Single-use disposable reference sensor

Abstract

A single-use disposable potentiometric reference sensor includes an insulating base substrate; a reference electrode disposed on the insulating base substrate, wherein the reference electrode is a silver-silver chloride electrode; an inner layer disposed on the reference electrode, wherein the inner layer is an amorphous salt layer including an amorphous polysaccharide and a salt having equally-mobility cations and anions; and a semi-permeable cover membrane disposed over the inner layer, wherein the semi-permeable cover membrane has water vapor and ion permeability.

Description

TECHNICAL FIELD

[0001] The present invention relates generally to electrochemical sensors. In particular, the present invention relates to electrochemical potentiometric reference sensors. More particularly, the present invention relates to planar electrochemical potentiometric reference sensors having a membrane coating. BACKGROUND

[0002] A basic prior art electrochemical sensor typically consists of an electrochemical cell having two electrodes. The first electrode responds to chemical species in a liquid sample and is commonly referred to as the indicating electrode. The second electrode is a reference electrode that does not respond to changes in the composition of the liquid sample and provides a constant potential against which the potential generated by the indicating electrode from the liquid sample is measured.

[0003] In the past, chemical analyzers for the quantitative measurement of chemical species in liquid samples including blood have typically included very complex fluidics for washing and calibrating multipurpose sensors. As a result, manufacturers of such chemical analyzers have attempted to produce sensors at relatively low cost so that the sensors are used as single-use devices. The technology that is suitable for such sensor devices is planar technology. Sensors made by planar technology include both thick film technology and thin film technology.

[0004] A typical component of prior art planar electrochemical sensor construction is a device that includes a plurality of metallic conductor elements on a planar insulating substrate. Prior art planar electrochemical sensors consist of a plurality of layers over the plurality of metallic conductor elements, with one end of the plurality of metallic conductor elements exposed for connection to external measurement circuitry and a second end of the plurality of metallic conductor elements exposed for receiving a plurality of coatings that form an overall electrolyte layer including a hydrophilic layer such as a gel material that serves as an aqueous electrolyte and other reagents selected for measuring a particular species in a liquid sample. Chemical species from the liquid sample undergo electrochemical reactions at the electrode surfaces, generating a current or voltage. The generated current or voltage is typically proportional to the concentration of the chemical species in the provided liquid sample, however, the reference electrode remains stable throughout the measurement process.

[0005] In order to provide electrodes that are useful in a plurality of processes, such as, for example, blood analysis operations used in hospitals, blood chemistry laboratories, and the like, it is desirable to provide a small, economically single-use electrode that has a long shelf life and is inexpensive. Most prior art electrodes employ a hydrophilic or aqueous reference electrolyte, making long shelf life difficult to achieve. Hydrophilic electrolytes are hydrated gels or the like that allow for ionic transport. Shipping and storing "wet" electrolytes involves relatively complex packaging.

[0006] Salt bridge potentiometric reference electrodes consist of a silver / silver chloride (Ag / AgCl) based electrode in contact with a concentrated salt solution, preferably with an equitransfer salt such as potassium chloride. The concentrated chloride saturates the Ag / AgCl potential, while the equitransfer of potassium and chloride prevents the development of a junction potential at the interface of the reference sensor and the sample. To maintain a stable reference potential over time, the amount of salt in the array reservoir is critical. In a planar sensor array, the size of the salt reservoir is very limited, and once it is in contact with an aqueous solution, it can be quickly (i.e. less than one second) washed away. There have been many attempts to solve this problem by trying various cover membranes, none of which have been very successful. Previous attempts have failed primarily due to the poor water vapor diffusion properties of the hydrophobic cover membrane polymer and the too slow (or too fast) permeability of the salt through the membrane.

[0007] A number of different single-use potentiometric reference sensor technologies have been reported. U.S. Patent No. 4,933,048 (Lauks, 1990) discloses an open junction reference electrode assembly. The reference electrode assembly includes a metal member coated with an electrode material reversible to an ion X and a layer of electrolyte containing the ion X formed over the electrode. The electrolyte extends beyond the perimeter of the electrode. The portion of the electrolyte extending beyond the perimeter of the electrode is covered by a membrane permeable to H2O molecules but not to the ion X. A portion of the electrolyte extends through the permeable membrane or is otherwise able to form a liquid junction with the sample solution at a location relatively far from the electrode. Thus, the ion must diffuse through the electrolyte between the liquid junction and the electrode along a relatively long path, providing a long time constant for ion diffusion, while the electrolyte can wet relatively quickly. As a result, there is a period of time after the electrolyte wets and before the ion diffusion affects the ion concentration in the vicinity of the electrode, during which the potential of the electrode is essentially constant. The electrolyte consists of a concentrated salt containing a hydrophilic polymer membrane as the inner layer, and is partially covered with a hydrophobic membrane that exposes a small portion of the inner layer directly to the sample solution. To achieve this type of configuration, the sensor manufacturing process is complex, which can result in increased sensor failure rates.

