, a pH electrode and a pH-analyte electrode. The pH-analyte electrode includes a magnesium sensing membrane having a porphyrin specific for magnesium and an enzyme that catalyzes insertion of a magnesium ion into the porphyrin. The pH sensing membrane includes an ionophore that senses protons alleviated from porphyrin upon insertion of the magnesium ion.

Description

POTENTIOMETRIC SENSOR, KIT AND METHOD OF USE

Background

[001 ] Magnesium assays are being increasingly requested in hospitals and clinical research institutions. A robust magnesium sensor capable of detecting a biological active portion ionized form may aid in the clinical diagnosis of patients.

[002] Current commercial and experimental Mg²⁺ sensors have issues with selectivity and/or specificity of the ionized magnesium. Generally, these sensors are based on neutral ionophore based potentiometric detection. As such, many of the sensors may need corrections (e.g., algorithmic corrections) for pH adjustments and/or selectivity adjustments of other interfering species. For example, many of the sensors may need corrections for pH adjustment and/or selectivity adjustments due to Ca²⁺, K⁺, and/or Na⁺.

[003] Another approach for magnesium sensing is based on cation recognition. For example, an optical signal may result from an analyte linkage wherein chromophore molecules have adapted to a magnesium ion. This optical signal may be identified, and then during post-processing, magnesium concentrations may be determined.

Brief Description of the Several Views of the Drawings

[004] To assist those of ordinary skill in the relevant art in making and using the subject matter hereof, reference is made to the appended drawings, which are not intended to be drawn to scale, and in which like reference numerals are intended to refer to similar elements for consistency. For purposes of clarity, not every component may be labeled in every drawing. [005] FIG. 1 illustrates a schematic diagram of an exemplary embodiment of a potentiometnc sensor in accordance with the present disclosure.

[006] FIG. 2 illustrates a partial cross sectional view of the exemplary embodiment of the potentiometnc sensor taken along lines 2-2 in FIG. 1 showing a pH electrode, a pH-analyte electrode and a reference electrode positioned within wells.

[007] FIG. 3 illustrates a partial cross sectional view of the exemplary embodiment of the potentiometric sensor taken along the lines 3-3 in FIG. 1 showing a pH electrode, a pH-analyte electrode and a reference electrode illustrated in FIG. 1 .

[008] FIG. 4 illustrates a block diagram of an exemplary embodiment of a potentiometric kit in accordance with the present disclosure.

[009] FIG. 5 illustrates a structural formula for the chemical compound Protoporphyrin IX.

[0010] FIG. 6 illustrates a flow chart of an exemplary method for determining a concentration of Mg²⁺ within a sample using a potentiometric sensor.

[0011 ] FIG. 7 illustrates a partial cross sectional view of an exemplary embodiment of a potentiometric sensor showing a reference electrode positioned within a flow channel in accordance with the present disclosure.

Detailed Description

[0012] Before explaining at least one embodiment of the disclosure in detail, it is to be understood that the disclosure is not limited in its application to the details of construction, experiments, exemplary data, and/or the arrangement of the components set forth in the following description or illustrated in the drawings unless otherwise noted.

[0013] The disclosure is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for purposes of description, and should not be regarded as limiting.

[0014] The following detailed description refers to the accompanying drawings. The same reference numbers in different drawings may identify the same or similar elements.

[0015] As used in the description herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, unless otherwise noted, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0016] Further, unless expressly stated to the contrary, "or" refers to an inclusive and not to an exclusive "or". For example, a condition A or B is satisfied by one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0017] In addition, use of the "a" or "an" are employed to describe elements and components of the embodiments herein. This is done merely for convenience and to give a general sense of the inventive concept. This description should be read to include one or more, and the singular also includes the plural unless it is obvious that it is meant otherwise. Further, use of the term "plurality" is meant to convey "more than one" unless expressly stated to the contrary. [0018] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example.

[0019] Circuitry, as used herein, may be analog and/or digital components, or one or more suitably programmed processors (e.g., microprocessors) and associated hardware and software, or hardwired logic. Also, "components" may perform one or more functions. The term "component," may include hardware, such as a processor (e.g., microprocessor), an application specific integrated circuit (ASIC), field programmable gate array (FPGA), a combination of hardware and software, and/or the like.

[0020] Software may include one or more computer readable instructions that when executed by one or more components cause the component to perform a specified function. It should be understood that the algorithms described herein may be stored on one or more non-transient memory. Exemplary non-transient memory may include random access memory, read only memory, flash memory, and/or the like. Such non-transient memory may be electrically based, optically based, and/or the like.

[0021 ] It is to be further understood that, as used herein, the term user is not limited to a human being, and may comprise, a computer, a server, a website, a processor, a network interface, a human, a user terminal, a virtual computer, combinations thereof, and the like, for example. [0022] Referring now to the Figures, and in particular to FIGS. 1 -3, shown therein are illustrations of hardware forming an exemplary embodiment of a potentiometric sensor 10. The potentiometric sensor 10 is generally a membrane- based magnesium sensor capable of quantitatively measuring a concentration of ionized magnesium species within a sample solution.

