Single-use disposable oxygen sensor

Abstract

To provide a single-use disposable oxygen sensor.SOLUTION: An electrochemical oxygen sensor is provided, comprising a sensing surface having a working electrode and a reference electrode, a hydrophilic layer formed from an oxygen diffusion-limiting layer emulsion overlaying the working electrode, and a hydrophobic membrane formed from a hydrophobic solution disposed over the hydrophilic layer. The hydrophilic layer contains an epoxy network and a hydrophilic polymer. The hydrophobic layer contains an acetate copolymer and a cross-linking agent that reacts with liquid epoxy resin in the hydrophilic layer forming the epoxy network, where the hydrophobic member is water vapor- and oxygen-permeable.SELECTED DRAWING: Figure 5

Description

Detailed Description of the Invention

[0001] [Background of the invention] 1. FIELD OF THE INVENTION

[0002] The present invention relates generally to planar electrochemical sensors with membrane coatings used to perform chemical analyses, and more particularly to disposable oxygen sensors.

[0003] 2. Description of Prior Art

[0004] One of the greatest challenges in single-use sensors is the fabrication of electrochemical planar oxygen sensors. The single-use sensor substrate consists of a metal-coated plastic substrate covered with an insulating layer. The electrode dimensions are determined by the opening dimensions in this layer. The dimensions of the oxygen working electrode in this format are usually larger than typical electrochemical oxygen sensors, and upon contact with the sample, it produces a high current that causes sample depletion and produces erratic values. The dimensions of the working electrode are proportional to the amount of current produced, which is based on the Cottrell equation. The Cottrell equation describes the response of the current in time as a function of a step in potential. The Cottrell equation is as follows:

number

[0005] Although it is technically possible to fabricate "small electrodes," they are not cost-effective for single-use disposable sensors.

[0006] Several different technologies for single-use oxygen sensors have been reported. One approach comes from i-Stat Systems by Abbott Diagnostics. The oxygen sensor uses microfabrication techniques to fabricate small electrodes in a silicon nitride substrate. A multi-step process involves masking, etching, and lifting to create a small-sized oxygen working electrode similar in size to a typical oxygen working electrode. Silicon nitride-based substrates are expensive and require complex processes for fabricating the oxygen sensors described above.

[0007] Another approach comes from Epocal, Inc. (a subsidiary of Siemens Healthineers) of Ottawa, Ontario, Canada, which introduced heterogeneous membranes. These membranes consist of a mixture of oil and water fractions. This mixture is emulsified in a cross-linkable polyvinyl alcohol aqueous medium with a salt and redox couple and a cross-linkable hydrophobic polymer to facilitate a manufacturing process using either dispensing or printing. The next step requires settling of the deposited layer, degassing, and final UV curing to immobilize all fractions. This procedure is a complex and time-dependent process due to the phase-separated nature of heterogeneous membranes. process, which can lead to variations between sensors.

[0008] Another related approach discloses a method for determining oxygen concentration in a biological sample. U.S. Patent No. 7,648,624 (Cai et al., 2010) discloses an oxygen sensor including a working electrode, a reference electrode, and a reagent matrix disposed on at least the working electrode. The reagent matrix contains a reduced form of a redox mediator, an oxidase, and a peroxidase to obtain an initial oxygen measurement. The oxygen sensor has a known correlation between oxygen and a predefined analyte, the correlation being expressed as a function of the oxygen sensor's response to both oxygen and the predefined analyte. The manufacturing process for this oxygen sensor is laborious and time-consuming, which is related to the reagent and mixing time required to provide a solution that can be dispensed onto the working electrode to form the reagent matrix.

[0009] [Summary of the Invention] Disposable arterial blood gas (ABG) sensors offer many advantages over conventional blood gas analyzers, including no maintenance, availability, ease of use, reduced contamination, cost-effectiveness, rapid analysis, and simplicity.

[0010] Because disposable oxygen sensors tend to use relatively large electrode surfaces that produce relatively large currents compared to oxygen electrodes found in conventional blood gas analyzers, an alternative method for producing sufficient "small currents" on large electrodes is to develop a diffusion layer that limits oxygen diffusion from the sample to the electrode. The requirements for the diffusion-limiting layer are a fixed path length, electrical conductivity, and linear oxygen permeability when hydrated.

[0011] In the present invention, a multilayer reagent matrix is formed above a working electrode to fabricate an oxygen sensor. The reagent matrix includes a hydrophilic polymer layer and a hydrophobic polymer layer. The hydrophilic polymer layer is formed by combining a liquid epoxy resin and a hydrophilic polymer emulsion, and the combination is used as an oxygen diffusion limiting layer. A hydrophobic polymer layer containing an epoxy curing agent is used for the cover membrane and to solidify the liquid epoxy in the hydrophilic polymer layer.