[0008] U.S. Patent No. 7,767,068 (Lauks et al., 2010) discloses a heterogeneous membrane electrode. The heterogeneous membrane consists of a mixture of an oil and a water-soluble compartment. The aqueous portion consists of a salt and a redox couple containing a cross-linkable hydrophilic polymer. The oil portion consists of a cross-linkable hydrophobic polymer. The mixture is emulsified in order to support a manufacturing process using dispensing or printing. The next steps require settling of the deposited layer, degassing, and final UV curing to fix all compartments. This process is a complex time-dependent process due to the phase separation characteristics of the heterogeneous membrane, which can cause sensor-to-sensor variability. SUMMARY

[0009] It is well known that potentiometric reference electrodes must be reliable and not susceptible to environmental influences by providing a stable potential. All potentiometric reference electrodes have a liquid junction potential. These are the boundary / interface potentials that are created between the reference electrode and the sample. Although all potentiometric reference electrodes have a junction potential, it is important that the junction potential be relatively constant and not influenced by the temperature or the local chemical composition around the reference electrode.

[0010] It is a key challenge in single-use planar sensors to integrate an electrochemical potentiometric reference electrode within the planar sensor array.

[0011] It is important that any single-use potentiometric reference electrode incorporated into a planar sensor array have a relatively constant and reproducible junction potential, where the concentration of various electrolytes in the blood sample can vary depending on the health status of the animal from which the blood sample is taken. As previously discussed, planar sensor arrays have potentiometric reference electrodes with salt reservoirs of very limited size. In order to prevent the limited size salt reservoir from being quickly washed away upon exposure to a blood sample, it is necessary to incorporate a hydrophobic component in the potentiometric reference electrode to prevent this from happening or to delay any change in the reference potential or the junction potential of the reference electrode for a period of time during which the blood sample measurement is made.

[0012] It is an object of the present invention to provide a single-use disposable electrochemical potentiometric reference electrode / sensor. It is another object of the present invention to provide a single-use disposable potentiometric reference electrode / sensor with a relatively long shelf life. It is yet another object of the present invention to provide a salt bridge electrochemical potentiometric reference electrode / sensor with dry reagents that reach an active state upon absorption of water vapor upon use.

[0013] The present invention achieves these and other objects by providing a single-use disposable potentiometric reference sensor comprising an amorphous polysaccharide / salt layer in combination with a semi-permeable cover membrane.

[0014] In one embodiment, the single-use disposable potentiometric reference sensor comprises: an insulating bottom substrate; a reference electrode disposed on the insulating bottom substrate, wherein the reference electrode is a silver-silver chloride electrode; an inner layer disposed on the reference electrode, wherein the inner layer is an amorphous salt layer comprising an amorphous polysaccharide and a salt having equally mobile cations and anions; and a semi-permeable cover membrane disposed over the inner layer, wherein the semi-permeable cover membrane has water vapor and ion permeability.

[0015] In one embodiment, the amorphous polysaccharide has amorphous properties such that the amorphous polysaccharide and the salt do not separate upon formation of the inner layer when supersaturated with the salt.

[0016] In one embodiment, the amorphous polysaccharide is selected from a variety of compounds including, but not limited to, pullulan, dextran, and starch sugar.

[0017] In one embodiment, the salt is one of potassium chloride, ammonium chloride, potassium nitrate, lithium acetate, and the like, wherein the cation and the anion have substantially equal mobility.

[0018] In one embodiment, the semi-permeable cover membrane is made of chlorosulfonated polyethylene or made of cellulose acetate butyrate polymer.

[0019] In another embodiment, a single-use disposable electrochemical sensor includes: an insulating base substrate having a sensing surface; a potentiometric working electrode formed on the sensing surface, wherein the potentiometric working electrode has a species-specific reagent matrix disposed thereon, wherein the species-specific reagent matrix has one or more layers selected for measuring a specific species in a liquid sample; and a potentiometric reference electrode formed on the sensing surface, wherein the reference electrode is a silver-silver chloride electrode having a multi-layer reference coating thereon. The multi-layer reference coating has an inner layer disposed on the Ag / AgCl reference electrode and a semi-permeable cover membrane disposed over the inner layer, wherein the inner layer includes an amorphous polysaccharide and a salt having a cation and an anion of equal mobility, the semi-permeable cover membrane is a semi-permeable hydrophobic polymer, wherein the semi-permeable cover membrane has water vapor permeability and ion permeability.

