Single-use disposable reference sensor

Abstract

To provide a single-use potentiometric reference sensor that is dependable by providing a stable potential and is not susceptible to environmental factors.SOLUTION: A single-use disposable potentiometric reference sensor includes an insulating base substrate 20, a reference electrode 70 disposed on the insulating base substrate, where the reference electrode is a silver-silver chloride electrode, a potentiometric reference electrode reagent matrix 60 disposed on the reference electrode, where the potentiometric reference electrode reagent matrix is an amorphous salt layer that includes an amorphous polysaccharide and a salt having equi-mobility cations and anions, and a semipermeable cover membrane disposed over the potentiometric reference electrode reagent matrix, where the semipermeable cover membrane has water vapor and ion permeability.SELECTED DRAWING: Figure 5

Description

Detailed Description of the Invention

[0001] [Background of the invention] 1. Field of the invention The present invention relates generally to electrochemical sensors. In particular, the present invention relates to electrochemical potentiometric reference sensors. More specifically, the present invention relates to planar electrochemical potentiometric reference sensors comprising a membrane coating.

[0002] 2. Description of the Prior Art A basic prior art electrochemical sensor typically consists of an electrochemical cell with two electrodes. The first electrode responds to chemical species in the liquid sample and is commonly referred to as the indicator electrode. The second electrode is a reference electrode that is insensitive to changes in the composition of the liquid sample and provides a constant potential against which the potential developed by the indicator electrode from the liquid sample is measured.

[0003] Traditionally, chemical analyzers for quantitatively measuring chemical species in liquid samples, including blood, typically have very complex fluidic structures for cleaning and calibration of sensors for multiple uses. For this reason, manufacturers of such chemical analyzers have attempted to produce relatively inexpensive sensors, resulting in the analyzers being used as single-use devices. A suitable technology for such sensor devices is planar technology. Sensors produced by planar technology have included both thick-film and thin-film technologies.

[0004] A typical component of the structure of a prior art planar electrochemical sensor is a device comprising a plurality of metallic conductive elements on a planar insulating substrate. Prior art planar electrochemical sensors consist of layers covering the metallic conductive elements, one end of which is exposed for connection to an external measurement circuit and the other end of which is exposed for receiving a plurality of coatings forming an integral electrolyte layer. This integral electrolyte layer includes a hydrophilic layer, such as a gel material, which acts as an aqueous electrolyte, and other reagents selected to measure a particular species in the liquid sample. Chemical species from the liquid sample undergo an electrochemical reaction on the electrode surface which generates a current or voltage. The generated current or voltage is typically proportional to the concentration of the chemical species in the provided liquid sample, while the reference electrode remains stable throughout the measurement process.

[0005] To provide an electrode that is useful for many processes, such as for blood analysis work in hospitals, blood chemistry laboratories, etc., it is desirable to provide a disposable electrode that is small, can be stored for a long time, and is economically inexpensive. Long-term storage is difficult to achieve because most of the prior art electrodes employ hydrophilic or aqueous reference electrodes. Hydrophilic electrolytes are hydrated gels that allow ion transport. The shipping and storage of "wet" electrolytes requires relatively complex packaging.

[0006] A salt bridge potentiometric reference electrode consists of a silver / silver chloride (Ag / AgCl) based electrode in contact with a high concentration aqueous salt solution, preferably an isomotile salt such as potassium chloride. The high concentration of chloride ions saturates the Ag / AgCl potential, while the isomotile potassium and chloride prevent the development of a liquid junction potential at the interface between the reference sensor and the sample. The amount of salt in the array reservoir is important to maintain a stable reference potential over time. In planar sensor arrays, the size of the salt reservoir is very limited and can be washed away immediately (i.e., in less than 1 second) upon contact with the aqueous solution. There have been many efforts to solve this problem, and various cover membranes have been tried, but without much success. Previous attempts have failed mainly due to the low water vapor diffusivity of the hydrophobic cover membrane polymer, which causes the salt to permeate through the membrane too slowly (or too quickly).