[0023] The potentiometric sensor 10 may include a housing 12 supporting and/or encompassing one or more working pH electrodes 14, one or more working pH-analyte electrodes 16, and one or more reference electrodes 18. Additionally, the housing 12 may house one or more additional electrodes for sensing other species within a sample solution. For example, the housing 12 may house electrodes sensing other species including, but not limited to, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, urea nitrogen, lactase dehydrogenase (LD), cholesterol, bilirubin, choline esterase, neutral lipid, glucose, hematocrit, and/or the like.

[0024] Referring to FIGS. 1 and 3, a sample may be provided to one or more sample inlets 20 of the housing 12 and pass through a flow channel 22 of the housing 12 to one or more wells 24 of the housing 12. In some embodiments, one or more reference electrodes 18 may be positioned in one or more flow channels 22 and/or the one or more wells 24 of the housing 12. In some embodiments, one or more reference electrode 18 may be positioned in one or more well 24 of the housing 12 as shown in FIG. 3. In some embodiments, one or more reference electrode 18 may be positioned in the one or more flow channel 22, but not disposed within any of the well(s) 24.

[0025] The sample may be any fluidic sample and/or sample capable of being fluidic (e.g., a biological sample mixed with a fluidic substrate). In some embodiments, the sample may include tissue, fluid, and/or other material derived from a patient. For example, the sample may include, but is not limited to, serum, heparinized plasma, throat swabs, sputum, urine, blood, surgical drain fluids, tissue biopsies, and/or the like. It should be noted that although the present disclosure is directed towards a biological sample, one skilled in art will appreciate that the concepts disclosed herein may be applied to any sample wherein a concentration of magnesium may be determined, and as such, the present disclosure is not limited to biological samples.

[0026] The sample may flow through the flow channel 22 by a driving force. The driving force may include, but is not limited to, capillary force, pressure, gravity, vacuum, electrokinesis, and/or the like.

[0027] In some embodiments, solvent may be used as a solution to deliver the sample to the one or more working pH electrodes 14, the one or more working pH- analyte electrodes 16, and/or the one or more reference electrodes 18. In some embodiments, the solvent may deliver the sample to the one or more working pH electrodes 14, the one or more working pH-analyte electrodes 16, and/or the one or more reference electrodes 18, and then the solvent may evaporate. Evaporation of the solvent may cure one or more membranes on the surface of the particular electrode 14, 16 and/or 18.

[0028] In some embodiments, the flow channel 22 may be a hollow channel. Alternatively, a portion and/or the entire flow channel 22 may be filled with a carrier material. For example, in some embodiments, a portion and/or the entire flow channel 22 may be filled with filter paper, gel, and/or beads.

[0029] Referring to FIG. 2, in some embodiments, the housing 12 is formed of a multi-layer stack including a fluid card layer 26. The flow channel 22 may be formed within the fluid card layer 26. The fluid card layer 26 may be formed of material including, but not limited to, plastic, ceramic, glass, and/or the like, for example. In some embodiments, the flow channel 22 may be formed through removal of a portion of the fluid card layer 26. Alternatively, the flow channel 22 may be formed via a mold having the flow channel 22 shaped therein. The shape of the flow channel 22 may be rectangular, circular, and/or any fanciful shape.

[0030] The flow channel 22 may deliver the sample to one or more wells 24, three of which are labeled in FIG. 3 as 24a, 24b and 24c. Each well 24 may intersect with the one or more working pH electrodes 14, the one or more working pH-analyte electrodes 16, or the one or more reference electrodes 18. In some embodiments, the wells 24 may intersect with two or more of the electrodes from the group of the working pH electrodes 14, the working pH-analyte electrodes 16 and/or the reference electrodes 18. For example, two or more working pH electrodes 14 may be positioned within a single well 24. FIG. 7 shows an example of a potentiometric sensor 10a that is constructed in a similar manner as the potentiometric sensor 10 discussed herein, with the exception that one or more reference electrode 18 may be positioned within the flow channel 22 as illustrated in FIG. 7. In some embodiments of the potentiometric sensor 10a, one or more reference electrode 18 may solely be positioned within the flow channel 22 and not within the well(s) 24.

[0031 ] Each well 24 may have a first end 31 and a second end 33, and may be shaped to house at least one type of electrode 14, 16 or 18. The electrode may be positioned at the second end 33 of the well 24, for example. The sample may flow through the flow channel 22 into the first end 31 of the well 24 and may then contact the electrode positioned on the second end 33 of the well 24. For example, as illustrated in FIG. 2, the sample may flow through the flow channel 22 and into the first end 31 a of the well 24a. The sample may then flow through the well 24a to the working pH electrode 14 positioned on the second end 33a of the well 24a.