[0020] In one embodiment, a method of forming a single-use disposable potentiometric reference sensor includes: providing a sensor body having an insulating base substrate with at least one electrically conductive path and an insulating and reagent retaining layer disposed onto the insulating base substrate, wherein the insulating and reagent retaining layer overlying the insulating base substrate has at least one reagent retaining opening, wherein the at least one reagent retaining opening exposes a portion of the at least one electrically conductive path; disposing an amorphous salt layer mixture including an amorphous polysaccharide and a salt having a cation and an anion of equal mobility into one of the at least one reagent retaining openings; drying the amorphous salt layer mixture, thereby forming an inner layer; disposing a cover membrane solution including a hydrophobic polymer over the inner hydrophilic layer; and drying the cover membrane solution, thereby forming a semi-permeable cover membrane, wherein the semi-permeable cover membrane has water vapor permeability and ion permeability.

[0021] In one embodiment, the amorphous salt layer mixture is formed by: adding together a plurality of components including a predefined amount of an amorphous polysaccharide and a predefined amount of a 3 mol / L salt solution, and mixing the plurality of components, thereby forming the amorphous salt layer mixture.

[0022] In one embodiment, the amorphous salt layer mixture is formed by measuring 750 milligrams of amorphous polysaccharide, measuring a 3 milliliter volume of a 3 mol / L salt solution, and mixing the components to form the amorphous salt layer mixture.

[0023] In one embodiment, the cover film solution is formed by measuring a pre-defined amount of a hydrophobic polymer, the hydrophobic polymer being one of chlorosulfonated polyethylene or cellulose acetate butyrate, and mixing the hydrophobic polymer into a pre-defined amount of THF / cyclohexanone to form the cover film solution.

[0024] In one embodiment, the cover film solution is formed by measuring 8-10 weight percent of one of chlorosulfonated polyethylene or cellulose acetate butyrate, and mixing the hydrophobic polymer into a pre-defined amount of THF / cyclohexanone to form the cover film solution.

[0025] In yet another embodiment, a method of forming a single-use, disposable electrochemical potentiometric reference sensor includes providing a sensor body having an insulating base substrate with at least one electrically conductive path and an insulating and reagent retaining layer disposed on the insulating base substrate, wherein the insulating and reagent retaining layer has at least one reagent retaining opening, wherein the at least one reagent retaining opening exposes a portion of the at least one electrically conductive path; disposing an amorphous salt layer mixture including an amorphous polysaccharide and a salt having equally mobile cations and anions into one of the at least one reagent retaining opening; drying the amorphous salt layer mixture to form an inner layer; disposing a cover film solution including a hydrophobic polymer over the hydrophilic inner layer; and drying the cover film solution to form a semi-permeable cover film, wherein the semi-permeable cover film has water vapor permeability and ionic permeability.

[0026] In one embodiment, a multi-layer reagent matrix for making a potentiometric reference electrode into a single-use, disposable reference sensor is disclosed. The multi-layer matrix includes an inner layer formed from an amorphous salt layer mixture including an amorphous polysaccharide and a salt, wherein the inner layer covers an Ag / AgCl reference electrode. The amorphous polysaccharide has amorphous properties such that the amorphous polysaccharide and the salt do not separate when formed in the inner layer when oversaturated with the salt. A hydrophobic cover film formed from a cover film solution is disposed over the inner layer, the cover film solution including a hydrophobic polymer, wherein the hydrophobic semi-permeable film polymer is permeable to water vapor and ions.

[0027] In one embodiment, the inner layer is a mixture of potassium chloride and at least one of pullulan, dextran, and starch sugar. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1is a perspective view of a potentiometric reference sensor that is an embodiment of the present invention.

[0029] Figure 2 is Figure 1 is an exploded view of a two component layer of a potentiometric reference sensor that is an embodiment.

[0030] Figure 3 is a top view of an electrically insulating bottom layer of a potentiometric reference sensor.

[0031] Figure 4 is a top view of an electrically insulating reagent holding layer.

[0032] Figure 5 is a magnified cross-sectional view of a potentiometric reference sensor taken along line 5-5 in Figure 1

[0033] Figure 6 is a magnified view of a multi-layer reagent matrix of a potentiometric reference electrode showing an inner layer and a hydrophobic cover film layer.

[0034] Figure 7 is an exemplary top view of a potentiometric reference sensor connected to a flow cell for testing the stability and reproducibility of the junction potential of the reference sensor.

[0035] Figure 8 is a graphical representation showing the stability of the junction potential readings of a potentiometric reference sensor of the present invention versus a double junction reference electrode in various ionic strength solutions. DETAILED DESCRIPTION

[0036] The present invention is shown in Figures 1 to 8 In one embodiment, the single-use disposable potentiometric reference sensor 10 of the present invention is part of a single-use disposable electrochemical sensor 5 made using a 2-layer construction (see Figures 1 to 4 ). The 2-layer construction has a laminate 12 that includes an electrode end portion 14, an electrical contact end portion 16, a working electrode 17, a reference electrode 18 at the electrode end portion 14, and electrical contact pads 16a and 16b at the electrical contact end portion 16. The laminate 12 also includes an electrically insulating bottom layer 20, and an electrically insulating and electrode defining layer 30. All layers of the laminate 12 are made of a dielectric material, preferably a plastic. Examples of preferred dielectric materials are polyvinyl chloride, polycarbonate, polysulfone, nylon, polyurethane, nitrocellulose, cellulose propionate, cellulose acetate, cellulose acetate butyrate, polyester, polyimide, polypropylene, polyethylene, polystyrene, and the like.