[0007] Currently, several different technologies for single-use potentiometric reference sensors have been reported. U.S. Patent No. 4,933,048 (Lauks, 1990) discloses an open-contact reference electrode assembly. The reference electrode assembly includes a metallic member coated with an electrode material that is reversible with ion X, and a layer of electrolyte containing ion X formed on the electrode. The electrolyte extends beyond the periphery of the electrode. The portion of the electrolyte that extends beyond the periphery of the electrode is charged with H 2The electrode is covered by a membrane that is permeable to O molecules but not to ions X. A portion of the electrolyte may extend through the permeable membrane or otherwise form a liquid junction with the sample solution at a location relatively far from the electrode. Thus, while ions must diffuse along a relatively long path through the electrolyte between the liquid junction and the electrode, providing a long time constant for ion diffusion, the electrolyte can "wet" relatively quickly. As a result, there is a period of time after the electrolyte is wetted before ion diffusion affects the ion concentration near the electrode, during which the electrode potential is substantially constant. The electrolyte consists of a high concentration of salt containing a hydrophilic polymer membrane as an inner layer, partially covered by a hydrophobic membrane that can directly expose a small portion of the inner layer to the sample solution. To achieve this type of configuration, the process of sensor fabrication is complex, which can increase the sensor failure rate.

[0008] US Patent No. 7,767,068 (Lauks et al., 2010) discloses a heterogeneous membrane electrode. The heterogeneous membrane consists of a mixture of oil and aqueous compartments. The aqueous part is made of salts and redox couples containing cross-linkable hydrophilic polymers. The oil part is composed of cross-linkable hydrophobic polymers. This mixture is emulsified to support the manufacturing process using either dispensing or printing. The next step requires settling, degassing and finally UV curing of the deposited layer to fix all compartments. This procedure is a complex time-dependent process due to the phase separation properties of the heterogeneous membrane, which can induce variability between sensors.

[0009] [Summary of the Invention] It is widely known that potentiometric reference electrodes must be reliable by providing a stable potential and be insensitive to environmental factors. All potentiometric reference electrodes have a liquid junction potential. This is the boundary / interface potential that occurs between the reference electrode and the sample. It is essential that all potentiometric reference electrodes have a liquid junction potential, if any, that is relatively constant and is not affected by the temperature or local chemical composition around the reference electrode.

[0010] The integration of electrochemical potentiometric reference electrodes within a planar sensor array is a significant challenge in single-use planar sensors.

[0011] Given that the concentrations of various electrolytes in a blood sample are likely to vary depending on the health of the animal from which the blood sample was taken, it is important that any single-use potentiometric reference electrode incorporated into a planar sensor array have a relatively constant and reproducible liquid junction potential. As discussed above, planar sensor arrays include potentiometric reference electrodes with salt reservoirs of very limited size. To prevent the limited size of the salt reservoir from being washed away quickly when exposed to a blood sample, it is necessary to incorporate a hydrophobic component into the potentiometric reference electrode to prevent it from being washed away or to delay for the time of any change in the reference potential or liquid junction potential of the reference electrode while the blood sample measurement is being taken.

[0012] It is an object of the present invention to provide a disposable, single-use electrochemical potentiometric reference electrode / sensor. Another object of the present invention is to provide a disposable, single-use potentiometric reference electrode / sensor that can be stored for relatively long periods. Yet another object of the present invention is to provide an electrochemical potentiometric reference electrode / sensor that can be stored for relatively long periods. Objective To provide a chemical potentiometric reference electrode / sensor.

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

[0014] In one embodiment, a single-use disposable potentiometric reference sensor includes an insulating base substrate, a reference electrode disposed on the insulating base substrate and being a silver-silver chloride electrode, an inner layer disposed on the reference electrode and being an amorphous salt layer including an amorphous polysaccharide and a salt having isomobility cations and anions, and a semipermeable cover membrane disposed over the inner layer and having water vapor and ion permeability.

[0015] In one embodiment, the amorphous polysaccharide has amorphous properties such that when supersaturated with the salt, separation of the amorphous polysaccharide and the salt does not occur when the inner layer is formed.

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

[0017] In one embodiment, the salt is one of potassium chloride, ammonium chloride, potassium nitrate, lithium acetate, and the like, in which the cations and anions have approximately equal mobility.

[0018] In one embodiment, the semipermeable cover membrane is made from a chlorosulfonated polyethylene polymer or a cellulose acetate butyrate polymer.