[0012] Liquid epoxy resins containing bisphenol A diglycidyl ether, i.e., epichlorohydrin and bisphenol A, can be mixed with hydrophilic polymers such as PVA and PVP to form aqueous epoxy emulsions. While epoxy limits oxygen diffusion, the water-soluble hydrophilic polymer allows oxygen diffusion and can create electrical connections in electrochemical sensor mechanisms. This epoxy / water-based hydrophilic polymer mixture results in a homogeneous emulsion that is easy to dispense and produces a uniform film. However, this layer can be washed away once it comes into direct contact with an aqueous solution. A hydrophobic cover film prevents this layer from being washed away while also allowing water vapor and oxygen to diffuse across the film. Unfortunately, if the liquid epoxy is left uncured, the diffusing water vapor will begin to dissolve (i.e., hydrate) the hydrophilic moieties in the layer, eventually increasing the path length (i.e., layer thickness) and thereby degrading sensor performance.

[0013] Liquid epoxies can be cured by reacting with a curing agent (e.g., an amine) that converts the water-miscible epoxy into a water-insoluble, rigid polymer network. However, epoxy-amines exhibit an extremely fast curing reaction upon contact with liquid epoxies. Once the epoxy-amine is mixed with the liquid epoxy, the reaction begins immediately. As a result, it is extremely difficult to add a curing agent to an epoxy / hydrophilic polymer emulsion prior to the dispensing process.

[0014] The present invention avoids this problem by incorporating an epoxy-amine curing agent into the hydrophobic cover film solution. The hydrophobic cover film solution contains an acetate copolymer along with the amine curing agent. The epoxy-amine curing reaction begins immediately after dispensing the cover film solution on top of the epoxy / hydrophilic polymer layer. As a result of dispensing the hydrophobic cover film solution containing the epoxy-amine curing agent, the liquid epoxy embedded in the hydrophilic layer emulsion becomes a rigid epoxy network that contains the hydrophilic portion of this layer. The rigid epoxy network formed in the diffusion layer, which is coupled with the hydrophobic cover film formed above the diffusion layer, prevents any change in the path length (i.e., thickness) of the multilayer reagent matrix upon hydration.

[0015] It is an object of the present invention to provide an electrochemical oxygen sensor that accurately measures dissolved oxygen in small fluid samples. It is another object of the present invention to provide an amperometric oxygen sensor that is disposable and measures oxygen with high accuracy and precision.

[0016] The present invention achieves these and other objects by providing, in one embodiment, an electrochemical oxygen sensor having a sensing surface having a working electrode and a reference electrode; a hydrophilic layer formed from an oxygen diffusion-limiting layer emulsion, the hydrophilic layer overlying the working electrode, the diffusion-limiting layer containing an epoxy network and a hydrophilic polymer; and a hydrophobic membrane formed from a hydrophobic solution disposed over the hydrophilic layer, the hydrophobic solution containing an acetate copolymer and a crosslinker that reacts with a liquid epoxy resin in the hydrophilic layer to form an epoxy network, the hydrophobic membrane being water vapor and oxygen permeable.

[0017] In another embodiment of the present invention, the hydrophilic polymer is polyvinyl alcohol.

[0018] In another embodiment of the present invention, the acetate copolymer of the hydrophobic membrane is an ethylene vinyl acetate copolymer.

[0019] In one embodiment, the epoxy network is formed by a reaction between the liquid epoxy in the hydrophilic layer emulsion and the crosslinker from the hydrophobic layer.

[0020] In one embodiment, the electrochemical oxygen sensor is a single-use oxygen sensor.

[0021] In one embodiment, the acetate copolymer is ethylene vinyl acetate, with fifty percent (50%) of the ethylene vinyl acetate polymerized into the ethylene vinyl polymer backbone.

[0022] In another embodiment, a method of forming an electrochemical oxygen sensing device is disclosed, the method including providing a sensor body having a base layer with at least two independent electrically conductive pathways and an insulating and reagent retention layer disposed on the base layer, the insulating and reagent retention layer having at least two reagent retention openings, one of the at least two reagent retention openings exposing a portion of one of the at least two independent electrically conductive pathways and another of the at least two reagent retention openings exposing a portion of another of the at least two independent conductive pathways, disposing an oxygen diffusion limiting emulsion containing a liquid epoxy and a hydrophilic polymer in one of the at least two reagent retention openings, drying the oxygen diffusion limiting emulsion to form a hydrophilic layer, disposing a cover film solution containing an acetate copolymer and an epoxy hardener over the hydrophilic layer, and drying the cover film solution to form a hydrophobic layer.

[0023] In one embodiment of this method, the oxygen diffusion limited emulsion comprises adding together a plurality of components comprising a predefined amount of a liquid epoxy resin, a predefined amount of polyvinyl alcohol, a predefined amount of a surfactant, and a predefined amount of distilled water, and mixing the plurality of components to form the emulsion.

