WO2006032284A1

WO2006032284A1 - Polymeric reference electrode

Info

Publication number: WO2006032284A1
Application number: PCT/DK2005/000607
Authority: WO
Inventors: Jennifer A. Samproni
Original assignee: Radiometer Medical Aps; Sendx Medical, Inc.
Priority date: 2004-09-24
Filing date: 2005-09-26
Publication date: 2006-03-30
Also published as: EP1792172A1; US20060065527A1; CN101052872A; JP2008514903A

Abstract

The invention is a polymeric reference electrode having properties equal to or superior to prior art electrodes without the presence of a plasticizer and in which such properties are achieved by incorporation in the membrane of a polymer with a sufficiently low glass transition temperature (Tg) to mimic the characteristics of a highly plasticized thermoplastic membrane. Preferred polymers are the polyacrylates, preferably with a linear backbone and pendant substituent groups. The membrane may further include lipophilic polymers and lipophilic additives, such as salts. In the reference electrode the membrane is overlaid on an internal electrode comprised of an internal contact optionally coated with an electrolyte and entrapped in a hydrophilic polymer. The polymeric reference electrode is preferably for use in the context of an ion selective electrode assembly.

Description

POLYMERIC REFERENCE ELECTRODE

FIELD OF THE INVENTION

[0001] The present invention relates to a polymeric reference electrode for use in conjunction with an ion selective electrode. More specifically, the invention relates to a polymeric membrane and electrode that comprise the reference electrode.

BACKGROUND

[0002] Ion selective electrodes (ISEs) are widely used to measure the concentration of ions in a variety of biological and non-biological fluids. The ions to be measured are in fluids that vary in their complexity from fluoride in drinking water, a relatively simple solution, to electrolytes in blood, a substantially more complex solution. Frequently in biological solutions, multiple ions are measured in a single sample using sensors that contain multiple ion selective electrodes.

[0003] Generally, ion selective electrodes are composed of an ion selective membrane, an internal electrolyte solution, and an internal reference electrode. The internal reference electrode is contained inside an ion selective electrode assembly, and typically consists of a silver/silver chloride electrode in contact with an appropriate solution containing fixed concentrations of chloride and the ion for which the membrane is selective. The ion- selective electrode must be used in conjunction with a reference electrode (i.e. "outer" or "external" reference electrode) to form a complete electrochemical cell. The configuration is commonly denoted as outer reference electrode | test solution | membrane | internal reference electrode or, outer reference electrode | test solution | ion selective electrode. The measured potential differences (ion-selective electrode vs. outer reference electrode potentials) are linearly dependent on the logarithm of the activity of a given ion in solution. The reference electrode maintains a relatively constant potential with respect to the solution under the conditions prevailing in an electrochemical measurement, and further serves to monitor the potential of the working reference electrode.

[0004] An example of a conventional reference electrode is silver/silver chloride

(Ag/AgCI) single junction reference electrodes such as those often used with pH meters. Such reference electrodes generally consist of a cylindrical glass tube containing an internal electrolyte solution of 4 M solution of potassium chloride (KCI) saturated with AgCI. The lower end of the glass tube is sealed with a porous ceramic frit that allows the slow passage of the internal electrolyte solution and forms a liquid junction with the external test solution. Dipping into the filling solution is a silver wire coated with a layer of silver chloride. The wire is joined to a low-noise cable that connects to the measuring system to allow voltage to be measured across the junction.