[0019] In another embodiment, a single-use disposable electrochemical sensor comprises an insulating base substrate having a sensor surface, a potentiometric working electrode formed on the sensor surface, the potentiometric working electrode having a species-specific reagent matrix disposed thereon, the species-specific reagent matrix having one or more layers selected for measuring a particular species in a liquid sample, and a potentiometric reference electrode formed on the sensor surface, the potentiometric reference electrode being a silver-silver chloride electrode having a multi-layer reference coating thereon, the multi-layer reference coating comprising an inner layer disposed on the Ag / AgCl reference electrode, the inner layer comprising an amorphous polysaccharide and a salt having isomobile cations and anions, and a semi-permeable cover membrane disposed over the inner layer, the semi-permeable cover membrane being a semi-permeable hydrophobic polymer, the semi-permeable cover membrane having 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 conductive pathway and an insulating reagent retaining layer disposed on the insulating base substrate, the insulating reagent retaining layer on the insulating base substrate having at least one reagent retaining opening, the at least one reagent retaining opening exposing a portion of the at least one conductive pathway; disposing an amorphous salt layer mixture containing an amorphous polysaccharide and a salt having isomobility cations and anions within one of the at least one reagent retaining openings; drying the amorphous salt layer mixture to form an inner layer; disposing a cover membrane solution containing a hydrophobic polymer over the inner hydrophilic layer; and drying the cover membrane solution to form a semipermeable cover membrane, the semipermeable cover membrane having 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 quantity of an amorphous polysaccharide and a quantity of a 3 mol / L salt solution and mixing the components to form the amorphous salt layer mixture.

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

[0023] In one embodiment, the cover membrane solution is formed by measuring a predetermined amount of a hydrophobic polymer, which is one of chlorosulfonated polyethylene and cellulose acetate butyrate, and mixing the hydrophobic polymer in a predetermined amount of THF / cyclohexanone to form the cover membrane solution.

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

[0025] In yet another 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 conductive pathway and an insulating reagent retaining layer disposed on the insulating base substrate, the insulating reagent retaining layer having at least one reagent retaining opening, the at least one reagent retaining opening exposing a portion of the at least one conductive pathway; disposing an amorphous salt layer mixture containing an amorphous polysaccharide and a salt having isomobility cations and anions within one of the at least one reagent retaining openings; drying the amorphous salt layer mixture to form the inner layer; disposing a cover membrane solution containing a hydrophobic polymer over the hydrophilic inner layer; and drying the cover membrane solution to form a semipermeable cover membrane, the semipermeable cover membrane being water vapor permeable and ion permeable.

[0026] In one embodiment, a multi-layered reagent matrix for forming a potentiometric reference electrode in a single-use disposable reference sensor is disclosed. The multi-layered matrix includes an inner layer formed from an amorphous salt layer mixture containing an amorphous polysaccharide and a salt, the inner layer overlying an Ag / AgCl reference electrode. The amorphous polysaccharide has amorphous properties such that when supersaturated with the salt, the amorphous polysaccharide and the salt do not separate when the inner layer is formed. A hydrophobic cover membrane made from a cover membrane solution is disposed over the inner layer, the cover membrane solution including a hydrophobic polymer, and the hydrophobic semi-permeable membrane 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 amylose. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a perspective view of one embodiment of the present invention showing a potentiometric reference sensor. [Diagram 2] FIG. 2 is an exploded view of the embodiment of FIG. 1 showing the two constituent layers of the potentiometric reference sensor. [Diagram 3] FIG. 2 is a top view of the electrically insulating base layer of the potentiometric reference sensor. [Figure 4] FIG. 2 is a top view of an electrically insulating reagent retaining layer. [Diagram 5] FIG. 5 is an enlarged cross-sectional view of the potentiometric reference sensor taken along line 5-5 of FIG. 1. [Figure 6] FIG. 2 is an enlarged view of the multi-layered reagent matrix of the potentiometric reference electrode showing the inner layer and the hydrophobic cover membrane layer. [Figure 7] FIG. 13 is an exemplary top view of a potentiometric reference sensor connected to a flow cell to test the stability and reproducibility of the reference sensor's liquid junction potential. [Figure 8] FIG. 1 is a graphical representation showing the stability of liquid 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 OF THE PREFERRED EMBODIMENTS

[0029] The present invention is illustrated in Figures 1-8. In one embodiment, the disposable single-use potentiometric reference sensor 10 of the present invention is part of a disposable single-use electrochemical sensor 5 made using a two-layer configuration (see Figures 1-4). The two-layer configuration has a laminate 12 that includes an electrode end 14, an electrical contact end 16, a working electrode 17, a reference electrode 18 at the electrode end 14, and electrical contact pads 16a and 16b at the electrical contact end 16. The laminate 12 also includes an electrically insulating base layer 20 and an electrically insulating 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, cellulose nitrate, cellulose propionate, cellulose acetate, cellulose acetate butyrate, polyester, polyimide, polypropylene, polyethylene, polystyrene, and the like.

