Patent Application
20220381721
The present invention relates generally to a planar electrochemical sensor with membrane coatings used to perform a chemical analysis. Particularly, the present invention relates to a disposable oxygen sensor.
One of the biggest challenges in single-use sensors is making electrochemical planar oxygen sensors. Single use sensor substrate consists of a metal coated plastic substrate covered with an insulating layer. The electrode size is determined by the opening size of this layer. Usually the size of an oxygen working electrode in this format is larger than a typical electrochemical oxygen sensor and generates high current which causes sample depletion and generates erratic values when in contact with a sample. The size of the working electrode is proportional to the amount of current generated, which is based on the Cottrell equation. The Cottrell equation describes the current response, in time, as a function of a step in potential. The Cottrell equation is
where i(t) is current, n is the number of electrons transferred in the half reaction, F is Faraday's Constant (96,485 C/mol), A is the area of the electrode, Do is the diffusion coefficient, Co is the initial concentration, and t is time. From the equation, it is seen that as A, the area of the electrode, increases so does the i, the current.
Although it is technically possible to make a “small electrode”, it is not cost effective as a single use disposable sensor.
A few different, single-use oxygen sensor technologies have been reported. One approach is from i-Stat Systems from Abbott Diagnostics. An oxygen sensor uses microfabrication technology to make a small electrode in a silicon nitride substrate. The multistep process involves masking, etching and lifting to produce a small size oxygen working electrode which is similar in size to a classic oxygen working electrode. Silicon nitride base substrates are expensive and require a complex process to produce the oxygen sensor mentioned above.
Another approach comes from Epocal, Inc., Ottawa, Ontario, Canada (an affiliate of Siemens Healthineers), where heterogeneous membranes are introduced. This membrane consists of a mixture of oil and water compartments. The mixture emulsifies a cross linkable polyvinyl alcohol aqueous media with salt and redox couple and cross linkable hydrophobic polymers to promote a manufacturing process using either dispensing or printing. The next steps require settling of the deposited layer, degassing and finally UV curing to immobilize all the compartments. This procedure is a complicated time reliant process due to the phase separation characteristics of the heterogeneous membrane, which could introduce sensor-to-sensor variations.
In another related approach, a method of determining the concentration of oxygen in a biological sample is disclosed. In U.S. Pat. No. 7,648,624 (Cai et al., 2010), there is a disclosed an oxygen sensor that includes a working electrode, a reference electrode and a reagent matrix disposed on at least the working electrode where the reagent matrix contains a reduced form of a redox mediator, an oxidase and a peroxidase to obtain an initial oxygen measurement where the oxygen sensor has a known correlation between oxygen and the predefined analyte expressed as a function of the response of the oxygen sensor to both oxygen and the predefined analyte. The process to manufacture the oxygen sensor is burdensome and time consuming, which is a function of the reagents and mixing times to provide a usable solution for dispensing onto the working electrode forming the reagent matrix.
A single use arterial blood gas (ABG) sensor has many advantages over a conventional blood gas analyzer including zero maintenance, accessibility, ease of use, reduction of contamination, cost effectiveness, quick analysis, convenience, etc. Because a disposable oxygen sensor tends to use a comparatively large electrode surface that generates a comparatively large current compared to the oxygen electrodes found in a conventional blood-gas analyzer, an alternative method to create adequate “small current” on a large electrode is to formulate a diffusion layer that limits oxygen diffusion from the sample to the electrode. Requirements for the diffusion limiting layer are fixed path-length upon hydration, electrical conductivity and linear oxygen permeability.
In the present invention, a multi-layer reagent matrix is formed over a working electrode to create the oxygen sensor. The reagent matrix includes a hydrophilic polymer layer and a hydrophobic polymer layer. The hydrophilic polymer layer is formed by the combination of liquid epoxy resin and hydrophilic polymer emulsions where the combination is used as the oxygen diffusion limiting layer. The hydrophobic polymer layer containing epoxy curing agents are used for the cover membrane and to solidify the liquid epoxy in the hydrophilic polymer layer.
Liquid epoxy resin, including bisphenol A diglycidylether, epichlorohydrin and bisphenol A can be formed into an aqueous-epoxy emulsion by mixing it with hydrophilic polymers such as PVA, PVP etc. Epoxy is a substance that restricts oxygen diffusion while water soluble hydrophilic polymers enable oxygen dispersion and can make electrical connections in an electrochemical sensor setup. This epoxy/aqueous hydrophilic polymer mixture turns into a homogeneous emulsion which is easy to dispense and produces a homogeneous film. However, this layer can wash away once directly in contact with aqueous solution. A hydrophobic cover membrane prevents the washing away of this layer and it also allows water vapor and oxygen diffusion across the membrane. Unfortunately, if the liquid epoxy remains uncured, diffused water vapor begins to dissolve hydrophilic parts in this layer (i.e. hydration) and eventually increases the path length (i.e. the layer thickness), thereby deteriorating sensor performance.