[0037] ​The insulating base layer 20 has a conductive layer 21 on which at least two conductive paths 22 and 24 are defined. The conductive paths 22 and 24 can be formed by scribing or scoring the conductive layer 21, or by screen printing the conductive paths 22 and 24 onto the insulating base layer 20. Scribing or scoring of the conductive layer 21 can be accomplished by mechanical scribing of the conductive layer 21 to form non-conductive score lines 28 sufficient to form at least two independent conductive paths 22 and 24. The preferred method of scribing or scoring in accordance with the present application is accomplished by using a carbon dioxide laser, a YAG laser, or an excimer laser. The conductive layer 21 can be made of any conductive material such as, for example, copper, gold, tin oxide / gold, palladium, other noble metals or oxides thereof, or carbon film compositions. The conductive material used in this embodiment is palladium. Acceptable thicknesses of the base layer 20 range from 0.002 inch (0.05 mm) to 0.010 inch (0.25 mm). One such useful material for the base layer 20 is a 0.005 inch (0.125 mm) palladium polyester film (stock number Melinex 329) sold by the Marian Company, Indianapolis, Indiana.

[0038] The insulating and electrode defining layer 30 has at least two openings 32 and 34. The opening 32 exposes a portion of the conductive path 22 and the opening 34 exposes a portion of the conductive path 24 to form a reagent holding well. In this embodiment, the insulating and electrode defining layer 30 is a medical grade single sided adhesive tape / film available from Transcendia Corporation, Franklin Park, Illinois. Acceptable thicknesses of the tape used in the present application range from about 0.001 inch (0.025 mm) to about 0.005 inch (0.13 mm). One such tape / film, stock number PE31280 (about 0.002 inch (0.045 mm)) is used because it is easy to handle and has good performance in the ability to hold sufficient amounts of chemical reagents. It should be understood that the use of tape is not essential. The insulating and electrode defining layer 30 can be made of a plastic sheet and can be coated with a pressure sensitive adhesive, photopolymer, ultrasonically bonded to the base layer 20, screen printed onto the base layer 20, or 3-D printed onto the base layer 20 to achieve the same results as using the polyester tape mentioned.

[0039] The at least two openings 32 and 34 define electrode areas W and R, respectively, forming a working electrode W and a reference electrode R. Typically, the working electrode W is loaded with a reagent matrix deposited directly onto the portion of the conductive layer 21 exposed in the electrode area W, where the reagent matrix is formulated for measuring a particular species in a liquid sample. It is contemplated that a second working electrode, a third working electrode, and more working electrodes can be incorporated into the electrochemical sensor in conjunction with one reference sensor 10. It is also contemplated that the reference sensor can be a separate, independent sensor from any of the one or more working electrodes used to measure a sample liquid, and the reference sensor still functions properly as long as the working electrode and the reference sensor 10 contact the same liquid sample.

[0040] In a combination sensor, the working electrode and the reference electrode are each in electrical contact with separate conductive paths 22 and 24, respectively. The separate conductive paths terminate and are exposed for electrical connection with a reading device on the end of the layer combination 12 opposite the electrode end portion 14.

[0041] The reagent holding openings are preferably made as small as possible while still being able to hold enough chemical reagent for the sensor to function properly. The reagent holding openings in this embodiment are circular in shape and have a diameter of about 0.03 inches (about 0.76 mm). The two reagent holding openings 32, 34 are aligned with each other and spaced about 0.0256 inches (0.65 mm) apart from each other. The circular reagent holding openings are for illustrative purposes only. It should be understood that the shape of the reagent holding openings is not critical, and the size of the openings is driven more by technical feasibility and other manufacturing limitations in dispensing the reagent matrix mixture into the openings.

[0042] The possible electrode arrangement when the reference sensor is coupled with the flow cell should be W-R. If two or more working electrode sensors are included, the arrangement should be W-W-R, where the arrangement listed as the electrode arrangement will occur based on the direction of sample flow through the working electrode sensors W first, and then through the reference electrode sensor R last. In other words, the fluid sample enters the flow cell 70, and the fluid sample will cover the working electrode sensors W first, and then the reference electrode sensor R. This position arrangement is important in this case because if the working electrode sensors are downstream from the reference sensor, the release of KCI ions from the reference sensor can contaminate the working electrode sensors.