[0030] 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 may be formed by scribing or scoring the conductive layer 21 or by silk-screening the conductive paths 22 and 24 onto the insulating base layer 20. Scribing or scoring the conductive layer 21 may be performed by mechanically scribing the conductive layer 21, which may produce sufficient non-conductive score lines 28 to produce at least two independent conductive paths 22 and 24. A preferred scribing or scoring method of the present invention is performed using a carbon dioxide laser, a YAG laser, or an excimer laser. The conductive layer 21 may be made of any conductive material, such as copper, gold, tin oxide / gold, palladium, other precious metals or their oxides, or carbon film compositions. The conductive material used in this embodiment is palladium. Acceptable thicknesses for the substrate 20 range from 0.002 inch (0.05 mm) to 0.010 inch (0.25 mm). One such material that can be used for the substrate 20 is a 0.005 inch (0.125 mm) palladium polyester film (product number Melinex 329) available from Marian Corporation of Indianapolis, Indiana.

[0031] The insulated electrode-defining layer 30 includes at least two openings 32 and 34. Opening 32 exposes a portion of conductive pathway 22, and opening 34 exposes a portion of conductive pathway 24 to create a reagent-holding well. In this embodiment, the insulated electrode-defining layer 30 is a medical grade single-sided adhesive tape / film available from Transcendia, Inc., Franklin Park, Illinois. Acceptable thicknesses of tape for use in the present invention range from about 0.001 inch (0.025 mm) to about 0.005 inch (0.13 mm). One such tape / film, part number PE31280 (about 0.002 inch (0.045 mm)), is used due to its ease of handling and good performance in terms of its ability to hold sufficient amounts of chemical reagents. It should be understood that the use of tape is not required. The insulated electrode-defining layer 30 may be made from a plastic sheet and may be coated with a pressure sensitive adhesive or photopolymer, ultrasonically bonded to the base layer 20, silk screened onto the base layer 20, or 3D printed onto the base layer 20 to achieve the same results as using the polyester tape described above.

[0032] 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 carries a reagent matrix deposited directly on the portion of the conductive layer 21 exposed to the electrode area W. In the electrode area W, the reagent matrix is ​​formulated for measuring a specific species in the liquid sample. It is also contemplated that a second, third, or more working electrodes may be mounted on the electrochemical sensor in combination with a reference sensor 10. It is also contemplated that the reference sensor may be a separate and independent sensor from any one of the one or more working electrodes for measuring the sample liquid and still function properly provided that the working electrode and the reference sensor 10 contact the same liquid sample.

[0033] In the combination sensor, the working and reference electrodes are in electrical contact with separate conductive paths 22 and 24, respectively, that are terminated and exposed for electrical connection to a reader at the end of the laminate 12 opposite the electrode end 14.

[0034] The size of the reagent holding opening is preferably as small as possible while still being able to hold enough chemical reagent for the sensor to function properly. The shape of the reagent holding opening is circular in this embodiment, with 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 apart by about 0.0256 inches (0.65 mm). The fact that the reagent holding openings are circular is 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 will further depend on the technical feasibility of dispensing the reagent matrix mixture into the openings and other manufacturing limitations.

[0035] A possible electrode configuration when a reference sensor is coupled with the flow cell should be WR. If more than one working electrode sensor is included, the configuration should be WWR, with the order appearing based on the sample flow direction crossing the working electrode sensor W first and the reference electrode sensor R last. In other words, as the fluid sample enters the flow cell 70, it first covers the working electrode sensor W and then the reference electrode sensor R. In this case, the positional configuration is important because if the working electrode sensor is located downstream of the reference sensor, the release of KCl ions from the reference sensor may contaminate the working electrode sensor.

[0036] Preferably, the potentiometric reference electrode 18 (electrode well 34) carries an Ag / AgCl layer (e.g., by applying an Ag / AgCl ink or by (a) sputter coating an Ag layer followed by chlorinating the Ag or (b) sputter coating an Ag / AgCl layer), or may carry other reference electrode materials that do 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 structural layer that is an amorphous salt layer. The amorphous salt layer comprises an amorphous polysaccharide and a salt with isomobile cations and anions.

[0037] 3 and 4, there is shown a top view of the base layer 20 and the insulating reagent holding layer 30. As shown in FIG. 3, the symmetry of the conductive pathways is such that either longitudinal end of the base layer 20 may be designated as either an electrode end 14 or an electrical contact end 16 depending on the orientation of the base layer 20 and the insulating reagent holding layer 30 relative to the assembly process. In this embodiment, the base layer 20 has scribe marks in the conductive layer 21 that define two separate conductive pathways. It should be understood that the insulating base layer may have one, two, or more conductive pathways and that the additional conductive pathways may be designated with similar or other analyte sensor reagents, making the electrochemical sensor a multi-analyte sensor.