[0024] In another embodiment of this method, the method includes measuring 1.6 grams of liquid epoxy resin, measuring 1.4 grams of 10% polyvinyl alcohol, and measuring a volume of 1 milliliter of distilled water.

[0025] In one embodiment, the method further comprises measuring a predefined amount of an antifoaming agent and adding the antifoaming agent to the hydrophilic emulsion.

[0026] In one embodiment, the method includes forming a cover film solution, where forming the cover film solution includes adding together cover film components including 50 wt % acetate copolymer, 3 wt % epoxy curing agent, and 1 wt % pentaerythritol tetrakis 3-mercaptopropionate, and mixing the cover film components in a predefined amount of THF / cyclohexanone to form the cover film solution.

[0027] In one embodiment, the method includes selecting 2% 2,4,6-tris(dimethylaminomethyl)phenol as the epoxy hardener.

[0028] In one embodiment, the method further includes forming a reference electrode in another of the at least two reagent-retaining openings.

[0029] In another embodiment, a reagent matrix for converting a working electrode into an oxygen sensor is disclosed, the reagent matrix including: a hydrophilic layer formed from an oxygen diffusion-limiting layer emulsion, the hydrophilic layer overlying the working electrode, the diffusion-limiting layer containing an epoxy network and a hydrophilic polymer; and a hydrophobic membrane formed from a hydrophobic solution disposed over the hydrophilic layer, the hydrophobic solution containing an acetate copolymer and a crosslinker that reacts with the liquid epoxy resin in the hydrophilic layer to form an epoxy network, the hydrophobic membrane being water vapor and oxygen permeable.

[0030] In one embodiment, the epoxy network is formed by a reaction between the liquid epoxy in the hydrophilic layer emulsion and the crosslinker from the hydrophobic layer. [Brief explanation of the drawings]

[0031] [Brief description of the drawing] [Figure 1] FIG. 1 is a perspective view of one embodiment of the present invention showing an oxygen sensor. [Figure 2] FIG. 2 is an exploded view of the embodiment of FIG. 1 showing the two component layers of the oxygen sensor. [Figure 3] FIG. 2 is a top view of the base layer of the oxygen sensor. [Figure 4] FIG. 2 is a top view of a reagent holding layer. [Figure 5] FIG. 5 is an enlarged cross-sectional view of the oxygen sensor taken along line 5-5 of FIG. 1. [Figure 6] FIG. 1 is an enlarged view of the multilayer reagent matrix of the working electrode showing the hydrophilic and hydrophobic layers. [Figure 7] FIG. 1 is an exemplary top view of an oxygen sensor connected to a flow cell. [Figure 8] 1 is a graphical representation showing the correlation between the readings of an oxygen sensor of the present invention and readings from a blood gas analyzer using blood samples measured with a tonometer. DETAILED DESCRIPTION OF THE INVENTION

[0032] [Detailed Description of the Invention] 1-8 illustrate several embodiments of the present invention. In one embodiment, an oxygen sensor 10 of the present invention is fabricated using a two-layer structure (see FIGS. 1-4). The two-layer structure includes a laminate 12 including an electrode end 14, an electrical contact end 16, at least a counter electrode 17, a working electrode 18, and a reference electrode 19 at the electrode end 14, and electrical contact pads 16a, 16b, and 16c at the electrical contact end 16. The laminate 12 also includes a base layer 20 and an insulating and electrode boundary definition layer 30. All layers of the laminate 12 are fabricated from a dielectric material, preferably a plastic. Examples of preferred dielectric materials include polyvinyl chloride, polycarbonate, polysulfone, nylon, polyurethane, nitrocellulose, cellulose propionate, cellulose acetate, cellulose acetate butyrate, polyester, polyimide, polypropylene, polyethylene, and polystyrene.

[0033] Base layer 20 has an electrically conductive layer 21 having at least three electrically conductive pathways 22, 24, and 26 defined thereon. The electrically conductive pathways 22, 24, and 26 may be formed by scribing or scoring the electrically conductive layer 21 or by silk screening the electrically conductive pathways 22, 24, and 26 onto base layer 20. Scribing or scoring the electrically conductive layer 21 may be performed by mechanically scribing the electrically conductive layer 21 to create non-electrically conductive score lines 28 sufficient to create at least three independent conductive pathways 22, 24, and 26. A preferred scribing or scoring method of the present invention is by using a carbon dioxide laser, a YAG laser, or an excimer laser. The electrically conductive layer 21 may be made of any electrically conductive material, such as copper, gold, tin oxide / gold, palladium, other noble metals or their oxides, or a carbon film composition. The electrically conductive material used in this embodiment is palladium. Acceptable thicknesses for base layer 20 are in the range of 0.002 inches (0.05 mm) to 0.010 inches (0.25 mm). One such usable material for base layer 20 is a 0.005 inch (0.125 mm) palladium polyester film (stock number: Melinex 329) sold by Marian, Inc. of Indianapolis, Indiana.