[0032] The height h and the diameter d of each well may be determined based on the desired flow of the sample through the well. For example, in some embodiments, the height h and the diameter d of each well may be determined to increase turbulent flow of the sample through the well 24 if mixing of the sample with a reagent is desired.

[0033] In some embodiments, size and/or shape of each well 24 may be formed based on effects of an electroactive area for each conductive electrode, effects of deposition volume for membrane dispensing, and/or containment of membrane components. For example, size and shape of wells 24 may be determined such that the migration and/or interference of membrane components discussed herein may be minimized.

[0034] In some embodiments, the one or more wells 24 may be a hollow channel. Alternatively, a portion and/or the entire well 24 may be filled with a carrier material. For example, in some embodiments, a portion and/or the entire well 24 may be filled with filter paper, gel, and/or beads. In some embodiments, the same material(s) may be used to fill the well 24 and the flow channel 22. Alternatively, different material(s) may be used to fill the well 24 and the flow channel 22.

[0035] Referring to FIG. 2, in some embodiments, the housing 12 may include a laminate layer 34. One or more wells 24 may be formed within the laminate layer 34. The laminate layer 34 may be formed of a dielectric material, including, but not limited to, plastic, ceramic, glass, and/or the like. In some embodiments, the laminate layer 34 may be patterned to form a part of well 24 intersecting the reference electrode 18 and may be formed through removal of portions of the laminate layer 34. Alternatively, the wells 24 may be formed via a mold having each well 24 shaped therein, or deposition of a material onto a substrate around each well 24.

[0036] Each electrode 14, 16 and 18 may include one or more conductive layers 36. The one or more conductive layer 36 may be formed of any suitable conductive material including, but not limited to, carbon, silver, silver chloride, gold, platinum, palladium, and/or the like. The one or more conductive layers 36 may be sputtered, electroplated, screen printed, inkjet printed, and/or any other technique capable of applying conductive material to the housing 12 associated with fabrication of a sensor array.

[0037] In some embodiments, the conductive layer 36 may be formed by laser ablation of a gold sputtered metal film on a backing with the laminate layer 34 defining wells 24 wherein the electrodes 14, 16, and 18 will be placed. Alternatively, in some embodiments, the conductive layer 36 may be formed of localized positioning of a carbon within the housing 12. As illustrated in FIGS. 3 and 4, electrodes 14, 16 and 18 may also include leads 38 for connection to voltmeters 40 and/or a computer system 42 for measurement.

[0038] Referring to FIGS. 2 and 3, the housing 12 may also include a substrate 44 which may be physically configured to receive electrodes 14, 16 and 18 upon a surface 46 of the substrate. In some embodiments, the substrate 44 may be formed of a rigid material. Alternatively, the substrate 44, or a portion of the substrate 44, may be formed of flexible material. The materiality of the substrate 44 may serve as the physical and electronic domain for measuring potential between the one or more working pH electrodes 14, the one or more working pH-analyte electrodes 16, and/or the one or more reference electrodes 18.

[0039] The substrate 44 may be formed of materials including, but not limited to, plastic, ceramic, glass, and/or any material capable of containing electrodes. For example, in some embodiments, the substrate 44 may be formed of polyethylene terephthalate (PET).

[0040] Referring to FIGS. 3 and 4, the one or more working pH electrodes 14 may include one or more pH sensing membranes 48. For example, the working pH electrode 14 illustrated in FIG. 3 includes pH sensing membrane 48a and the working pH-analyte electrode 16 includes a pH sensing membrane 48b.

[0041 ] The one or more pH sensing membranes 48a may be deposited on the conductive layer 36 such that the pH sensing membrane 48a may be exposed to the environment (e.g., sample) having an unknown Mg²⁺ concentration. The pH sensing membrane 48a may be formed of material including, but not limited to, ionophore(s), ionic salt(s), a polymer backbone, plasticizer(s), and/or solvent. For example, in some embodiments, the pH sensing membrane 48 may include: an ionophore (2 wt% tridodecylamine (TDDA), ionic salt (21 mole% potassium tetrakis (4- chlorophenyl) borate) (KTpCIPB)), a polymer backbone (33 wt% polyvinyl chloride (PVC)), a plasticizer (65% Di-n-octyl phthalate (DOP)), and a solvent (10w/v% tetrahydrofuran (THF)).

[0042] lonophores of the pH sensing membrane 48a may include, but are not limited to, triaurylammonium chloride, 4-nonadecylpyridine, N,N- Dioctadecylmethylamine, and/or the like. In some embodiments, membranes formed of pH sensitive dyes may be used to measure changes in pH in addition to, or in lieu of ionophore(s). The pH sensitive dyes may measure proton changes, hydrogen concentration and/or the like. For example, pH sensitive dyes such as polyaniline and/or polypyrrole films may be used to form the pH sensing membrane 48a.