[0005] More recently, an area of particular interest has been planar miniature reference electrodes for use with electrochemical systems. A polymeric reference electrode provides the benefits of reduced cost, ease of manufacture and microfabrication. Whereas various miniature planar electrochemical sensors have been successfully commercialized, a stable and reliable miniature planar reference electrode has yet to be introduced. The basic structure of a polymeric reference electrode is an inert membrane enclosing a known reference, such as Ag/AgCI. Nolan et. al., Anal. Chem. 1997, (60), 1244-1247, have disclosed a polymeric reference electrode comprising an internal electrolyte covered with a polyurethane or Nafion^® membrane. However, the usefulness of the membrane is limited by the long conditioning time required. Yoon et. al., Sensors and Actuators B, (64) 8-14, have described a polymeric reference electrode comprising a hydrophilic polyurethane membrane doped with equirnolar concentrations of cationic and anionic lipophilic additives over Ag/AgCI. This reference electrode has the limitation of a long preconditioning time and ion sensitivity. Choi et. al., U.S. Publ. Pat. Appl. 2002/0065332, have disclosed a polymeric reference electrode membrane comprising 1 ) a porous polymer or a hydrophilic plasticizer, such as cellulose acetate and 2) a lipophilic polymer, such as polyvinyl chloride or polyurethane, which forms a highly plasticized thermoplastic membrane and which has the advantage of a short condition time, however, the limitations of such membrane formulations are that plasticizer leaching may occur, thus changing the characteristics of the membrane. Further, undoped polyvinyl chloride membranes often exhibit sensitivity to ions due to impurities in the polymer. While these teachings demonstrate that reasonable results may be obtained in the construction of a reference electrode using a polymeric membrane, substantial limitations such as long preconditioning time, changes in the membrane due to plasticizer leaching and potential ion interference due to impurities in the membrane still exist. SUMMARY OF THE INVENTION

[0006] The invention herein is a significant improvement on the prior art electrodes described above. In its principal embodiment, the invention is a polymeric reference electrode which contains an internal electrode comprising a contact having a stable electrical potential and a membrane comprising a membrane polymer with a glass transition temperature (T₉) of less than about 25°C, wherein the membrane polymer comprises lipophilic plasticizing groups pendant from a polymeric backbone. It is critical that the T₉ is below the operating temperature of the measuring chamber (normally room temperature, 25°C); i.e. that it is flexible at the operating temperature in the absence of a plasticizing agent. Thus the membrane will be soft both during use and during storage at room temperature. The T₉ should preferably also be lower than the storage temperature such that the plasticnature of the membrane is preserved during storage. Thus, it is preferred that the T₉ be ≤0°C and more preferably that it be <-10°C. A T₉ range of -10^°C to -100^°C is preferred, and a range of -1O⁰C to -60⁰C is more preferred. The membrane must behave as if it were plasticized to allow for at least an operable level of membrane motility. Otherwise, the impedance of the membrane will be too great and it cannot be used to make electrochemical measurements.

[0007] The invention is therefore a polymeric reference electrode with a basic structure comparable to prior art electrodes but in which the previously required plasticizer component has been eliminated from the membrane and has been replaced by a plasticizer-free polymer which has a sufficiently low T₉ so that performance equal to or superior to the prior art devices is achieved without the detrimental properties that presence of a plasticizer causes. Thus according to the invention a reference electrode is provided, which has a suitable membrane motility for an extended period of time, has low impedance and ion interference, and provides for rapid hydration and/or fast conditioning of the membrane.

[0008] It is preferred but not required that the polymer have a linear portion and branched portion. The preferred membrane polymers are typically methacrylic-acrylic copolymers, but any suitable polymer that possesses the requisite T₉ property and otherwise has the appropriate electrode membrane properties may be used. Additionally, the electrode may contain additional polymers suitable for biosensors such as polyvinyl chloride, polyurethane, or silicone rubber, and lipophilic or hydrophilic additives. [0009] One may characterize another suitable (and preferred) plasticizer-free membrane as one comprising a copolymer of methacrylate monomers with R₁ and R₂ pendant alkyl groups where R₁ is any Ci_-3 alkyl group and R₂ is any C_4-12 alkyl group. The use of methacrylate monomers of different pendant alkyl groups (different length and degree of branching) allows one to achieve a polymer material with not only a plasticizer-free plasticizing effect but also a better mechanical strength for a desired T₉. Thus it is possible to taylor and optimise the resulting T₉ and mechanical strength by choosing more or less branching of the monomers and longer or shorter chain lengths and combine these in a number of ways. The preferred membrane polymers comprise segments, which may be summarized in the following formula:

where the lipophilic plasticizing groups R₁ and R₂ are the same or different and selected from C-i to C₁₆ alkyl groups, preferably C-i to C₁₂ alkyl groups, and R₃ and R₄ are the same or different and selected from H and CH₃.