[0034] Insulating and electrode demarcation layer 30 has at least three openings 32, 34, and 36. Opening 32 exposes a portion of conductive pathway 22, opening 34 exposes a portion of conductive pathway 24 to create a reagent retention well, and opening 36 exposes a portion of conductive pathway 26. In this embodiment, insulating and electrode demarcation layer 30 is a medical single-sided adhesive tape / film available from Transcendia, Inc. of Franklin Park, Illinois. Acceptable thicknesses of this 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, stock number PE31280 (about 0.002 inch (0.045 mm)), is used because of its ease of handling and good performance in terms of its ability to retain sufficient amounts of chemical reagents and promote capillary action through the sensor's fluid sample flow path. It should be understood that the use of tape is not required: insulation and electrode demarcation layer 30 may be made from a plastic sheet and coated with a pressure sensitive adhesive, a photopolymer, ultrasonically bonded to base layer 20, silk screened onto base layer 20, or 3D printed onto base layer 20 to achieve the same results as the use of the mentioned polyester tape.

[0035] Three openings 32, 34, and 36 define electrode areas C, W, and R, respectively, forming a counter electrode C, a working electrode W, and a reference electrode R. Generally, the working electrode W contains a hydrophilic polymer deposited directly on the portion of the conductive layer 21 exposed within the electrode area W. The hydrophilic polymer layer is loaded with a polymer layer and a hydrophobic polymer layer on top of the hydrophilic polymer layer, the hydrophobic polymer layer forming a cover membrane.

[0036] The counter electrode, working electrode, and reference electrode each make electrical contact with a separate conductive path 22, 24, and 26, respectively, that terminates on the end of the laminate 12 opposite the electrode end 14 and is exposed for making electrical connection to a reading device.

[0037] The dimensions of the reagent retention openings are preferably made as small as possible to allow the oxygen sensor's fluid sample flow path to be as short as possible while still retaining enough chemical reagent to function properly. The shape of the reagent retention openings in this embodiment is round and has a diameter of approximately 0.03 inches (0.75 mm). The two reagent retention openings 32 and 34 are aligned with each other and spaced approximately 0.0256 inches (0.65 mm) apart from each other. These circular reagent retention openings are for illustrative purposes only. It should be understood that the shape of the reagent retention openings is not critical, and the dimensions of the openings are dictated more by the technical feasibility of dispensing the reagent matrix mixture into the openings and other manufacturing constraints.

[0038] The positional arrangement of the counter, working, and reference electrodes is not critical to obtaining usable results from the oxygen sensor. Possible electrode arrangements for the oxygen sensor when coupled to a flow cell may be CWR, WCR, or any arrangement of the three electrodes, with the listed electrode arrangements occurring based on the direction of sample flow across the counter electrode C, working electrode W, and reference electrode R. The preferred position has been found to be CWR. That is, as the fluid sample enters the flow cell 70, it first covers the counter electrode C, then the working electrode W, and then the reference electrode R.

[0039] Preferably, the reference electrode 19 (electrode well 36) is loaded with an Ag / AgCl layer (e.g., by applying an Ag / AgCl ink, or by (a) sputter coating an Ag layer and then chlorinating 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. It should be noted that the positional arrangement of the working, reference, and counter electrodes within the flow path is not critical to obtaining usable results from the sensor.

[0040] 3 and 4, top views of base layer 20 and insulating and reagent holding layer 30 are illustrated. As illustrated in FIG. 3, the symmetry of the conductive pathways is such that one longitudinal end of base layer 20 can be designated as either the electrode end 14 or the electrical contact end 16, depending on the orientation of insulating and reagent holding layer 30 relative to base layer 20 and the assembly process. In this embodiment, base layer 20 has scribe marks in conductive layer 21 that demarcate three separate conductive pathways. It should be understood that the base layer may have more than two conductive pathways, and additional conductive pathways may be designated for similar or other analyte sensor reagents, making this oxygen sensor a multi-analyte sensor.

[0041] 4 is a top view of insulating and reagent holding layer 30. Insulating and reagent holding layer 30 has three or more openings spaced apart from one another such that each opening corresponds to one of the conductive paths defined on base layer 20. It should be understood that the electrically conductive paths disclosed herein may be made from any non-corroding metal. Carbon deposits, such as carbon paste or carbon ink, may also be used as electrically conductive paths, all of which are well known to those skilled in the art. It should also be understood that counter electrode C is used to pass current from working electrode W to counter electrode C. Although a two-electrode system (i.e., working and reference electrodes) can be used, the use of a relatively large working electrode presents a disadvantage compared to conventional oxygen sensors. The challenge is that the reference electrode in an amperometric system is required to maintain a high charge density. In a two-electrode system, if there is a high current flow from the working electrode to the reference electrode, the Ag-AgCl reference electrode can deteriorate due to the changing current density. This, in turn, causes a shift in the bias potential window and a decrease in the accuracy of the determined oxygen concentration level.