[0038] 4 is a top view of the insulating reagent-holding layer 30. The insulating reagent-holding layer 30 has two or more openings, which are spaced apart from one another and each coincide with one of the conductive paths defined on the base layer 20. It is clearly understood that if only a reference electrode is fabricated, the insulating reagent-holding layer will only include one conductive path on the base layer 20. It is understood that the conductive path(s) disclosed herein may be made from any non-corroding metal. Carbon deposits, such as carbon paste or carbon ink, may be used as conductive paths, all of which are well known to those skilled in the art.

[0039] 5, there is shown an enlarged cross-sectional view of the reference sensor 10 taken along line 5-5 of FIG. 1. Layers 20, 30, metal coating 21, electrode well 34, potentiometric reference electrode It should be understood that the relative sizes of the reagent matrix 60 are not actual sizes, but are merely indicative of the various components of the reference sensor 10. As seen in Figure 5, the insulating base layer 20 has a conductive layer 21 disposed thereon and an Ag / AgCl layer 70 formed on the conductive layer 21. The insulating reagent retaining layer 30 has a reagent retaining opening 34 which contains the potentiometric reference electrode reagent matrix 60.

[0040] 6 is an expanded view of a potentiometric reference electrode reagent matrix 60. The multi-layered 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. The hydrophobic cover membrane layer 40, as the name suggests, is not water soluble but is permeable to water vapor and ions.

[0041] The polymer used as the inner layer 50 should be sufficiently soluble in water and capable of stabilizing all other chemicals in the reagent to the conductive surface layer 21 in the electrode region. Suitable polymers include, but are not limited to, amorphous polysaccharides, including, but not limited to, pullulan, dextran, amylose, and the like. The inner layer 50 may be a single polymer or a combination of polymers, preferably in a concentration range of about 0.02% (by weight) to about 7.0% (by weight). The preferred hydrophilic moiety in the inner layer of the present invention is pullulan.

[0042] The inner layer also contains isomobile salts such as potassium chloride, potassium nitrate, ammonium chloride, and lithium acetate.

[0043] Acceptable polymers for use in the semipermeable membrane layer include chlorosulfonated polyethylene polymers and cellulose acetate butyrate polymers. The polymer used in the examples is chlorosulfonated polyethylene, available from Scientific Polymer Products, Inc., Ontario, NY, USA.

[0044] 7 is a top view showing a potentiometric reference sensor 10 connected to a flow cell 70 for determining the liquid junction potential of the reference sensor 10. The flow cell 70 has a test chamber 74 therein in which one or more reference electrodes 18 are disposed. The test chamber 74 has a test chamber inlet 72 connected to a six-way valve 100 to provide five test samples, each having a different ionic strength. A predetermined amount of each of the five test samples is sequentially delivered to the test chamber 74 to determine the liquid junction potential of the reference sensor(s) 10 in each of the five test samples. It should be understood that the reference sensor 10 is electrically connected to a standard double junction reference electrode 200, both of which are connected to suitable electronics to perform the liquid junction potential measurements.

[0045] Preparation of Inner and Semipermeable Membrane Layer Compositions The reagent layer composition for the hydrophilic mixture used to produce the inner layer is preferably prepared in two steps, but may also be prepared in one step.

[0046] Step 1: Add 750 mg of pullulan (amorphous polysaccharide) and 3 ml of 3 mol / L KCl solution together.

[0047] Step 2: Mix the ingredients from step 1 above until the amorphous polysaccharide is completely dissolved in the KCl solution.

[0048] The reagent layer composition for the cover membrane solution used to prepare the semipermeable membrane is also preferably prepared in two steps, but may be prepared in one step.

[0049] Step 1: Add 8-10% by weight of one of chlorosulfonated polyethylene polymer and cellulose acetate butyrate to THF / cyclohexanone.

[0050] Step 2: Mix the ingredients from Step 1 together to make the cover membrane solution.

[0051] Sensor structure Assembly of the various embodiments of the present invention is relatively simple: generally, after the insulating base layer 20 and insulating reagent holding layer 30 are laminated together, a suitable reagent mixture is dispensed into the reagent holding opening.

[0052] More specifically, in the two-layer configuration shown in FIG. 1, a piece of palladium coated polyester film (coated on only one side) is cut to the shape shown in FIG. 2 to form the base layer 20 of the sensor 10. Although mechanical scribing is optional, preferably a laser is used to scribe the conductive palladium polyester film. As shown in FIG. 2, the film is scribed with a laser to create at least two electrode areas at the sample fluid end 14 and at least two contact points 22 and 24 at the electrical contact end 16. If only a potentiometric reference sensor 10 is being made, only one electrode area at the sample fluid end 14 and one contact point at the electrical contact end 16 are created. The scribe lines are very thin, but sufficient to create two separate and distinct conductive paths. If only a potentiometric reference sensor 10 is being made, an optional scribe line may be made along the perimeter of the reference sensor 10 to reduce the possibility of electrostatic potential effects on the reference sensor 10. A piece of single-sided adhesive tape is then cut to a predetermined size and shape, thereby forming an insulated electrode-defining layer 30 that covers most of the conductive layer 21 of the base layer 20 except for exposing a small electrical contact area, shown in FIG. 1 by reference numeral 16.