[0043] Preferably, the potentiometric reference electrode 18 (electrode well 34) can be loaded with an Ag / AgCl layer (e.g., by applying Ag / AgCl ink or by (a) sputter coating an Ag layer followed by chloridizing the Ag or (b) sputter coating an Ag / AgCl layer) or other reference electrode material that does not require a redox mediator to function properly. Disposed / deposited on the Ag / AgCl layer is a hydrophilic inner layer. The inner layer is an amorphous structure layer as an amorphous salt layer. The amorphous salt layer includes an amorphous polysaccharide and a salt having an isomobility cation and anion.

[0044] Turning now to Figure 3 and Figure 4 , a top view of the bottom layer 20 as well as the insulating and reagent holding layer 30 is shown. As Figure 3 indicated, the symmetry of the conductive paths is such that either longitudinal end of the bottom layer 20 can be designated as an electrode end portion 14 or an electrical contact end portion 16 depending on the orientation of the insulating and reagent holding layer 30 relative to the bottom layer 20 and the assembly process. In this embodiment, the bottom layer 20 has scribe marks in the conductive layer 21 defining two separate conductive paths. It should be understood that the insulating bottom layer can have one, two or more conductive paths, with additional conductive paths can be designated for similar or other analyte sensor reagents, thereby making the electrochemical sensor a multi-analyte sensor.

[0045] Figure 4 is a top view of the insulating and reagent holding layer 30. The insulating and reagent holding layer 30 has two or more openings spaced apart from one another such that each opening coincides with one of the conductive paths defined on the bottom layer 20. It can be clearly understood that if only a reference electrode is made, the insulating and reagent holding layer would include only one conductive path on the bottom layer 20. It should be understood that the conductive paths disclosed herein can be made of any non-corrosive metal. Carbon deposits such as, for example, carbon paste or carbon ink can also be used as the conductive paths, all of which are well known to those of ordinary skill in the art.

[0046] Turning now to Figure 5 , an enlarged cross-sectional view of the reference sensor 10 taken along line 5-5 in Figure 1 is shown. It should be understood that the relative sizes of the layers 20, 30, metal coating 21, electrode well 34, and potentiometric reference electrode reagent matrix 60 are not to scale, but merely to illustrate the various components of the reference sensor 10. As Figure 5 indicated, the insulating bottom layer 20 has a conductive layer 21 disposed thereon and an Ag / AgCl layer 70 formed onto the conductive layer 21. The insulating and reagent holding layer 30 has a reagent holding opening 34 containing a potentiometric reference electrode reagent matrix 60.

[0047] Figure 6is an enlarged view of the potentiometric reference electrode reagent matrix 60. The multi-layer reagent matrix 60 includes a hydrophilic polymer layer 50 and a hydrophobic polymer layer 40. The inner layer 50 includes an amorphous polysaccharide 52 and a salt 54. As the name implies, the hydrophobic cover film layer 40 is not water soluble, but is water vapor and ion permeable.

[0048] The polymer used as the inner layer 50 should have sufficient water solubility and should also be able to stabilize all other chemicals in the reagent to the conductive surface layer 21 in the electrode area. Suitable polymers include, but are not limited to, amorphous polysaccharides (including, but not limited to, pullulan, dextran, starch sugar, etc.). The inner layer 50 can be a single polymer or a combination of polymers, preferably in a concentration range of about 0.02% (w / w) to about 7.0% (w / w). The preferred hydrophilic portion in the inner layer of the present invention is pullulan.

[0049] The inner layer also contains an isokinetic salt such as potassium chloride, potassium nitrate, ammonium chloride, lithium acetate, etc.

[0050] Acceptable polymers for use in the semi-permeable membrane layer include chlorosulfonated polyethylene polymers and cellulose acetate butyrate polymers. The polymer used in the example is chlorosulfonated polyethylene. It is available from Scientific Polymer Products, Ontario, NY, USA.

[0051] Figure 7 is a top view illustration showing the potentiometric reference sensor 10 connected to a flow cell 70 for determining the junction potential of the reference sensor 10. The flow cell 70 has a test chamber 74 and one or more reference electrodes 18 are provided. The test chamber 74 has a test chamber inlet 72 which is connected to a six-way valve 100 to provide five test samples each having a different ionic strength. A predefined amount of each of the five test samples is supplied in series to the test chamber 74 for determining the junction potential of the reference sensor 10 in each of the five test samples. It will be appreciated that the reference sensor 10 is electrically connected to a standard double junction reference electrode 200 and both are connected to appropriate electronics to perform the junction potential measurements.

[0052] Preparation of the inner layer and semi-permeable membrane layer compositions

[0053] The reagent layer composition for creating the hydrophilic mixture of the inner layer is preferably prepared in two steps (although it can be prepared in one step):

[0054] Step 1 : 750 mg of pullulan (amorphous polysaccharide) and 3 ml of 3 mol / L KCI solution are added together.

[0055] Step 2: Mix the components from Step 1 until the amorphous polysaccharide is completely dissolved in the KCI solution.