[0042] Turning now to Figure 5, an enlarged cross-sectional view of oxygen sensor 10 taken along line 5-5 in Figure 1 is illustrated. It should be understood that the relative dimensions of layers 20 and 30 and electrode wells 32, 34, and 36, and the thicknesses of the working and reference electrode reagent matrices, are not drawn to scale but are merely for the purpose of illustrating the various components of oxygen sensor 10. As can be seen in Figure 5, base layer 20 has an electrically conductive layer 21 disposed thereon, with score lines 28 creating non-electrically conductive breaks in conductive layer 21. Insulating and reagent-retaining layer 30 has reagent-retaining opening 34 containing working electrode oxygen-diffusion-limiting multilayer reagent matrix 34a and reagent-retaining opening 36 containing reference electrode reagent matrix 60.

[0043] 6 is an enlarged view of multi-layered reagent matrix 32a. Multi-layered reagent matrix 32a includes hydrophilic polymer layer 50 and hydrophobic polymer layer 40. Hydrophilic layer 50 includes epoxy network 52 and hydrophilic portion 54. Hydrophobic layer 40, as its name suggests, is not water-soluble but is permeable to water vapor and oxygen.

[0044] The polymer used as the hydrophilic moiety in the hydrophilic layer should be sufficiently water-soluble and also capable of stabilizing all other chemicals in the reagent to the conductive surface layer in the electrode area. Suitable polymers include, but are not limited to, low and high molecular weight polyethylene oxide (PEO), polyethylene glycol, polyvinylpyrrolidone (PVP), starch, methyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), and polyamino acids. The hydrophilic moiety 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 moiety in the hydrophilic layer of the present invention is PVA. PVA is available from Scientific Polymer Products, New York, USA.

[0045] The hydrophilic layer also contains an epoxy network that is not water soluble, however, the epoxy network is made from a hydrophilic emulsion containing a water soluble liquid epoxy resin.

[0046] The polymer used in the hydrophobic layer is an ethylene vinyl acetate copolymer, which is 50 wt% vinyl acetate, available from Scientific Polymer Products, New York, USA.

[0047] A surfactant may optionally be included in the hydrophilic emulsion (used to fabricate the hydrophilic layer) to facilitate dispensing of the hydrophilic emulsion into the working electrode area W. The surfactant also aids in the rapid dissolution of the dry chemical reagent (i.e., PVA) as the fluid enters the sample chamber 17 of the oxygen sensor 10, 430. The amount and type of surfactant are selected to ensure the previously mentioned functions. The surfactant can be selected from, but is not limited to, a variety of anionic, cationic, nonionic, and zwitterionic detergents. Examples of acceptable surfactants are polyoxyethylene ether, Tween 20, sodium cholate hydrate, and hexadecylpyridinium chloride monohydrate. The preferred surfactants are t-octylphenoxypolyethoxyethanol, available from Sigma-Aldrich under the brand name Triton X-100. The concentration of the surfactant in the reagent matrix is preferably from about 0.01% (w / w) to about 2%.

[0048] FIG. 7 is a top view representation of the oxygen sensor 10 connected to a flow cell 70. The flow cell 70 includes a test chamber 74 in which a working electrode 18 and a reference electrode 19 are disposed. The test chamber 74 has a test chamber inlet 72 connected to a calibrant supply line 90 and a blood sample supply line 100. A predefined amount of calibrant is supplied to the test chamber 74 for a single-point calibration. After calibration, the calibrant is moved out of the test chamber 74, followed by a predefined amount of blood sample. The blood sample is measured for the amount of oxygen (i.e., oxygen concentration) in the blood sample. It is understood that the oxygen sensor 10 is electrically connected to appropriate electronic circuitry to perform chronoamperometric measurements.

[0049] [Preparation of Hydrophilic Layer Composition and Hydrophobic Layer Composition]

[0050] The preferred reagent layer composition for the hydrophilic emulsion used in fabricating the hydrophilic layer may be prepared in one step, but is preferably prepared in two steps.

[0051] Step 1: Add together 1.6 gm of liquid epoxy resin available under the trademark DER331 (DER331) by The Dow Chemical Company, 1.4 g of 10% polyvinyl alcohol (Mw. 130K), 1 ml of 1% Triton X-100 and distilled water, and 100 mg of antifoaming agent.

[0052] Step 2: Mix all ingredients in step 1 above using a homogenizer at 9,000 rpm per minute. The prepared hydrophilic emulsion is stable for several days when stored at room temperature.

[0053] The reagent layer composition for the cover membrane solution used in fabricating the hydrophobic layer may also be prepared in one step, but is preferably prepared in two steps.