[0010] The internal contact may be any suitable contact material including, but not limited to Ag/AgCI. The conductive electrolyte may be any suitable salt such as KCI, sodium formate, sodium chloride or the like. The internal electrolyte may be entrapped in any suitable hydrophilic inert polymer which may be, but is not limited to, hydrophilic polyurethane (PU), polyhexylethylmethacrylate (pHEMA), polyvinyl pyrollidone (PVP), polyvinyl alcohol (PVA) or other hydrophilic polymers.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] The Figures of the drawings are graphical representations of the data resulting from the various experiments set forth below and are exemplary in nature. In each experiment illustrated, a specific ion or compound in a solution was detected by a sensor which included an ion selective electrode and a reference electrode of the present invention and that detection was compared with the simultaneous detection of the same ion or compound in of the same solution by a sensor using a reference electrode of known properties. For each figure, the Y-axis is the value of the respective conventional sensor referenced against a reference electrode of the present invention and the X-axis is the value of the respective conventional sensor referenced against a conventional reference electrode (of the ABL™725 analyzer, Radiometer Medical ApS, Denmark). The solid line boundaries of the performance interval incorporate uncertainties of both the experimental analyzer and the ABL 725 reference analyzer, and are calculated from published performance test results. The combined performance interval defines the confidence interval (2 standard deviations) performance band for the analyzers. The figures show the comparative data and also indicate the ranges of error of the data.

[0012] Figure 1 depicts the response of a pH electrode referenced against a reference electrode of the present invention compared to a pH electrode referenced against a conventional reference electrode on different days and demonstrates that the values are consistent with use/exposure to blood.

[0013] Figure 2 depicts the response of a pCO2 electrode referenced against a reference electrode of the present invention compared to a pCO2 electrode referenced against a conventional reference electrode on different days and demonstrates that the values are consistent with use/exposure to blood.

[0014] Figure 3 depicts the response of a sodium (Na+) ion selective electrode referenced against a reference electrode of the present invention compared to a Na+ ion selective electrode referenced against a conventional reference electrode on different days and demonstrates that the values are consistent with use/exposure to blood.

[0015] Figure 4 depicts the response of a potassium (K+) ion selective electrode referenced against a reference electrode of the present invention compared to a K+ ion selective electrode referenced against a conventional reference electrode on different days and demonstrates that the values are consistent with use/exposure to blood.

[0016] Figure 5 depicts the response of a calcium (Ca++) ion selective electrode referenced against a reference electrode of the present invention compared to a Ca++ ion selective electrode referenced against a conventional reference electrode on different days and demonstrates that the values are consistent with use/exposure to blood.

[0017] Figure 6 depicts the response of a pH electrode separately referenced against exemplary reference electrodes #1 and #2 of the present invention compared to a control (Ctrl) which is a pH electrode referenced against a conventional reference electrode. The figure demonstrates that the values obtained for #1 and #2 are equivalent to the values obtained using a conventional reference electrode.

[0018] Figure 7 depicts the response of a Na+ electrode separately referenced against exemplary reference electrodes #1 and #2 of the present invention compared to a control (Ctrl) which is a Na+ electrode referenced against a conventional reference electrode. The figure demonstrates that the values obtained for #1 and #2 are equivalent to the values obtained using a conventional reference electrode.

[0019] Figure 8 depicts the response of a K+ electrode separately referenced against exemplary reference electrodes #1 and #2 of the present invention compared to a control (Ctrl) which is a K+ electrode referenced against a conventional reference electrode. The figure demonstrates that the values obtained for #1 and #2 are equivalent to the values obtained using a conventional reference electrode.

[0020] Figure 9 depicts the response of a Ca++ electrode separately referenced against exemplary reference electrodes #1 and #2 of the present invention compared to a control (Ctrl) which is a Ca++ electrode referenced against a conventional reference electrode. The figure demonstrates that the values obtained for #1 and #2 are equivalent to the values obtained using a conventional reference electrode.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

[0021] The overall nature of the invention will be evident from the following descriptions of the various materials and aspects of the invention.