[0053] Prior to attaching the insulated electrode-defining layer 30 to the base layer 20 in the combined sensor, at least two openings 32 and 34 of approximately the same size are drilled by mechanical means, such as a laser or a die punch assembly, to form the electrode openings 32 and 34 in the insulated electrode-defining layer 30. The electrode openings may be of any shape. In the illustrated embodiment, the openings are circular. A preferred hole size for the openings 32 and 34 is typically about 0.030 inches (0.76 mm) in diameter, but may be any size. As shown in FIG. 2, the electrode openings 32 and 34 are aligned with one another and have a spacing between adjacent openings of about 0.020 inches (0.508 mm) to about 0.050 inches (1.27 mm). The circular shape of the openings is for illustrative purposes only. It should be understood that the shape and size of the openings, and the distance between the openings, is not critical. The circular openings do not need to be approximately the same in dimensions, so long as the surface area ratio remains approximately constant. While the electrode arrangement may be any combination, a preferred arrangement of electrodes formed in openings 32 and 34 is W (working electrode) and R (potentiometric reference electrode) positioned from test chamber entrance 72. Insulated electrode-defining layer 30 is then attached to base layer 20 to define electrode wells for creating working electrode W and reference electrode R. If only reference sensor 10 is fabricated, it would be contemplated that the location of reference sensor 10 within sample chamber 70 would be similar to the positional arrangement as previously described.

[0054] To prepare the potentiometric reference sensor, a predetermined amount of the hydrophilic mixture is dispensed into the potentiometric reference electrode well 34, completely covering the Ag / AgCl electrode and allowed to dry, e.g., by air drying at room temperature for several minutes or by allowing to dry at 37 o C for a shorter period to form the inner layer. Drying at temperatures above room temperature for a shorter period allows for a more efficient manufacturing process. The inner layer mixture and its composition are described above. During this drying process, amorphous polysaccharide(s) and potassium chloride are deposited on top of the metal layer as water evaporates from the 3 mol / L KCl solution. The amorphous nature of the polysaccharide(s) is due to the fact that the polysaccharide(s) / salt solution dries. When the polysaccharide(s) is mixed with a high concentration of salt (i.e., supersaturated) without separation of the polysaccharide(s) and the salt, a uniform inner layer is formed. When the inner layer thus formed comes into contact with water vapor diffusing through the cover membrane, it accelerates the dissolution of the salt, rapidly generates ions, and begins to compensate for the liquid junction potential at the interface of the semipermeable membrane and stabilizes the potential of Ag / AgCl on the planar array.

[0055] The cover membrane solution is then dispensed onto the inner layer, ensuring that the solution completely covers the inner layer. The cover membrane solution is allowed to air dry at room temperature overnight or at 37 o C for at least 30 seconds. During this process, the components of the cover membrane (i.e., chlorosulfonated polyethylene) form a hydrophobic layer that is permeable to water vapor and ions. In the case of this potentiometric reference sensor, water vapor diffuses from the sample solution to which the potentiometric reference sensor is exposed into the salt-dissolved inner layer, which transports cations and anions from the isomobility salts through the semipermeable membrane layer into the sample solution, preventing fluctuations in the liquid junction potential at the sample / cover membrane interface when the inner layer is hydrated with a calibrant or sample. As previously described, the semipermeable membrane layer allows diffusion of water vapor and ions through the cover membrane, while the inner layer contains water-soluble hydrophilic polymers and salts, which provide electrical connection between the working and reference electrodes in a sensor array when measuring a particular species in a sample, or with a standard double junction reference electrode as shown in FIG. 7 when determining the stability of the potentiometric reference sensor liquid junction potential.

[0056] The amount of time required to dry the reagents depends on the temperature at which the drying process is carried out.

[0057] Stability Test of Liquid Junction Potential of Potentiometric Reference Sensors One or more potentiometric reference sensors 10, along with a standard double junction reference electrode 200, are connected to a flow cell as shown in Figure 7. When a fluid sample is applied to the potentiometric reference sensor of the present invention shown in Figure 1, the fluid sample enters the flow cell 70, flows past electrodes W and R, crosses the double junction reference electrode 200, and is stopped for a predetermined period of time.