[0056] The reagent layer composition for creating the covering membrane solution of a semi-permeable membrane is also preferably prepared in two steps (although it can also be prepared in one step):

[0057] Step 1: Add 8-10% by weight of either chlorosulfonated polyethylene polymer or cellulose acetate butyrate to THF / cyclohexanone.

[0058] Step 2: Mix the ingredients from Step 1 together to form a coating solution.

[0059] Sensor Construction

[0060] Assembly of the various embodiments of the present invention is relatively simple. Typically, the insulating bottom layer 20 and the insulating and reagent holding layer 30 are laminated to each other, and then a suitable reagent mixture is dispensed into the reagent holding opening.

[0061] for Figure 1 The two-layer configuration shown, more specifically, involves cutting a single sheet of palladium-coated polyester film (coated only on one side) into the form shown. Figure 2 The shape shown forms the bottom layer 20 of sensor 10. While mechanical scribing is an option, it is preferable to use a laser to scribble the conductive palladium polyester film. Figure 2 As shown, the membrane is laser-etched to form at least two electrode regions at the sample fluid end 14 and at least two contact points 22 and 24 at the electrical contact end 16. If only the potentiometer reference sensor 10 is fabricated, only one electrode region at the sample fluid end 14 and one contact point at the electrical contact end 16 are formed. The etch lines are very fine, but sufficient to create two separate and distinct conductive paths. If only the potentiometer reference sensor 10 is fabricated, optional etch lines can be fabricated along the periphery of the reference sensor 10 to reduce the potential influence of static potential on the reference sensor 10. A single-sided adhesive tape is then cut to a specific size and shape to form an insulating and electrode defining layer 30, such that it will cover most of the conductive layer 21 of the bottom layer 20, except for the exposed portion. Figure 1 The small electrical contact area is shown by reference numeral 16 in the attached figure.

[0062] Before attaching the insulating and electrode defining layer 30 to the bottom layer 20 in the combined sensor, at least two openings 32 and 34 of substantially equal size are punched out by laser or by a mechanical means such as a die assembly, thereby creating openings 32 and 34 in the insulating and electrode defining layer 30. The shape of the electrode openings can be any shape. In the illustrated embodiment, the openings are circular. The preferred hole size of openings 32 and 34 has a typical diameter of about 0.030 inches (0.76 mm), but can be any size.Figure 2 As shown, the electrode openings 32 and 34 are aligned with each other and have a spacing between adjacent openings of about 0.020 inch (0.508 mm) to about 0.050 inch (1.27 mm). The circular openings are for illustrative purposes only. It should be understood that the shape and size of the openings or the distance between them is not critical. The circular openings need not be substantially equal in size as long as the ratio of surface area remains substantially constant. Although the arrangement of electrodes can be any combination, the preferred arrangement of electrodes formed in the openings 32 and 34 is W (working electrode) and R (potentiometric reference electrode) as positioned from the test chamber inlet 72. The insulation and electrode defining layer 30 is then attached to the bottom layer 20 in such a way as to define electrode wells for creating the working electrode W and the reference electrode R. It is contemplated that if only a reference sensor 10 is made, the position of the reference sensor 10 in the sample chamber 70 will be placed in a similar position arrangement as the foregoing position arrangement.

[0063] To create a potentiometric reference sensor, a predefined amount of a hydrophilic mixture is dispensed into the potentiometric reference electrode well 34 to completely cover the Ag / AgCl electrode and dried. For example, it can be air dried at room temperature for a few minutes or dried at 37 °C for a shorter time, forming an inner layer. Drying at a temperature higher than room temperature for a shorter time makes the manufacturing process more efficient. The inner layer mixture and its composition are as described above. During this drying process, as the water in the 3 mol / L KC1 solution evaporates, the amorphous polysaccharide and potassium chloride are deposited onto the metal layer. The amorphous nature of the polysaccharide allows the polysaccharide to be mixed with a high concentration of salt (i.e., supersaturated) without any separation of the polysaccharide and salt when the polysaccharide / salt solution is dried into a uniform inner layer film. This formed inner layer accelerates the dissolution of the salt when in contact with water vapor diffusing through the covering film, which quickly generates ions and starts to compensate the junction potential at the interface of the semi-permeable membrane, while stabilizing the Ag / AgCl potential on the planar array.

[0064] Next, the cover film solution is dispensed onto the inner layer such that the solution completely covers the inner layer. The cover film solution is air dried overnight at room temperature or dried for 30 seconds or more at 37°C. During this process, the cover film components (i.e., chlorosulfonated polyethylene) form a hydrophobic layer that is permeable to water vapor and ions. In the case of this potentiometric reference sensor, when the inner layer is hydrated by the calibrant or sample, water vapor from the sample solution in which the potentiometric reference sensor is exposed diffuses into the inner layer, dissolving the salt, whereby cations and anions from the isopotential salt are transported through the semipermeable membrane layer to the sample solution, thereby preventing a junction potential fluctuation at the sample / cover film interface. As previously discussed, the semipermeable membrane layer allows diffusion of water vapor and ions through the cover film, while the inner layer contains a water-soluble hydrophilic polymer and salt, which forms an electrical connection between the working electrode and the reference electrode in the sensor array when measuring a particular species in the sample, or with the Figure 7 standard dual junction reference electrode as shown.