[0022] The membrane is comprised of a membrane polymer with a polymeric backbone and pendant lipophilic plasticizing groups that provide the polymer with a sufficiently low glass transition temperature (T₉) to mimic the characteristics of a highly plasticized thermoplastic membrane for use in a polymeric reference electrode. The membrane has a short conditioning time. The membrane does not contain plasticizers which are known to leach out of membranes over time. Additionally, the membrane is quite hydrophobic. This can slow the migration of the internal electrolyte from the reference electrode, and furthermore limit biofouling.

[0023] The glass transition temperature (T₉) marks the onset of segmental mobility for a polymer. It is the temperature below which the polymer segments do not have sufficient energy to move past one another. Several factors affect the T₉. Bond interaction, molecular weight, functionality, branching, and chemical structure all affect T₉ and other characteristics of the membrane such as membrane motility and mechanical strength. Accordingly the characteristics of the membrane may be tailored somewhat by the choice of pendant lipophilic plasticizing groups. For instance decreased mobility of polymer chains, increased chain rigidity, and a resulting higher T₉ are obtained where the polymers have many small and rigid substituents as in polymethyl methacrylate (PMMA) or bulky substituents as in polystyrene. Polymers with low glass transition temperatures (e.g., T₉ of -10°C to -75°C) are known and commercially available (e.g., from vendors such as Sartomer Co., Exton, PA.) Such polymers include, but are not limited to, numerous polyacrylates, such as mono- and di-methacrylates. Those skilled in the art will be readily able to select the specific polymers which are best suited for their particular applications, either directly or with the assistance of the vendors.

[0024] The T₉ of the polymer can be measured directly on the polymer using any suitable method, f.ex. "Differential Scanning Calorimetry". Preferably the polymer T₉ is in a range from about -1 O⁰C to about -100⁰C, and a range of -1O⁰C to about -6O⁰C is more preferred.

[0025] The polymeric backbone of the membrane polymer may for instance be a polyvinyl chloride or a polyacrylate backbone. The polyacrylate backbone is preferred. Thus the preferred membrane polymer has an acrylate backbone and is a homopolymer or copolymer of one or more of the following monomers: methyl methacrylate, methacrylate, ethylacrylate, propylacrylate, butyl acrylate, pentyl acrylate, hexylacrylate and heptylacrylate. The methacrylate backbone may be preferred. The polymer must have a moderately rigid backbone. Depending on the T₉ required for the specific application, the polymer may be a homopolymer, a functionalized homopolymer or a copolymer including two or more different monomer units. In general the polymethacrylates yield a relatively higher T₉ in comparison with the corresponding polyacrylates.

[0026] Methods to adjust the T₉ of polymers are well known to those skilled in the art. Accordingly some tailoring of the characteristics of the membrane polymer may take place. Branched chain alkyl acrylates or a- or /?-substituted monomers tend to produce a polymer with a higher T₉ than polymers produced from the corresponding straight chain or non-substituted monomer. Commonly the pendant branch substituents will be C₁-C₁₆ alkyl groups, preferably C₁-Ci₂ alkyl groups and more preferred C₃-C₇ alkyl groups. In a preferred embodiment, the lower alkyl acrylates (C₁ to C₄) are used. Additionally, properties of the membrane polymers can be adjusted by including minor amounts of other monomers. Thus, it may be desirable to adjust the hydrophobic/lipophilic balance by including hydroxyl groups such as hydroxymethyl acrylate. The strength and rigidity of the membrane can also be modified by selection of the type (e.g. difunctional vs. polyfunctional) and quantity of cross-linking reagent.

[0027] A branched alkyl acrylate monomer is an acrylate monomer wherein the alkyl group is non-linear and non-aromatic. Examples of such compounds include methyl methacrylate and /-butylacrylate. A lower alkyl acrylate monomer is an acrylate monomer wherein the alkyl group is a C₁ to C₄. Examples of such compounds include methacrylate, methyl methacrylate, ethylacrylate, propylacrylate, and butyl acrylate.