Liquid epoxy can be cured by reacting with curing agents (e.g., amines) which turn a water miscible epoxy into a non-water soluble rigid polymer network. Epoxy-amines, however, show very fast curing reactions when contacting liquid epoxy. Once the epoxy-amines are mixed with the liquid epoxy, the reaction initiates immediately. As a result, it is very difficult to add curing agent to an epoxy/hydrophilic polymer emulsion before the dispensing process.
The present invention circumvents this problem by incorporating epoxy-amine curing agents into the hydrophobic cover membrane solution. The hydrophobic cover membrane solution also contains an acetate copolymer along with the amine curing agents. An epoxy-amine curing reaction initiates immediately after the cover membrane solution is dispensed onto the top of the epoxy/hydrophilic polymer layer. As a result of dispensing the hydrophobic cover membrane solution containing the epoxy-amine curing agent, liquid epoxy which is embedded in the hydrophilic layer emulsion turns into a rigid epoxy network containing the hydrophilic part of this layer. The rigid epoxy network formed within the diffusion layer coupled with the hydrophobic cover membrane that is formed over the diffusion layer prevents any change in the path length (i.e. the thickness) of the multi-layer reagent matrix when hydrated.
It is an object of the present invention to provide an electrochemical oxygen sensor that measures dissolved oxygen accurately in a small amount of fluid sample. It is another object of the present invention to provide an amperometric oxygen sensor that is disposable and that measures oxygen with high accuracy and precision.
The present invention achieves these and other objectives by providing, in one embodiment, an electrochemical oxygen sensor having a sensing surface with a working electrode and a reference electrode, a hydrophilic layer formed from an oxygen diffusion-limiting layer emulsion, the hydrophilic layer overlaying the working electrode where the diffusion-limiting layer contains 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 cross-linking agent that reacts with the liquid epoxy resin in the hydrophilic layer forming the epoxy network where the hydrophobic member is water vapor and oxygen permeable.
In another embodiment of the present invention, the hydrophilic polymer is a polyvinyl alcohol.
In another embodiment of the present invention, the acetate copolymer of the hydrophobic membrane is an ethylenevinyl acetate copolymer.
In one embodiment, the epoxy network is formed by a reaction between liquid epoxy in the hydrophilic layer emulsion and the cross-linking agent from the hydrophobic layer.
In one embodiment, the electrochemical oxygen sensor is a single-use oxygen sensor.
In one embodiment, the acetate copolymer is an ethylenevinyl acetate where fifty percent (50%) of the ethylenevinyl acetate is polymerized in an ethylene vinyl polymer backbone.
In another embodiment, there is disclosed a method of forming an electrochemical oxygen sensing device. The method includes providing a sensor body having a base layer with at least two independent electrically conductive paths, an insulating and reagent holding layer disposed onto the base layer where the insulating and reagent holding layer has at least two reagent holding openings where one of the at least two reagent holding openings exposes a portion of one of the at least two independent electrically conductive paths and the other of the at least two reagent holding openings exposes a portion of another of the at least two independent conductive paths, disposing an oxygen diffusion-limiting emulsion containing a liquid epoxy and a hydrophilic polymer into one of the at least two reagent holding openings, drying the oxygen diffusion-limiting emulsion forming a hydrophilic layer, disposing a cover membrane solution containing an acetate copolymer and epoxy curing agents over the hydrophilic layer, and drying the cover membrane solution forming a hydrophobic layer.
In one embodiment of the method, the oxygen diffusion-limiting emulsion includes adding together a plurality of components that includes a predefined amount of 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 forming an emulsion.
In another embodiment of the method, the method includes measuring 1.6 grams of the liquid epoxy resin, measuring 1.4 grams of a 10% polyvinyl alcohol, and measuring a volume of 1 milliliter of distilled water.
In one embodiment, the method further includes measuring a predefined amount of an anti-foaming agent and adding the anti-foaming agent to the hydrophilic emulsion.
In one embodiment, the method includes forming the cover membrane solution that includes adding together a plurality of cover membrane components comprising 50wt % of an acetate copolymer, 3 wt % of epoxy curing agent, and 1 wt % of pentaerythritol tetrakis 3-mercaptopropionate, and mixing the plurality of cover membrane components in the predefined amount of THF/cyclohexanone forming the cover membrane solution.