[0065] The length of time required to dry the reagents depends on the temperature at which the drying process is performed.

[0066] Testing the stability of the junction potential of the potentiometric reference sensor

[0067] One or more potentiometric reference sensors 10 are connected to a flow cell as shown in Figure 7 When a fluid sample is supplied to the potentiometric reference sensor of the present invention as shown in Figure 1 The fluid sample enters the flow cell 70 and flows through the electrodes W and R, and through the dual junction reference electrode 200 and stops for a pre-defined period of time.

[0068] Potentiometry is used to measure the junction potential of the reference sensor 10 using a constant potential device such as a Lawson Labs EMF 16 constant potential device, Malvern, PA. Those potentiometric reference sensors made as shown in Figure 1 The potentiometric reference sensors made as those shown in and described above are used to test the junction potential of the reference sensor 10 of the present invention when exposed to one of various ionic strength solutions (80-200 mmol / L) after being hydrated for an initial 80 seconds.

[0069] Example 1

[0070] Demonstration of the stability and reproducibility of the potentiometric reference electrode junction potential at different ionic strength solution levels

[0071] Liquid samples with different ionic strengths (IS1 through IS5) were used to determine the stability of the junction potential of single-use, disposable reference sensors of the present application. The junction potential was measured using the potentiometric reference sensors of the present application relative to a standard double junction reference electrode. A potentiostat was used to measure the junction potential between the single-use disposable reference sensors 10 of the present application and the standard double junction reference electrode 200. The potentiostat was a Lawson Labs EMF 16 potentiostat, Malvern, PA.

[0072] The procedure included flowing an initial solution of 140 mmol / L ionic strength into the flow cell, to each of the reference sensors being tested as well as the double junction reference electrode and stopping, allowing the sample solution to reside in the flow cell for 80 seconds to hydrate the inner layer of the potentiometric reference sensors. At the end of the 80 second hydration period, the junction potential was measured. Following the initial ionic strength sample, four additional sequential samples each flowed into the flow cell at approximately forty (40) second intervals, stopped and the potential was measured. At approximately every 40 second interval, the junction potential was measured, as shown in Table 1 below.

[0073] In this example, a number of potentiometric reference sensors using a palladium substrate were made for testing the junction potential of the reference sensors. The results are shown in Table 1.

[0074] Table 1

[0075]

[0076] Five disposable potentiometric reference sensors 10 were used to test each ionic strength solution for durations from about 80 seconds to about 240 seconds. The average values were calculated and are shown in Table 1. The standard deviation values for each tested reference sensor are also provided.

[0077] Figure 8 The potentiometric reference electrode / sensor (i.e. inner layer / half permeable cover membrane layer electrode) of the present application was shown to respond to different ionic strength aqueous solutions of 140 mmol / L (IS1), 80 mmol / L (IS2), 100 mmol / L (IS3), 160 mmol / L (IS4) and 200 mmol / L (IS5). As shown in Table 1, the millivolt change in the single-use potentiometric reference sensors appeared to be independent of the ionic strength of the solution. The total measured millivolt change for a particular reference sensor was less than + / - 0.1 mV. Figure 8

[0078] ​The junction potential was relatively stable and appeared to not fluctuate throughout the measurement in the range of the five ionic strength solutions described above. The data indicates that the cover film allows the inner layer to hydrate relatively quickly, a sufficient rate of ion release (which is sufficient to prevent any anomalies in the junction potential of the reference sensor), and to maintain a high ion concentration for a relatively long period of time (approximately 4 minutes) when in contact with an aqueous solution. The data further indicates that the junction potential of one reference sensor 10 to other similar reference sensors 10 is relatively consistent between reference sensors. The difference in junction potential from one reference sensor 10 to another reference sensor 10 is less than + / - 0.33 mV for a given ionic strength solution. This indicates that the potentiometric reference electrode 10 of the present application can be made and used without significant variation in the junction potential from one reference electrode 10 to another reference electrode 10, making it suitable as a single-use, disposable, potentiometric reference sensor.

[0079] Advantages of the present application over prior art single-use potentiometric reference sensors include zero maintenance, accessibility, ease of use, reduced contamination, cost efficiency, rapid analysis, convenience, etc.

[0080] While preferred embodiments of the application have been described herein, the above description is merely illustrative. Modifications of the application disclosed herein will occur to those skilled in the art and all such modifications are believed to be within the scope of the application as defined by the appended claims.