[0028] In summary the preferred membrane polymer comprises segments of the following formula:

where the lipophilic plasticizing groups R1 and R2 are the same or different and selected from C1 to C16 alkyl groups, preferably C1 to C12 alkyl groups, and R3 and R4 are the same or different and selected from H and CH3. In one preferred embodiment the lipophilic plasticizing groups R1 and R2 are the same and selected from C1 to C7 alkyl groups, preferably C1 to C4 alkyl groups, and R3 and R4 are the same and selected from H and CH3. In another preferred embodiment the lipophilic plasticizing groups R1 are selected from C1 to C3 alkyl groups, the lipophilic plasticizing groups R2 are selected from C4 to C12 alkyl groups, preferably C4 to C7 alkyl groups, and R3 and R4 are the same or different and selected from H and CH₃.. Examples of preferred membrane polymers are poly(butylmethylmethacrylate), poly(butylethylmethacrylate), poly(methylmethacrylate), poly(ethylmethacrylate), poly(butylmethacrylate) and poly(butylacrylate)

[0029] The membrane may also comprise a lipophilic polymer or polymer substituent. The lipophilic component plays an important role in increasing the adhesion and controlling the porosity. The lipophilic polymer is preferably selected from the group consisting of silicone rubber, polyvinyl chloride, polyurethane, polyvinyl chloride carboxylated copolymer or polyvinyl chloride-co-vinyl acetate-co-vinyl alcohol and mixtures thereof. A separate lipophilic additive, such as a lipophilic salt, may be present in the membrane, which lowers the impedance and improves the selectivity over counter ions. Adding cationic and/or anionic lipophilic additives to the membrane is believed to cancel out the effect of positively and/or negatively charged ions in a test solution. The membrane should preferably be equally resistant to diffusion of positively and negatively charged ions. Thus, if the membrane polymer or other added components do not have an inherent higher selectivity to positively or negatively charged ions it is preferred that anionic and cationic lipophilic additives are added in substantially equimolar concentrations. If the membrane polymer or other added components do have an inherent higher selectivity to positively or negatively charged ions this may to a certain degree be cancelled out or accounted for by adding only one of the anionic or cationic lipophilic additives or more of the relevant one. Examples of such additives include the cationic salt potassium tetrakis(4-chlorophenyl)borate (KtpCIPB) and the anionic salt tridodecylmethyl ammonium chloride (TDMAC).

[0030] The membrane may be encased in a protective polymeric layer. The protective layer is used to screen out interfering substances or to improve biocompatibility. Examples of such a protective layer include but are not limited to hydrophilic polyurethane and cellulose acetate.

[0031] A hygroscopic component readily absorbs moisture from the surrounding environment. It improves the wetting and thus provides for a shorter conditioning time and thus a faster establishment of a stable potential. Examples of such materials include glycerol and sorbitol. Examples of such polymers include hydrophilic polyurethane (PU), polyhydroxyethylmethacrylate (pHEMA), polyvinylpyrrolidone (PVP) and polyvinyl- acrylate (PVA).

[0032] An internal electrical contact is typically a thin, flat piece of an appropriate metal, metal alloy, metal oxide or metal salt, for example silver/silver chloride or sodium vanadium bronze such as disclosed in International Patent Application WO 01/65247. The internal electrical contact provides either alone or in electrolytic correspondence with an electrolyte a stable electrical potential. The internal electrical contact is optionally disposed on an inert support material such as a polymer, ceramic, glass or silicon wafer. This provides the possibility of miniaturization of the sensor. [0033] An internal electrode is an internal electrical contact which is optionally in electrolytic correspondence with an internal electrolyte. The internal electrolyte is preferably coated on at least one of its flat surfaces.

[0034] An internal electrolyte is a salt, typically potassium chloride (KCI), sodium chloride (NaCI) or sodium formate, that is applied to at least one flat surface of the internal electrode to enter into electrolytic correspondence with the internal electrical contact. Other salts can also be used, as long as they have substantially equitransferent ions, i.e., cation and anion are of similar size. The preference that the ions of the salt be of similar size is so that they have substantially similar mobilities within the membrane of the invention. The internal electrolyte may be encased in a protective layer of a hydrophilic polymer, such as polyhydroxyethylmethacrylate (pHEMA), polyvinylpyrr lidone (PVP) and polyvinylacrylate (PVA). The electrolyte may also be mixed with a hygroscopic element before application to the contact.

[0035] The reference electrodes of the present invention are stable in substantially all media of interest, notably in complex media such as physiological fluids. Particular interesting media are blood media, such as whole blood serum and plasma. Other interesting media are urine, spinal and interstitial fluids as well as milk.