[0043] In some embodiments, optically active molecular indicators may additionally be used to monitor changes in pH. For example, optically active molecular indicators may include, but are not limited to, 6-carboxyfluorescein, 2', 7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, 1 ,4-dihydroxyphthalonitrile, 8- hydroxypyrene-1 ,3,6-trisulfonic acid (HPTS)1 , seminaphthorhodafluor (SNARF)/seminaphthofluorescein (SNAFL) dyes, boron-dipyrromethene (KBH-01 ), and/or the like.

[0044] Ionic salts of the pH sensing membrane 48a may include, but are not limited to, sodium tetraphenylborate, sodium tetrakis (4-fluorophenyl)borate dehydrate, and/or the like.

[0045] Polymers of the pH sensing membrane 48a may include, but are not limited to, polyurethane (hydrophilic), silicone rubber, and/or the like.

[0046] Plasticizers of the pH sensing membrane 48a may include, but are not limited to, dibutyl sebacate, Bis(1 -butylpentyl)decan-1 ,10-diyl diglutarate, bis(l- butylpentyl)adipate, bis(2-ethylhexyl)sebacate, 2-nitrophenyl octyl ether, and/or the like.

[0047] The magnesium sensing membrane 52 may be formed of one or more enzymes and one or more porphyrins. The porphyrin(s) is specific for magnesium, while the enzyme(s) catalyzes insertion of a magnesium ion into the porphyrin.

[0048] The one or more working pH-analyte electrodes 16 may include the pH sensing membrane 48b and a magnesium sensing membrane 52. The pH sensing membrane 48b may be formed of materials similar to pH sensing membrane 48a as described herein. In particular, the ionophore(s) senses protons alleviated from porphyrin upon insertion of the magnesium ion. In some embodiments, the pH sensing membrane 48b may be deposited and/or formed on the magnesium sensing membrane 52 such that the pH sensing membrane 48b is exposed to the environment (e.g., sample) having an unknown Mg²⁺ concentration. Alternatively, the pH sensing membrane 48b may be deposited and/or formed on the conductive layer 36 with the magnesium sensing membrane 52 deposited and/or formed on the pH sensing membrane 48b.

[0049] In one non-limiting example, the magnesium sensing membrane 52 may be formed of the enzyme, magnesium protoporphyrin chelatase (6.6.1 .1 .), and the porphyrin, protoporphyrin IX (Mg). The enzyme magnesium protoporphyrin chelatase may include three subunits, Chi I , ChID, and ChlH. This enzyme may be isolated/reconstituted (e.g., soybean) from plants and/or cloned/expressed through gene encoding.

[0050] In some embodiments, active and stable enzymes for the magnesium sensing membrane 52 may be found using methods and techniques as described in at least Ribo Guo, Meizhong Luo, and Jon D. Weinstein. "Magnesium-Chelatase from Developing Pea Leaves." Plant Physiology, 1 16(2). (1998): 605-615, and/or L.C. Gibson, P.E. Jensen, C.N. Hunter, "Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a Bchl-BchD complex." Journal of Biochemistry, 337(Pt. 2). (1999): 243-251 , which are hereby incorporated by reference in their entirety.

[0051 ] In certain embodiments, the enzyme may be obtained from a commercial source and/or synthesized within a lab. In other embodiments, the enzyme may be extracted and isolated from any source that natively and/or recombinantly produces the enzyme. That is, the enzyme may be natively produced by the source from which it is obtained, or the source may be genetically engineered to recombinantly produce the enzyme.

[0052] The enzyme of the magnesium sensing membrane 52 may be immobilized on and/or within the membrane. Immobilization of the enzyme on and/or within the magnesium sensing membrane 52 may be through adsorption, covalent binding, cross-linking of the enzyme, entrapment within gels, entrapment within polymer matrices, and/or the like.

[0053] In some embodiments, porphyrin of the magnesium sensing membrane 52 may be obtained from a commercial source. For example, the porphyrin may be obtained as Protoporphyrin IX (SKU-P8293-1 G) shown in FIG. 5. Protoporphyrin IX is manufactured and distributed by the Sigma-Aldrich Corporation, having a corporate office location in St. Louis, MO. Protoporphyrin IX is generally a biochemically used carrier molecule for divalent cations such as iron, magnesium and zinc.

[0054] Alternatively, porphyrin may be extracted and isolated. For example, porphyrin may be extracted and isolated using techniques as described in Michelle L. Dean, Tyson A. Miller, and Christian Bruckner. "Egg-Citing! Isolation of Protoporphyrin IX from Brown Eggshells and Its Detection by Optical Spectroscopy and Chemiluminescence." J. Chem. Edu., 88(6). (201 1 ): 788-792, which is hereby incorporated by reference in its entirety.