In one embodiment, the method includes selecting 2% 2,4,6-tri(dimethylaminomethyl)phenol as the epoxy curing agent.
In one embodiment, the method further includes forming a reference electrode in another of the at least two reagent holding openings.
In another embodiment, a reagent matrix for making a working electrode into an oxygen sensor is disclosed. The reagent matrix includes a hydrophilic layer formed from an oxygen diffusion-limiting layer emulsion, the hydrophilic layer overlaying the working electrode where the diffusion-limiting layer contains 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 cross-linking agent that reacts with the liquid epoxy resin in the hydrophilic layer forming the epoxy network where the hydrophobic member is water vapor and oxygen permeable.
In one embodiment, the epoxy network is formed by a reaction between liquid epoxy in the hydrophilic layer emulsion and the cross-linking agent from the hydrophobic layer.
Several embodiments of the present invention are illustrated in
Base layer 20 has an electrically conductive layer 21 on which is delineated at least three electrically conductive paths 22, 24 and 26. The electrically conductive paths 22, 24 and 26 may be formed by scribing or scoring electrically conductive layer 21, or by silk-screening electrically conductive paths 22, 24 and 26 onto base layer 20. Scribing or scoring of conductive layer 21 may be done by mechanically scribing the electrically conductive layer 21 creating a non-electrically conductive scoring line 28 sufficiently to create the at least three independent conductive paths 22, 24 and 26. The preferred scribing or scoring method of the present invention is done by using a carbon dioxide laser, a YAG laser or an excimer laser. Conductive layer 21 may be made of any electrically conductive material such as, for example, copper, gold, tin oxide/gold, palladium, other noble metals or their oxides, or carbon film compositions. The electrically conductive material used in this embodiment is palladium. An acceptable thickness for base layer 20 is in the range of 0.002 in (0.05 mm) to 0.010 in (0.25 mm). One such usable material for base layer 20 is a 0.005 in (0.125 mm) palladium polyester film (Stock. No. Melinex 329) sold by Marian, Inc., Indianapolis, Ind.
The insulating and electrode delineating layer 30 has at least three openings 32, 34 and 36. Opening 32 exposes a portion of conductive path 22, opening 34 exposes a portion of conductive path 24 creating reagent holding wells, and opening 36 exposes a portion of conductive path 26. In this embodiment, insulating and electrode delineating layer 30 is a medical grade one-sided adhesive tape/film available from Transcendia, Inc., Franklin Park, Ill. Acceptable thicknesses of the tape for use in the present invention are in the range of about 0.001 in. (0.025 mm) to about 0.005 in. (0.13 mm). One such tape/film, Stock No. PE31280 (about 0.002 in. (0.045 mm)), is used due to its ease of handling and good performance in terms of its ability to hold a sufficient quantity of chemical reagents and to promote capillary action through the fluid sample channel of the sensor. It should be understood that the use of a tape is not required. Insulating and electrode delineating layer 30 may be made from a plastic sheet and may be coated with a pressure sensitive adhesive, a photopolymer, ultrasonically-bonded to base layer 20, silk-screened onto base layer 20, or 3-D printed onto base layer 20 to achieve the same results as using the polyester tape mentioned.
The 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, working electrode W is loaded with a hydrophilic polymer layer deposited directly onto a portion of the conductive layer 21 exposed in electrode area W and a hydrophobic polymer layer on top of the hydrophilic polymer layer where the hydrophobic polymer layer forms a cover membrane.
The counter electrode, the working electrode and the reference electrode are each in electric contact with separate conductive paths 22, 24, and 26 respectively. The separate conductive paths terminate and are exposed for making an electric connection to a reading device on the end opposite the electrode end portion 14 of laminated body 12.
The size of the reagent holding openings is preferably made as small as possible in order to make the fluid sample channel of the oxygen sensor as short as possible while still being capable of holding sufficient chemical reagent to function properly. The shape of the reagent holding openings in this embodiment is round and has a diameter of about 0.03 in. (0.75 mm). The two reagent holding openings 32, 34 are aligned with each other and are spaced about 0.0256 in. (0.65 mm) from each other. The circular reagent holding openings are for illustrative purposes only. It should be understood that the shape of the reagent holding openings is not critical and that the size of the openings is driven more by the technical feasibility of dispensing the reagent matrix mixture into the openings and other manufacturing limitations.