[0058] A potentiostat, such as an EMF16 potentiostat from Lawson Labs, Malvern, Pa., was used to measure the liquid junction potential of reference sensor 10. A potentiometric reference sensor constructed similarly to that shown in FIG. 1 and described above was used to test the liquid junction potential of reference sensor 10 of the present invention when exposed to various ionic strength solutions (80-200 mmol / L) after an initial 80-second hydration in one of the various ionic strength solutions.

[0059] Example 1 Demonstration of stability and reproducibility of liquid junction potentials of potentiometric reference electrodes at various ionic strength solution levels. The stability of the liquid junction potential of the single-use disposable reference sensor of the present invention was determined using liquid samples of various ionic strengths (IS1 to IS5). The liquid junction potential was measured using a potentiometric reference sensor of the present invention against a standard double junction reference electrode. A potentiostat was used to measure the liquid junction potential between the disposable single-use reference sensor 10 of the present invention and a standard double junction reference electrode 200. The potentiostat was an EMF16 potentiostat from Lawson Labs, Malvern, PA.

[0060] The procedure involved flowing an initial solution with an ionic strength of 140 mmol / L through the flow cell and then through the double junction reference electrode along with each reference sensor being tested, allowing the sample solution to reside in the flow cell for 80 seconds to hydrate the inner layer of the potentiometric reference sensor. At the end of the 80 second hydration period, the liquid junction potential was measured. Following the ion strength sample, four additional consecutive samples were each flowed through the flow cell at approximately 40 second intervals and stopped to measure the potential. The liquid junction potentials at each approximately 40 second interval were measured as shown in Table 1 below.

[0061] In this example, several potentiometric reference sensors using palladium substrates were fabricated to test the liquid junction potential of the reference sensors. The results are shown in Table 1.

[0062] [Table 1] Each ionic strength solution was tested with five disposable potentiometric reference sensors 10 for times ranging from about 80 seconds to about 240 seconds. Mean values ​​were calculated and are shown in Table 1. Standard deviation values ​​for each reference sensor tested are also provided.

[0063] FIG. 8 shows the liquid junction potential response of a potentiometric reference electrode / sensor of the present invention (i.e., inner layer / semipermeable cover membrane layer electrode) to aqueous solutions of various ionic strengths: 140 mmol / L (IS1), 80 mmol / L (IS2), 100 mmol / L (IS3), 160 mmol / L (IS4), and 200 mmol / L (IS5). As shown in FIG. 8, the millivolt changes in the single-use potentiometric reference sensor do not appear to be related to the ionic strength of the solution. All millivolt variations measured for a particular reference sensor were less than + / - 0.1 mV.

[0064] The liquid junction potential appears to be relatively stable and does not vary over the measurements in the range of five ionic strength solutions mentioned above. The data indicates that the cover membrane provides relatively rapid hydration of the inner layer and sufficient ion release rate (fast enough to prevent abnormalities in the liquid junction potential of the reference sensor) to maintain a high ion concentration for a relatively long time (about 4 minutes) when in contact with an aqueous solution. The data further indicates that the liquid junction potential from one reference sensor 10 to another similar reference sensor 10 is relatively consistent between reference sensors. The liquid junction potential difference from one reference sensor 10 to another reference sensor 10 for a given ionic strength solution is less than + / - 0.33 mV. This indicates that the potentiometric reference electrode 10 of the present invention can be made and used without significant changes in the liquid junction potential from one reference sensor 10 to another reference sensor 10, making it suitable as a single-use disposable potentiometric reference sensor.

[0065] Advantages of the present invention over prior art single-use potentiometric reference sensors include zero maintenance, ready availability, ease of use, reduced contamination, cost-effectiveness, rapid analysis, convenience, and the like.

[0066] While preferred embodiments of the present invention have been described herein, the foregoing are by way of example only. Those skilled in the art will naturally envision further modifications of the invention disclosed herein, and all such modifications are deemed to be within the scope of the invention as defined by the appended claims.

Claims

1. 1. A single-use disposable potentiometric reference sensor comprising: An insulating base substrate; a reference electrode disposed on the insulating base substrate and being a silver-silver chloride electrode; an inner layer disposed on the reference electrode, the inner layer being an amorphous salt layer comprising an amorphous polysaccharide and a salt having isomobility cations and anions; and a semipermeable cover membrane disposed to cover the inner layer, the semipermeable cover membrane having water vapor permeability and ion permeability. Single use disposable potentiometric reference sensor.

2. The amorphous polysaccharide, when supersaturated with the salt, has amorphous properties such that the amorphous polysaccharide and the salt do not separate when the inner layer is formed.