[0055] Certain biological samples utilized with the magnesium sensing membrane (such as but not limited to, blood) will contain an ATP concentration sufficient to catalyze the enzymatic reaction of insertion of a magnesium ion into the porphyrin. Other biological samples (such as but not limited to, urine) will not contain ATP or will contains a lower concentration of ATP that is not sufficient to catalyze the enzymatic reaction. Therefore, in some embodiments, adenosine triphosphate (ATP) may be included within the magnesium sensing membrane 52. For example, ATP may be immobilized on and/or within the magnesium sensing membrane 52. Alternatively, in some embodiments, ATP may be within the sample. For example, within the sample, ATP concentrations of approximately 1 mM and greater may enable design of the potentiometric sensor 10 without the inclusion of ATP within the magnesium sensing membrane 52.

[0056] In some embodiments, a polymer may be used to immobilize the enzyme, ATP, and/or porphyrin on and/or within the magnesium sensing membrane 52. For example, polymers such as polyvinyl alcohol (PVA), mesoporous silica, hydrogels, sol-gel precursor mixtures of 3- glycidoxypropyltrimethoxysilane with methyltrimethoxysilane or tetraethoxysilane, ionic liquid methylimidazolium hexafluophosphate, photopolymerization of poly(ethylene glycol) diacrylate (PEG- DA) with 2-hydroxy-2-methyl phenyl-propanone as photoinitiator protoporphyrin IX, and/or the like, may be used to immobilize components into the magnesium sensing membrane 52.

[0057] Referring to FIG. 4, the one or more working pH electrodes 14, the one or more working pH-analyte electrodes 16, and the one or more reference electrodes 18 may be connected to one or more voltmeters 40. For example, in FIG. 4, working pH electrode 14 and reference electrodes 18 are connected to voltmeter 40a, and working pH-analyte electrode 16 and reference electrode 18 are connected to voltmeter 40b. Voltmeters 40a and 40b may measure changes in electrical potential between the reference electrode 18 and the working pH electrode 14, and the reference electrode 18 and the working pH-analyte electrode 16, respectively. [0058] The voltmeters 40 may be in communication with one or more computer systems 42. The one or more computer systems 42 may be a system or systems that are able to embody and/or execute the logic of the processes described herein. Logic embodied in the form of software instructions and/or firmware may be executed on any appropriate hardware. For example, logic embodied in the form of software instructions and/or firmware may be executed on dedicated system or systems, on a personal computer system, on a distributed processing computer system, and/or the like. In some embodiments, logic may be implemented in a stand-alone environment operating on a single computer system and/or logic may be implemented in a networked environment such as a distributed system using multiple computers and/or processors.

[0059] The computer system 42 may include one or more processors 54 working together, or independently to, execute processor executable code, one or more memories 56 capable of storing processor executable code, one or more input devices 58, and one or more output devices 60.

[0060] Each element of the computer system 42 may be partially or completely network-based or cloud based, and may or may not be located in a single physical location. In some embodiments, the one or more processors 54 may communicate with the voltmeters 40 via a network. As used herein, the terms "network-based", "cloud-based", and any variations thereof, are intended to include the provision of configurable computational resources on demand via interfacing with a computer and/or computer network, with software and/or data at least partially located on the computer and/or computer network. The network may permit bidirectional communication of information and/or data between the one or more processors 54 and/or the voltmeters 40. The network may interface with the one or more processors 54 and the voltmeters 40, in a variety of ways. For example, the network may interface by optical and/or electronic interfaces, and/or may use a plurality of network topographies and/or protocols including, but not limited to, Ethernet, TCP/IP, circuit switched paths, combinations thereof, and/or the like. For example, in some embodiments, the network may be implemented as the World Wide Web (or Internet), a local area network (LAN), a wide area network (WAN), a metropolitan network, a wireless network, a cellular network, a GSM-network, a CDMA network, a 3G network, a 4G network, a satellite network, a radio network, an optical network, a cable network, a public switch telephone network, an Ethernet network, combinations thereof, and/or the like. Additionally, the network may use a variety of protocols to permit bi-directional interface and/or communication of data and/or information between the one or more processors 54 and the voltmeters 40.

[0061 ] In some embodiments, the network may be the Internet and/or other network. For example, if the network is the Internet, a primary user interface of the computer system 42 may be delivered through a series of web pages (e.g., pH and/or Mg²⁺ concentration determination webpages). It should be noted that the primary user interface of the computer system 42 may also be another type of interface including, but not limited to, Windows-based application.

[0062] The one or more processors 54 may be implemented as a single processor or multiple processors working together, or independently, to execute the logic as described herein. It is to be understood, that in certain embodiments using more than one processor 54, the processors 54 may be located remotely from one another, located in the same location, or comprising a unitary multi-core processor. The processors 54 may be capable of reading and/or executing processor executable code and/or capable of creating, manipulating, retrieving, altering and/or storing data structure into the one or more memories 56.