The positional arrangement of the counter electrode, the working electrode, the reference electrode is not critical for obtaining usable results from the oxygen sensor. The possible electrode arrangements when the oxygen sensor is coupled with a flow cell may be C-W-R or W-C-R or any arrangement of the three electrodes, with the arrangement listed as the arrangement of electrodes would appear based on the sample flow direction across the counter electrode C, the working electrode W, and the reference electrode R. The preferred position was found to be C-W-R; that is, as the fluid sample enters the flow cell 70, the fluid sample would cover the counter electrode C first, then the working electrode W and then the reference electrode R.
Preferably, the reference electrode 19 (electrode well 36) may be loaded with a Ag/AgCl layer (e.g., by applying Ag/AgCl ink or by sputter-coating (a) a Ag layer followed by chloridizing the Ag or (b) a Ag/AgCl layer) or other reference electrode materials that do not require a redox mediator to function properly. It should be noted that the positional arrangement of the working, the reference and the counter electrodes in the channel is not critical for obtaining usable results from the sensor.
Turning now to
Turning now to
The polymer used as the hydrophilic portion in the hydrophilic layer should be sufficiently water-soluble and should also be 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, polyvinyl pyrrolidone (PVP), starch, methylcellulose, hydroxypropylcellulose, polyvinyl alcohol (PVA), carboxy methyl cellulose (CMC), and polyamino acids. The hydrophilic portion may be a single polymer or a combination of polymers preferable in a concentration range of about 0.02% (w/w) to about 7.0% (w/w). The preferred hydrophilic portion in the hydrophilic layer of the present invention is PVA. PVA is available from Scientific Polymer Products, NY, USA.
The hydrophilic layer also contains an epoxy network that is not water soluble. The epoxy network, however, is created from a hydrophilic emulsion containing a water-soluble, liquid epoxy resin.
The polymer used in the hydrophobic layer is an ethylenevinyl acetate copolymer. It is a 50 wt % vinyl acetate available from Scientific Polymer Products, NY, USA.
A surfactant may be optionally included in the hydrophilic emulsion (used to create the hydrophilic layer) to facilitate dispensing of the hydrophilic emulsion into the working electrode area W. The surfactant also helps in quickly dissolving the dry chemical reagents (i.e. the PVA) when a fluid enters the sample chamber 17 of the oxygen sensor 10, 430. The amount and type of surfactant is selected to assure the previously mentioned functions. Surfactants can be selected from, but are not limited to, various anionic, cationic, non-ionic, and zwitterionic detergents. Examples of acceptable surfactants are polyoxyethylene ether, Tween 20, sodium cholate hydrate, hexadecylpyridinium cholide monohydrate and CHAPs. The preferred surfactant is a polyoxyethylene ether. More preferably, it is t-octylphenoxypolyethoxyethanol and is available under the brand name Triton X-100 from
Sigma-Aldrich. The concentration of surfactant in the reagent matrix is preferably about 0.01% (w/w) to about 2%.
The preferred reagent layer composition for the hydrophilic emulsion used to create the hydrophilic layer is preferably prepared in two steps, although it may be prepared in one step:
Step 1: Adding together 1.6 gm of liquid epoxy resin available under the trademark D.E.R. 331 from The Dow Chemical Company (DER 331), 1.4 g of 10% polyvinyl alcohol (Mw. 130K), 1% Triton X-100 and 1 ml of distilled water, and 100 mg of an anti-foaming agent.
Step 2: Mixing all the components in Step 1 above using a homogenizer at 9,000 rpm for 1 minute. The prepared hydrophilic emulsion is stable for several days when stored at room temperature.
Reagent layer composition for the cover membrane solution used to create the hydrophobic layer is also preferably prepared in two steps although it too may be prepared in one step:
Step 1: Adding 2 wt % ethylenevinyl acetate copolymer where the copolymer is an ethylenevinyl acetate where 50% acetate is copolymerized in ethylenevinyl polymer backbone, 3% epoxy curing agents (2% 2,4,6-tri(dimethylaminomethyl)phenol, and 1% pentaerythritol tetrakis(3-mercaptopropionate)) in THF/Cyclohexanone.
Step 2: Mixing the ingredients in Step 1 together forming the cover membrane solution. The prepared cover membrane solution is stable for several weeks stored at room temperature.
Assembly of the various embodiments of the present invention is relatively straightforward. Generally, the base layer and insulating and reagent holding layer are laminated to each other followed by dispensing the appropriate reagent mixture into each of the reagent holding openings.