[0054] Step 1: Add 2 wt% of ethylene vinyl acetate copolymer, where the copolymer is ethylene vinyl acetate and 50% of the acetate is copolymerized into the ethylene vinyl polymer backbone, and 3% of epoxy hardener (2% 2,4,6-tris(dimethylaminomethyl)phenol and 1% pentaerythritol tetrakis(3-mercaptopropionate)) in THF / cyclohexanone.

[0055] Step 2: The raw materials in Step 1 are mixed together to form a cover membrane solution. The prepared cover membrane solution is stable for several weeks when stored at room temperature.

[0056] [Sensor structure]

[0057] Assembly of various embodiments of the present invention is relatively simple: generally, the base layer and insulating and reagent holding layer are laminated together, and then the appropriate reagent mixture is dispensed into each of the reagent holding openings.

[0058] More specifically, for the two-layer configuration shown in FIG. 1, a strip of palladium film is shown in FIG. The illustrated shape is cut to form the base layer 20 of the sensor 10. The conductive palladium polyester film is preferably scored using a laser, although mechanical scribing is optional. As illustrated in FIG. 2, the film is scored with a laser so that three electrode areas are formed at the sample fluid end 14 and three contact points 22, 24, and 26 are formed at the electrical contact end 16. The score lines are very thin, but sufficient to create three distinct and distinct electrically conductive paths. A piece of single-sided adhesive tape is then cut to size and shape to form the insulating and electrode demarcation layer 30, covering most of the conductive layer 21 of the base layer 20 except for exposing a small electrical contact area, illustrated by reference numeral 16 in FIG. 1.

[0059] Prior to attaching the insulating and electrode boundary-defining layer 30 to the base layer 20, at least three openings 32, 34, and 36 having substantially equal dimensions are punched in the insulating and electrode boundary-defining layer 30 by a laser or mechanical means such as a die-punch assembly to create the electrode openings 32, 34, and 36. The electrode openings may be any shape. In the illustrated embodiment, the openings are circular. Preferred hole sizes for the openings 32 and 34 have a typical diameter of about 0.030 inches (0.75 mm), but may be any size. As illustrated in FIG. 2, the electrode openings 32 and 34 (and optional electrode opening 36, if included) are aligned with one another, with a spacing between adjacent openings of about 0.020 inches (0.508 mm) to about 0.050 inches (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 the openings, are not critical. The circular openings do not need to be substantially equal in size, so long as the surface area ratio remains substantially constant. While any combination of electrode arrangements is possible, a preferred arrangement of electrodes formed within openings 32, 34, and 36, as positioned from test chamber inlet 72, is C (counter electrode), W (working electrode), and R (reference electrode). Insulation and electrode demarcation layer 30 is then attached to base layer 20 in a manner that defines electrode wells for fabricating counter electrode C, working electrode W, and reference electrode R.

[0060] A predefined amount of hydrophilic emulsion is dispensed into the working electrode well and allowed to dry. The hydrophilic emulsion may be air-dried for several minutes at room temperature or dried for 30 seconds at 37°C to form a hydrophilic layer. A shorter drying time above room temperature allows for a more efficient manufacturing process. The hydrophilic emulsion and its composition are described above.

[0061] Next, the cover membrane solution is dispensed onto the hydrophilic layer, allowing the solution to completely cover the hydrophilic layer, and then either air-dried overnight at room temperature or dried at 37°C for at least 30 seconds. During this process, the epoxy curing agent in the cover membrane solution reacts with the liquid epoxy resin in the hydrophilic layer to form a rigid epoxy network containing the hydrophilic portion of the layer (i.e., polyvinyl alcohol), which maintains the predefined path length (i.e., thickness) of the hydrophilic layer when it is hydrated with the calibrant. As previously discussed, the hydrophobic layer allows the diffusion of water vapor and oxygen across the cover membrane, while the hydrophilic layer is an oxygen-diffusion-limiting layer containing a water-soluble hydrophilic polymer that allows oxygen diffusion and makes an electrical connection to the working electrode.

[0062] The amount of time required for the reagent to dry depends on the temperature at which the drying process is carried out.

[0063] [Oxygen sensor test]

[0064] The oxygen sensor 10 was connected to a flow cell as illustrated in Figure 7. When a fluid sample is applied to the oxygen sensor of the embodiment of the invention shown in Figure 1, the fluid sample enters the flow cell 70 and flows over electrodes C, W, and R, where it remains for a predefined period of time, the length of time depending on whether the fluid is a calibrant or a blood sample.