[0063] Exemplary embodiments of the one or more processors 54 may include, but are not limited to, a digital signal processor (DSP), a central processing unit (CPU), a field programmable gate array (FPGA), a microprocessor, a multi-core processor, combinations thereof, and/or the like, for example. In some embodiments, additional processors 54 may include, but are not limited to implementation as a personal computer, a cellular telephone, a smart phone, network-capable television set, a television set-top box, a tablet, an e-book reader, a laptop computer, a desktop computer, a network-capable handheld device, a video game console, a server, a digital video recorder, a DVD-player, a Blu-Ray player, and/or combinations thereof, for example.

[0064] The one or more processors 54 may be capable of communicating with the one or more memories 56 via a path (e.g., data bus). The one or more processors 54 may also be capable of communicating with the input devices 58 and/or the output devices 60.

[0065] The one or more processors 54 may be capable of interfacing and/or communicating with the voltmeters 40. For example, the one or more processors 54 may be capable of communicating by exchanging signals (e.g., analog, digital, optical, and/or the like) using a network protocol.

[0066] The one or more memories 56 may be capable of storing processor executable code. Additionally, the one or more memories 56 may be implemented as a conventional non-transient memory, such as, for example, random access memory (RAM), a CD-ROM, a hard drive, a solid state drive, a flash drive, a memory card, a DVD-ROM, a floppy disk, an optical drive, combinations thereof, and/or the like.

[0067] In some embodiments, one or more memories 56 may be located in the same physical location as the processor 54, and/or one or more memories 56 may be located remotely from the processor 54. For example, one or more memories 56 may be located remotely from the processor 54 and communicate with other processors via the network. Additionally, when more than one memory 56 is used, a first memory may be located in the same physical location as the processor 54, and additional memories 56 may be located in a remote physical location from the processor 54. Additionally, one or more memories 56 may be implemented as a "cloud memory" (i.e., one or more memories 56 may be partially or completely based on or accessed using the network).

[0068] The one or more input devices 58 may be capable of receiving information input from a user and/or processor(s), and may be capable of transmitting such information to the one or more processors 54, network, and/or voltmeters 40. The one or more input devices 58 may include, but are not limited to, implementation as a keyboard, touchscreen, mouse, trackball, microphone, fingerprint reader, infrared port, slide-out keyboard, flip-out keyboard, cell phone, PDA, video game controller, remote control, fax machine, network interface, combinations thereof, and the like, for example.

[0069] The one or more output devices 60 may be capable of outputting information in a form perceivable by a user and/or processors(s). For example, the one or more output devices may include, but are not limited to, implementation as a computer monitor, a screen, a touchscreen, a speaker, a website, a television set, a smart phone, a PDA, a cell phone, a fax machine, a printer, a laptop computer, combinations thereof, and/or the like, for example. It is to be understood that in some exemplary embodiments, the one or more input devices 58 and the one or more output devices 60 may be implemented as a single device, such as, for example, a touchscreen or a tablet. It is to be further understood that as used herein the term user is not limited to a human being, and may comprise, a computer, a server, a website, a processor, a network interface, a human, a user terminal, a virtual computer, combinations thereof, and/or the like, for example.

[0070] Referring to FIG. 4, the one or more memories 56 may store processor executable code and/or information comprising one or more database 64 and program logic 66. In some embodiments, the processor executable code may be stored as a data structure, such as a database and/or a data table, for example. In some embodiments, outputs of one or more voltmeters 40 may be stored in one or more databases and/or data tables 64 within the one or more memories 56.

[0071 ] FIG. 6 illustrates a flow chart 70 of an exemplary method for determining Mg²⁺ concentration within a sample using the potentiometric sensor 10.

[0072] In a step 72, the sample (e.g., blood) may be provided into the sample inlet 20 of the potentiometric sensor 10. In a step 74, the sample may flow through the flow channel(s) 22. Flow through the flow channels 22 may be by any force such as capillary, pressure, and/or the like. In a step 76, the sample may flow through the flow channels 22 to one or more wells 24, and come into contact with one or more working pH electrodes 14, one or more working pH-analyte electrodes 16, and one or more reference electrodes 18.

[0073] As the sample comes into contact with the magnesium sensing membrane 52 of the working pH-analyte electrode 16. The selective insertion of magnesium ions into porphyrin of the magnesium sensing membrane 52 may enable a way for detection of Mg²⁺ from fluid samples. In a step 78, the enzyme within the magnesium sensing membrane 52 may catalyze the insertion of magnesium into the porphyrin of the magnesium sensing membrane 52. For example, in some embodiments, magnesium protoporphyrin chelatase (6.6.1 .1 ) may catalyze the insertion of magnesium into protoporphyrin IX. The enzyme-catalyzed insertion of magnesium may include the use of hydrolyzable adenosine triphosphate (ATP), the sample may react with the enzyme such that:

ENZYME

ATP + porphyrin + Mg²⁺ + H₂0 > ADP + Mg - porphyrin + 2H⁺

wherein two protons, H⁺, may be produced in the reaction.