Claims

1. A single-use, disposable potentiometer reference sensor, comprising: Insulating bottom substrate; A reference electrode, wherein the reference electrode is disposed on the insulating bottom substrate, and wherein the reference electrode is a silver-silver chloride electrode; An inner layer disposed on the reference electrode, wherein the inner layer is an amorphous salt layer comprising an amorphous polysaccharide and salts having cations and anions with equal mobility; as well as A semi-permeable covering membrane is disposed on the inner layer, and the semi-permeable covering membrane has water vapor permeability and ion permeability.

2. The single-use disposable potentiometer reference sensor as claimed in claim 1, wherein the amorphous polysaccharide has amorphous properties such that when supersaturated with the salt, the amorphous polysaccharide and the salt do not separate during the formation of the inner layer.

3. The single-use disposable potentiometer reference sensor as described in claim 1, wherein the amorphous polysaccharide is selected from the group consisting of pullulan, dextran, and starch sugar.

4. The single-use disposable potentiometer reference sensor as described in claim 1, wherein the salt is potassium chloride.

5. The single-use disposable potentiometer reference sensor as claimed in claim 1, wherein the semi-permeable covering membrane is made of one of chlorosulfonated polyethylene or cellulose acetate butyrate.

6. A single-use, disposable electrochemical sensor, comprising: An insulating bottom substrate with a sensing surface; A potentiometer working electrode is formed on the sensing surface, wherein the working electrode has a species-specific reagent matrix disposed thereon, wherein the species-specific reagent matrix has one or more layers selected for measuring a specific species in a liquid sample; as well as A potentiometer reference electrode is formed on the sensing surface, wherein the reference electrode is a silver-silver chloride electrode having a multilayer reference coating thereon, the coating comprising: Inner layer, wherein the inner layer is an amorphous salt layer comprising amorphous polysaccharides and salts having cations and anions with equal mobility; as well as A semi-permeable covering membrane is disposed on the inner layer, and the semi-permeable covering membrane has water vapor permeability and ion permeability.

7. The single-use, disposable electrochemical sensor of claim 6, wherein the amorphous polysaccharide has amorphous properties such that when supersaturated with the salt, the amorphous polysaccharide and the salt do not separate during the formation of the inner layer.

8. The single-use, disposable electrochemical sensor of claim 6, wherein the amorphous polysaccharide is selected from the group consisting of pullulan, dextran, and starch sugar.

9. The single-use, disposable electrochemical sensor of claim 6, wherein the salt is potassium chloride.

10. The single-use, disposable electrochemical sensor of claim 6, wherein the semi-permeable covering membrane is made of chlorosulfonated polyethylene or cellulose acetate butyrate.

11. A method for forming a single-use, disposable electrochemical potentiometer reference sensor, comprising: A sensor body is provided, the sensor body having an insulating bottom substrate with at least one conductive path and an insulating and reagent holding layer disposed on the insulating bottom substrate, wherein the insulating and reagent holding layer has at least one reagent holding opening, wherein the at least one reagent holding opening exposes a portion of the at least one conductive path; An amorphous salt layer mixture comprising an amorphous polysaccharide and salts having cations and anions with equal mobility is disposed in one of the at least one reagent holding opening; The amorphous salt layer mixture is dried to form an inner layer as an amorphous structural layer. A coating film solution containing a hydrophobic polymer is disposed on the inner layer; and The coating solution is dried to form a semi-permeable coating membrane, wherein the semi-permeable coating membrane is permeable to water vapor and ions.

12. The method of claim 11, further comprising: The amorphous salt layer mixture comprising: Multiple components containing predetermined amounts of amorphous polysaccharides and predetermined amounts of 3M salt solution are added together; and The various components are mixed to form the amorphous salt layer mixture.

13. The method of claim 12, further comprising: Weigh 750 mg of the amorphous polysaccharide. as well as Weigh 3 ml of the 3M salt solution.

14. A multilayer reagent matrix for fabricating a reference electrode into a reference sensor, the reagent matrix comprising: An inner layer formed of an amorphous salt layer mixture comprising an amorphous polysaccharide and a salt, the inner layer covering the reference electrode, wherein the amorphous polysaccharide has amorphous properties such that when supersaturated with the salt, the amorphous polysaccharide and the salt do not separate during the formation of the inner layer; as well as A semi-permeable cover membrane formed from a cover membrane solution, the semi-permeable cover membrane being disposed on the inner layer, the cover membrane solution comprising a hydrophobic polymer, wherein the hydrophobic polymer is permeable to water vapor and ions.

15. The reagent matrix of claim 14, wherein the inner layer is a mixture of potassium chloride and at least one of pullulan, dextran and starch sugar.

16. The reagent matrix of claim 14, wherein the semi-permeable covering membrane is made of chlorosulfonated polyethylene or cellulose acetate butyrate.

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