[0065] The current response of the oxygen sensor 10 was measured by chronoamperometry using a Nova Biomedical blood gas analyzer, although a potentiostat may also be used. The oxygen sensor shown in FIG. 1 and constructed as described above was used to test the response of the oxygen sensor 10 of the present invention to the concentration of oxygen in a sample. A blood sample containing a predetermined amount of oxygen was applied to the sample inlet 18 of the sensor strip and entered the sensor strip while a potential of −0.10 V to −0.70 volts (depending on the reference electrode used) was applied between the working and reference electrodes. In the specific example described here, the applied potential was −0.65 V versus an Ag-AgCl reference electrode. The output response current is proportional to the concentration in the blood sample. Using the Nova Biomedical blood gas analyzer, measurements were given as the partial pressure of oxygen in mmHg.

[0066] [Example 1]

[0067] [Illustration of reference pO2 vs. sensor pO2 measured at different levels of pO2]

[0068] Blood samples with different pO2 were tested with the oxygen sensor of the present invention using a pHox blood gas analyzer from Nova Biomedical. Alternatively, an electrochemical analyzer (Model 812, CH Instruments, Austin, Texas, USA) may be used to measure the current response directly from the oxygen sensor strip 10. Oxygen concentration (pO2) was controlled using a tonometer (Precision Gas Mixer, PGM-3, Medicor, Inc., Salt Lake City, Utah, USA). Two milliliters of blood sample were placed in a temperature-controlled (37°C) cylindrical rotating cuvette and measured with the tonometer for 15 minutes.

[0069] A single-point calibration was required prior to measuring a blood sample to hydrate the hydrophilic layer and establish a reference point for the oxygen sensor 10. The single-point calibrator used was a 110 mmHg pO2 solution. This single-point calibrator was flowed through each oxygen sensor via the flow cell and allowed to dwell within the flow cell for 90 seconds to hydrate the hydrophilic layer. At the end of the 90-second hydration period, a single-point calibration measurement was performed. Following calibration, a tonometer-measured blood sample was introduced into the oxygen sensor to replace the calibrator, and a reading was taken 30 seconds after blood sample introduction. Concentration calculations were based on the single-point calibration and used the Cottrell equation. Performed in this manner, the pO2 measurement response was found to be linear over a range of pO2 at the working electrode from 50 mmHg to 170 mmHg. Six blood samples with tonometer-measured oxygen levels ranging from 49 mmHg to 243 mmHg were tested. These tonometer-measured blood samples were also measured using a Nova Biomedical pHox blood gas analyzer to obtain reference readings for each blood sample for comparison with the readings obtained from the oxygen sensor of the present invention.

[0070] In this example, several biosensors using palladium substrates were fabricated to test the response of the oxygen sensor. The results are shown in Table 1.

[0071] [Table 1]

[0072] Each blood concentration was tested at least 10 times (i.e., 10 disposable oxygen sensors 10 were used for each oxygen concentration level, and the average values were calculated and displayed in Table 1). Standard deviation values are also provided for each concentration level tested.

[0073] 8 shows the measured pO2 responses of a working electrode of the present invention (i.e., a hydrophilic layer / cover membrane layer electrode) to various pO2 levels of 49 mmHg, 82 mmHg, 103 mmHg, 122 mmHg, 167 mmHg, and 243 mmHg. These responses (i.e., current responses) are linear with respect to oxygen concentration throughout the first five pO2 ranges described above.

[0074] Advantages of the present invention over conventional blood gas analyzers include no maintenance, availability, ease of use, reduced contamination, cost effectiveness, rapid analysis, and simplicity.

[0075] While preferred embodiments of the invention have been described herein, the above description is by way of example only. Further modifications of the invention disclosed herein will occur to those skilled in the relevant art, and all such modifications are deemed to fall within the scope of the invention as defined by the appended claims.

Claims

1. a sensing surface having a working electrode and a reference electrode; a hydrophilic layer formed from an oxygen diffusion-limiting layer emulsion, the hydrophilic layer overlying the working electrode, the diffusion-limiting layer comprising an epoxy network and a hydrophilic polymer; and a hydrophobic membrane formed from a hydrophobic solution disposed over the hydrophilic layer, the hydrophobic solution containing an acetate copolymer and a crosslinking agent that reacts with the epoxy network, the hydrophobic material being water vapor permeable and oxygen permeable; An electrochemical oxygen sensor comprising:

2. 2. The electrochemical oxygen sensor of claim 1, wherein the hydrophilic polymer is polyvinyl alcohol.

3. 2. The electrochemical oxygen sensor of claim 1, wherein the acetate copolymer of the hydrophobic membrane is an ethylene vinyl acetate copolymer.

4. 2. The electrochemical oxygen sensor of claim 1, wherein the epoxy network is formed by a reaction between a liquid epoxy in the hydrophilic layer emulsion and the crosslinker from the hydrophobic layer.

5. 10. The electrochemical oxygen sensor of claim 1, which is a single-use oxygen sensor.

6. 4. The electrochemical oxygen sensor of claim 3, wherein said acetate copolymer is ethylene vinyl acetate, and 50 percent of said ethylene vinyl acetate is polymerized into an ethylene vinyl polymer backbone.