[0074] In a step 80, the protons produced in the reaction may be detected by the use of the pH sensing membranes 48a and 48b via electrical potential measurements of electrodes 14, 16 and 18, obtained by voltmeters 40a and 40b. For example, the potential difference between the working pH electrode 14 and the reference electrode 18 may be obtained by the voltmeter 40a. Similarly, the potential difference between the working pH-analyte electrode 16 and the reference electrode 18 may be obtained by the voltmeter 40b.

[0075] In a step 82, the potential difference measurements may be communicated to and/or stored in one or more data tables 64 within the computer system 42. In a step 84, using program logic 66 of the computer system 42, the processor 54 may analyze the potential difference measurements and determine an indirect measurement of magnesium within the sample using the differential potentiometric measurements. For example, based on the kinetics of the reaction, the concentration of components may be selected for an expected analyte range within the sample and as such, an increase of H⁺ production may be directly dependent on an increase in Mg²⁺ concentration, and vice versa. In some embodiments, the expected analyte range may be between approximately 0.1 mmol/L to 10 mmol/L, for example. In other embodiments, the expected analyte range may be between approximately 0.45 mmol/L to 0.66 mmol/L.

[0076] Variations in proton concentration may change the potential of the pH sensing membrane layers 48a and 48b, and as such, the electrical potential measurements of electrodes 14 and 16, obtained by voltmeters 40a and 40b may change accordingly. For example, in a sample having minimal or no Mg²⁺, the electrical potential measured through the voltmeter 40a and the electrical potential measured through the voltmeter 40b may be substantially similar (e.g., 200 mV). If there is an increase in pH about the working pH electrode 14 and the working pH- analyte electrode 16, the electrical potential measured through the voltmeter 40a may still be substantially similar to the electrical potential measured through the voltmeter 40b. However, if Mg²⁺ is present within the sample, the reaction of the Mg²⁺ on and/or within the magnesium sensing membrane 52 of the working pH- analyte electrode 16 may provide additional H⁺ production about the working pH- analyte electrode 16. This additional H⁺ production may decrease the pH about the working pH-analyte electrode 16, and thus may decrease the electrical potential measured through the voltmeter 40b as compared to voltmeter 40a. The resulting decrease in electrical potential measured by voltmeter 40b as compared to voltmeter 40a may be analyzed to determine the amount of Mg²⁺ within the sample.

[0077] From the above description, it is clear that the inventive concept(s) disclosed herein are well adapted to carry out the objects and to attain the advantages mentioned herein, as well as those inherent in the inventive concept(s) disclosed herein. While the embodiments of the inventive concept(s) disclosed herein have been described for purposes of this disclosure, it will be understood that numerous changes may be made and readily suggested to those skilled in the art which are accomplished within the scope and spirit of the inventive concept(s) disclosed herein.

Claims

What is claimed is:

1 . A potentiometric sensor, comprising:

a housing defining at least one well and at least one sample inlet in fluid communication with the at least one well, the at least one sample inlet sized and configured to receive a sample;

a reference electrode supported by the housing and in fluid communication with the sample inlet such that the sample contacts the reference electrode upon introduction of the sample into the sample inlet;

a pH electrode positioned within the at least one well such that the sample contacts the pH electrode upon introduction of the sample into the at least one well; and

a pH-analyte electrode positioned within the at least one well such that the sample contacts the pH-analyte electrode upon introduction of the sample into the at least one well, the pH-analyte electrode having a pH sensing membrane and a magnesium sensing membrane, the magnesium sensing membrane comprising a porphyrin specific for magnesium and an enzyme that catalyzes insertion of a magnesium(clai ion into the porphyrin, and the pH sensing membrane comprising an ionophore that senses protons alleviated from porphyrin upon insertion of the magnesium ion.

2. The potentiometric sensor of claim 1 , wherein the at least one well includes at least two wells, and further comprising at least one flow channel providing fluid communication between the at least two well.

3. The potentiometnc sensor of claim 2, wherein the flow channel is a hollow channel, and wherein the potentiometric sensor further comprises a carrier material positioned within the hollow channel.

4. The potentiometric sensor of claim 2, wherein the reference electrode is positioned within the at least one flow channel.

5. The potentiometric sensor of any one of claims 1 -4, further comprising a carrier material positioned within at least one of the at least one well.

6. The potentiometric sensor of any one of claims 1 -5, wherein the magnesium sensing membrane further includes adenosine triphosphate (ATP).

7. The potentiometric sensor of claim 6, wherein the ATP is immobilized within the magnesium sensing membrane.

8. The potentiometric sensor of any one of claims 1 -7, wherein the porphyrin is immobilized within the magnesium sensing membrane.

9. The potentiometric sensor of any one of claims 1 -8, wherein the enzyme is immobilized within the magnesium sensing membrane.