10. The single-use disposable potentiometric reference sensor of claim 1.

3. The amorphous polysaccharide is selected from the group consisting of pullulan, dextran, and amylose.

10. The single-use disposable potentiometric reference sensor of claim 1.

4. The salt is potassium chloride.

10. The single-use disposable potentiometric reference sensor of claim 1.

5. The semipermeable cover membrane is made from one of chlorosulfonated polyethylene and cellulose acetate butyrate.

10. The single-use disposable potentiometric reference sensor of claim 1.

6. 1. A disposable, single-use electrochemical sensor comprising: an insulating base substrate having a sensor surface; a potentiometric working electrode formed on the sensor surface, the potentiometric working electrode having a species-specific reagent matrix disposed thereon, the species-specific reagent matrix having one or more layers selected for measuring specific species in a liquid sample; a potentiometric reference electrode formed on the sensor surface, the potentiometric reference electrode being a silver-silver chloride electrode having a multi-layer reference coating disposed thereon; The multi-layer reference coating comprises: an inner layer that is an amorphous salt layer comprising an amorphous polysaccharide and a salt having isomobility cations and anions; a semipermeable cover membrane disposed to cover the inner layer, the semipermeable cover membrane having water vapor permeability and ion permeability. Disposable, single-use electrochemical sensor.

7. The amorphous polysaccharide, when supersaturated with the salt, has amorphous properties such that the amorphous polysaccharide and the salt do not separate when the inner layer is formed.

7. A disposable, single-use electrochemical sensor according to claim 6.

8. The amorphous polysaccharide is selected from the group consisting of pullulan, dextran, and amylose.

7. A disposable, single-use electrochemical sensor according to claim 6.

9. The salt is potassium chloride.

7. A disposable, single-use electrochemical sensor according to claim 6.

10. The semipermeable cover membrane is made from one of chlorosulfonated polyethylene and cellulose acetate butyrate.

7. A disposable, single-use electrochemical sensor according to claim 6.

11. 1. A method of forming a single-use disposable electrochemical potentiometric reference sensor, comprising: providing a sensor body having an insulating base substrate with at least one conductive pathway and an insulating reagent retaining layer disposed on said insulating base substrate, said insulating reagent retaining layer having at least one reagent retaining opening, said at least one reagent retaining opening exposing a portion of said at least one conductive pathway; disposing an amorphous salt layer mixture containing an amorphous polysaccharide and a salt having isomobility cations and anions within one of the at least one reagent retaining opening; drying the amorphous salt layer mixture to form an inner layer that is an amorphous structure layer; disposing a cover membrane solution containing a hydrophobic polymer over the inner layer; and drying the cover membrane solution to form a semipermeable cover membrane, the semipermeable cover membrane having water vapor permeability and ion permeability. A method for making a single-use disposable electrochemical potentiometric reference sensor.

12. forming the amorphous salt layer mixture; forming the amorphous salt layer mixture adding together a number of ingredients including a quantity of the amorphous polysaccharide and a quantity of a 3M salt solution; and mixing the components forming the amorphous salt layer mixture. The method of claim 11.

13. Measuring 750 milligrams of said amorphous polysaccharide; measuring 3 milliliters of the 3M salt solution. The method of claim 11.

14. forming the cover membrane solution; Making the cover membrane solution comprises: measuring a quantity of the hydrophobic polymer, the hydrophobic polymer being one of a chlorosulfonated polyethylene polymer and a cellulose acetate butyrate polymer; mixing the hydrophobic polymer in a volume of THF / cyclohexanone to form the cover membrane solution. The method of claim 11.

15. measuring 8-10% by weight of said chlorosulfonated polyethylene or said cellulose acetate butyrate. The method of claim 11.

16. A multi-layer reagent matrix that creates a reference electrode in a reference sensor, comprising: an inner layer formed from an amorphous salt layer mixture containing an amorphous polysaccharide and a salt, said inner layer overlying said potentiometric reference electrode, said amorphous polysaccharide having amorphous properties such that when supersaturated with a salt, separation of said amorphous polysaccharide and said salt does not occur when said inner layer is formed; a semipermeable cover membrane formed from a cover membrane solution disposed over the inner layer, the cover membrane solution including a hydrophobic polymer that is permeable to water vapor and ions. Multi-layered reagent matrix.

17. The inner layer is a mixture of potassium chloride and at least one of pullulan, dextran, and amylose. The reagent matrix of claim 16.

18. The semi-permeable membrane is made from one of chlorosulfonated polyethylene and cellulose acetate butyrate. The reagent matrix of claim 16.

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