7. 1. A method of forming an electrochemical oxygen sensor, comprising: providing a sensor body having a base layer with at least two independent conductive pathways, and an insulating and reagent holding layer disposed on the base layer, the insulating and reagent holding layer having at least two reagent holding openings, one of the at least two reagent holding openings exposing a portion of one of the at least two independent conductive pathways, and another of the at least two reagent holding openings exposing a portion of another of the at least two independent conductive pathways; disposing an oxygen diffusion limiting emulsion containing a liquid epoxy and a hydrophilic polymer in one of the at least two reagent holding openings; drying the oxygen diffusion-limiting emulsion to form a hydrophilic layer; disposing a cover film solution containing an acetate copolymer and an epoxy hardener over the hydrophilic layer; and drying the cover film solution to form a hydrophobic layer; Including, the one of the at least two reagent-retaining openings containing the hydrophilic and hydrophobic layers forms a working electrode; method.

8. forming the oxygen diffusion limited emulsion; forming the oxygen diffusion limited emulsion, a plurality of components including a predetermined amount of liquid epoxy resin, a predetermined amount of polyvinyl alcohol, a predetermined amount of surfactant, and a predetermined amount of distilled water; and mixing the components to form an emulsion; 8. The method of claim 7, comprising:

9. measuring 1.6 grams of said liquid epoxy resin; Measure out 1.4 grams of 10% polyvinyl alcohol, and Measuring a volume of 1 milliliter of distilled water; 9. The method of claim 8, further comprising:

10. 10. The method of claim 8, further comprising adding a predefined amount of an antifoaming agent to the hydrophilic emulsion.

11. forming the cover membrane solution; forming the cover membrane solution adding together a plurality of cover film ingredients including 50 wt % acetate copolymer, 3 wt % epoxy hardener, and 1 wt % pentaerythritol tetrakis 3-mercaptopropionate; and mixing the cover film components in the predetermined amount of THF / cyclohexanone to form the cover film solution; 8. The method of claim 7, comprising:

12. 12. The method of claim 11, further comprising selecting 2% 2,4,6-tris(dimethylaminomethyl)phenol as the epoxy hardener.

13. The method of claim 7 , further comprising forming a reference electrode in another of the at least two reagent-holding openings.

14. 1. A multi-layer reagent matrix for making a working electrode into an oxygen sensor, comprising: a hydrophilic layer formed from an oxygen diffusion-limiting layer emulsion containing a liquid epoxy resin, the hydrophilic layer overlying the working electrode, the diffusion-limiting layer containing an epoxy network and a hydrophilic polymer; and a hydrophobic membrane formed from a cover membrane solution disposed over the hydrophilic layer, the cover membrane solution containing an acetate copolymer and a crosslinker that reacts with the liquid epoxy resin in the hydrophilic layer to form the epoxy network, the hydrophobic member being water vapor permeable and oxygen permeable; A multi-layer reagent matrix comprising:

15. The reagent matrix of claim 14 , wherein the hydrophilic polymer is polyvinyl alcohol.

16. 15. The reagent matrix of claim 14, wherein the acetate copolymer of the hydrophobic membrane is an ethylene vinyl acetate copolymer.

17. 1. A method of making a multi-layered reagent matrix that converts a working electrode into an oxygen sensor, comprising: forming an oxygen diffusion limited emulsion for deposition on a working electrode surface, the oxygen diffusion limited emulsion comprising: adding together a plurality of components including a predefined amount of a liquid epoxy resin, a predefined amount of polyvinyl alcohol, a predefined amount of a surfactant, and a predefined amount of distilled water; and mixing the plurality of components to form an emulsion; and forming a cover film solution for depositing on the oxygen diffusion-restricting emulsion after drying the oxygen diffusion-restricting emulsion, the cover film solution comprising: adding together a plurality of cover film components including a predetermined amount of acetate copolymer, a predetermined amount of epoxy hardener, and a predefined amount of pentaerythritol tetrakis 3-mercaptopropionate; and mixing the plurality of cover film components in a predefined amount of THF / cyclohexanone to form the cover film solution; Including, A method of making a multilayer reagent matrix, wherein the oxygen diffusion limiting emulsion is used to fabricate a hydrophilic layer on the working electrode, and the cover membrane solution is used to fabricate a hydrophobic layer on the hydrophilic layer.

18. 18. The method of claim 17, wherein the step of forming the oxygen diffusion-limiting emulsion comprises selecting 1.6 grams of the liquid epoxy resin, selecting 1.4 grams of the 10% polyvinyl alcohol, and selecting 1 milliliter of distilled water.

19. 18. The method of claim 17, wherein forming the cover film solution comprises selecting 50 wt % of the acetate copolymer, selecting 3 wt % of the epoxy hardener, and selecting 1 wt % of pentaerythritol tetrakis 3-mercaptopropionate.

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