10. The potentiometric sensor of any one of claims 1 -9, wherein the pH-analyte electrode includes the pH sensing membrane, the magnesium sensing membrane, and a conductive layer, the magnesium sensing membrane positioned between the pH sensing membrane and the conductive layer.

1 1 . The potentiometric sensor of any one of claims 1 -10, wherein the pH electrode includes a pH sensing membrane deposited on a conductive layer.

12. The potentiometric sensor of any one of claims 1 -1 1 , wherein the enzyme is magnesium protoporphyrin chelatase.

13. The potentiometric sensor of any one of claims 1 -12, wherein the porphyrin is Protoporphyrin IX.

14. The potentiometric sensor of any one of claims 1 -13, wherein the reference electrode is positioned outside of the at least one well.

15. The potentiometric sensor of any one of claims 1 -13, wherein the reference electrode is positioned within the at least one well.

16. The potentiometric sensor of any one of claims 1 -13, wherein the housing defining at least one well defines a first well, a second well and a third well, and the at least one sample inlet is in fluid communication with the first well, the second well and the third well and wherein the reference electrode is positioned within the first well, the pH electrode is positioned with the second well, and the pH-analyte electrode is positioned within the third well.

17. A potentiometric kit, comprising:

a potentiometric sensor having:

a reference electrode;

a pH electrode; and,

a pH-analyte electrode having a pH sensing membrane and a magnesium sensing membrane, the magnesium sensing membrane comprising a porphyrin specific for magnesium, an enzyme that catalyzes insertion of a magnesium ion into the porphyrin, and the pH sensing membrane comprising an ionophore that senses protons alleviated from porphyrin upon insertion of the magnesium ion;

a first voltmeter for measuring a first potential difference value between the reference electrode and the pH electrode; and,

a second voltmeter for measuring a second potential difference value between the reference electrode and the pH-analyte electrode.

18. The potentiometric kit of claim 17, further comprising a computer system configured to communicate with the first and second voltmeters to obtain the first and second potential difference values, store the first and second potential difference values in at least one data table, and to execute instructions to determine a concentration of magnesium within a sample using the first and second potential difference values.

19. The potentiometric kit of any one of claims 17-18, wherein the magnesium sensing membrane further includes adenosine triphosphate (ATP).

20. The potentiometric kit of claim 19, wherein the ATP is immobilized within the magnesium sensing membrane.

21 . The potentiometric kit of any one of claims 17-20, wherein the porphyrin is immobilized within the magnesium sensing membrane.

22. The potentiometric kit of any one of claims 17-21 , wherein the enzyme is immobilized within the magnesium sensing membrane.

23. The potentiometric kit of any one of claims 17-22, wherein the pH-analyte electrode includes the pH sensing membrane, the magnesium sensing membrane, and a conductive layer, the magnesium sensing membrane positioned between the pH sensing membrane and the conductive layer.

24. The potentiometric kit of any one of claims 17-23, wherein the enzyme is magnesium protoporphyrin chelatase.

25. The potentiometric kit of claim 24, wherein the porphyrin is Protoporphyrin IX.

26. An electrode, comprising:

a conducting layer;

a magnesium sensing membrane deposited on the conducting layer, the magnesium sensing membrane comprising a porphyrin specific for magnesium and an enzyme that catalyzes insertion of a magnesium ion into the porphyrin, whereby magnesium is adsorbed within the magnesium sensing membrane during an enzymatic reaction; and,

a pH sensing membrane deposited on the magnesium sensing membrane, the pH sensing membrane in contact with an environment configured to receive a sample having an unknown Mg²⁺ concentration, the pH sensing membrane comprising an ionophore that senses protons alleviated from porphyrin upon insertion of the magnesium ion.

27. A method, comprising:

providing a sample into an inlet of a potentiometric sensor, the potentiometric sensor having a reference electrode, a pH electrode, and a pH-analyte electrode for reacting with Mg²⁺ within the sample;

measuring a first potential difference between the reference electrode and the pH electrode;

measuring a second potential difference between the reference electrode and the pH-analyte electrode; and,

determining a concentration of Mg²⁺ within the sample by evaluating the first potential difference and the second potential difference.

28. The method of claim 27, wherein the pH-analyte electrode includes a magnesium sensing membrane comprising a porphyrin specific for magnesium, an enzyme that catalyzes insertion of a magnesium ion into the porphyrin, and a pH sensing membrane comprising an ionophore that senses protons alleviated from porphyrin upon insertion of the magnesium ion.

29. The method of any one of claims 28, wherein the sample contains an ATP concentration sufficient to catalyze an enzymatic reaction of insertion of magnesium ion into the porphyrin.

28. The method of any one of claims 28, wherein the sample does not contain an ATP concentration sufficient to catalyze an enzymatic reaction of insertion of magnesium ion into the porphyrin, and wherein the magnesium sensing membrane further comprises a sufficient concentration of ATP to catalyze the enzymatic reaction of insertion of magnesium ion into the porphyrin.