More particularly for the 2-layer configuration shown in
Before attaching insulating and electrode delineating layer 30 to base layer 20, at least three openings 32, 34 and 36 of substantially equal size are punched by laser, or by mechanical means such as a die-punch assembly, creating electrode openings 32, 34 and 36 in insulating and electrode delineating layer 30. The shape of the electrode openings may be any shape. In the illustrated embodiment, the openings are circular. The preferred hole size for openings 32 and 34 has a typical diameter of about 0.030 in. (0.75 mm) but may be any size. As illustrated in
A predefined amount of hydrophilic emulsion is dispensed into the working electrode well and dried. For example, it may be air dried for several minutes at room temperature or dried for thirty seconds at 37° C. forming the hydrophilic layer. Drying for a shorter time period at a temperature above room temperature allows for a more efficient manufacturing process. The hydrophilic emulsion and its composition is as described above.
Next, the cover membrane solution is dispensed onto the hydrophilic layer so that the solution completely covers the hydrophilic layer and either air dried overnight at room temperature or dried for 30 seconds or more at 37° C. During this process, the epoxy curing agents in the cover membrane solution react with the liquid epoxy resin in the hydrophilic layer forming a rigid epoxy network containing the hydrophilic portions of the layer (i.e. polyvinyl alcohol), where the epoxy network maintains a predefined path length (i.e. thickness) of the hydrophilic layer when the hydrophilic layer is hydrated by calibrant. As discussed previously, the hydrophobic layer allows diffusion of water vapor and oxygen across the cover membrane while the hydrophilic layer is an oxygen diffusion-limiting layer that contains water soluble hydrophilic polymers, which enables oxygen dispersion and makes electrical connections to the working electrode.
The length of time required to dry the reagents is dependent on the temperature at which the drying process is performed.
The oxygen sensor 10 was connected to a flow cell as illustrated in
Chronoamperometry was used to measure the current response of the oxygen sensor 10 using a Nova Biomedical blood gas analyzer but a potentiostat could also be used. The oxygen sensor made like those shown in
Blood samples with different pO2 were tested with the oxygen sensor of the present invention using the Nova Biomedical pHOx blood gas analyzer. In the alternative, an Electrochemical Analyzer (CH Instruments, Model 812, Austin, Tex., USA) may also be used to measure the current response directly from the oxygen sensor strips 10. Oxygen concentration (pO2) was controlled using a Tonometer (Precision Gas Mixer, PGM-3, Medicor, Inc., Salt Lake City, Utah, USA). Two milliliters of the blood sample were placed into a temperature-controlled (37° C.) cylindrical rotating cuvette and tonometered for 15 minutes.
A one-point calibration before measuring a blood sample is required to hydrate the hydrophilic layer and to establish a reference point for the oxygen sensor 10. The one-point calibrant used was 110 mmHg pO2 solution. The one-point calibrant is flowed through the flow cell to each oxygen sensor and allowed to stay in the flow cell for 90 seconds to hydrate the hydrophilic layer. At the end of the 90-second hydration period, the one-point calibration measurement is performed. Following the calibration, a tonometered blood sample was introduced to the oxygen sensor replacing the calibrant and a reading was taken 30 seconds after introducing the blood sample. The concentration calculation was based on the one-point calibration and using the Cottrell equation. When done in this fashion, it was found that the pO2 measured responses are linear over a range of 50 mm Hg to 170 mm Hg pO2 at the working electrode. Six blood samples with tonometered oxygen levels in a range of 49 mmHg to 243 mmHg were tested. The tonometered blood samples were also measured with the Nova Biomedical pHOx blood gas analyzer to obtain a reference reading for each blood sample for comparison with the readings obtained from the oxygen sensor of the present invention.
In this example, multiple biosensors using the palladium substrate were made for testing the response of the oxygen sensor. The results are shown in Table 1.
Each blood level was tested at least ten (10) times (i.e. using 10 disposable oxygen sensors 10 for each oxygen concentration level and the average value calculated and displayed in Table 1. The standard deviation value for each concentration level tested is also provided.
The advantages of the present invention over a conventional blood gas analyzer includes zero maintenance, accessibility, ease of use, reduction of contamination, cost effectiveness, quick analysis, convenience, etc.
Although the preferred embodiments of the present invention have been described herein, the above description is merely illustrative. Further modification of the invention herein disclosed will occur to those skilled in the respective arts and all such modifications are deemed to be within the scope of the invention as defined by the appended claims.
This patent application is a Continuation of U.S. patent application Ser. No. 16/695,902, filed on Nov. 26, 2019, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16695902 | Nov 2019 | US |
| Child | 17879310 | US |