[0036] The membranes used in the reference electrode are made using methods well known to those skilled in the art. The exact method of preparation of the membrane is not a limitation of the instant invention. A suitable membrane is made by thoroughly mixing n-butyl acrylate (nBA) and methyl methacrylate (MMA) preferably in about a 50:50 to 95:5 rnolar ratio, and more preferably on the order of 80:20. The mixture is aliquotted into vials before polymerization. If the polymerizing agent requiring an initiator is used (e.g. benzoin methyl ether [BME] requires UV light; 2,2'-azobisisobutyronitrile requires heat), the polymerizing agent is added before aliquotting. The mixture is then exposed to the activator, for sufficient time to promote polymerization. Examples of crosslinkers requiring UV initiators include 2,2-dimethoxy-2-phenylacetophenone, benzopheone, bezoyl peroxide and related compounds. Examples of crosslinkers requiring heat as an initiator include benzoyl peroxide and related compounds. If no activation of the polymerizing agent is required, the mixture is aliquotted before addition of the polymerizing agent. The crosslinked polymer is then dissolved using vigorous agitation in an organic solvent, such as cyclohexanone or other organic solvent, to produce a solution of the desired viscosity.

[0037] Optionally the membrane polymer can be blended with one or more additional polymers such as polyvinylchloride, polyurethane, or polyurethane-silicone at varying ratios. Further, the incorporation of lipophilic additives such as potassium tetrakis(4- chlorophenyl)borate (KtpCIPB) and tridodecylmethylammonium chloride (TDMAC) is possible, preferably at about equimolar concentrations. The membrane is prepared by dispensing multiple layers onto the internal electrode, after application of the internal electrical contact and optionally the electrolyte, and allowing the solvent to completely dry between application of each of the layers. The thickness of the membrane can vary, with a preferred thickness of about 3μm. Such considerations are well known to those skilled in the art.

[0038] It is also possible to form the membrane in situ directly on the internal electrode to which the electrolyte has optionally been applied. For example, the monomer mixture, optionally in a suitable solvent, can be placed in the desired position and polymerized by directing the initiator (e.g. UV light) to the portions to be polymerized. Alternatively the membrane polymer can be polymerized in sheets, cut to the desired size and incorporated into an electrode. It is also possible to apply the polymer by methods such as spin coating, inkjet or screen printing. The reference electrode according to the invention may be disposed on a substrate such as a polymer, ceramic, glass or silicon wafer support material. Photopatterning allows for a plurality of different measuring sensors to be incorporated into a single test strip or sensor board with the polymeric reference electrode of the invention. Such methods are well known to those skilled in the art. For instance sensor boards may be prepared, which comprise measuring sensors, which are selective towards one or more parameters selected from the group consisting of pH, pCO₂, pθ₂, electrolytes such as Li⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, cr, HCO₃ ^" and NH₄ ⁺, haemoglobin, haemoglobin derivatives, hematocrit (Hct), and metabolites, such as bilirubin, glucose, lactate, urea, blood urea nitrogen (BUN), creatine or creatinine.

[0039] The internal electrode of the invention comprises an internal electric contact. It is preferably composed of Ag/AgCI, but may be composed of other appropriate materials as mentioned before. Such materials are well known to those skilled in the art. An internal electrolyte, such as KCI or sodium formate, is optionally applied to create a submembrane by dispensing a solution of the electrolyte onto the desired portions of the internal contact to form the internal reference electrode. The use of other electrolytes is possible; however, it is preferred that the ions are of similar size such that their migration rate through the membrane is similar. Hygroscopic elements such as glycerol and sorbitol may also be added to the solution before dispensing the electrolyte solution. After application of the electrolyte solution, the solvent is allowed to evaporate, leaving the electrolyte on the internal contact. The concentration of the electrolyte solution can vary depending on the electrolyte used. Typically a 1-4 M dispensing solution of KCI is used.

[0040] To protect and stabilize the electrolyte coated on the internal contact, the internal electrolyte may be entrapped in a protective layer of hydrophilic polyurethane (PU), polyhydroxyethylmethacrylate (pHEMA), polyvinylpyrrollidone (PVP), polyvinylacrylate (PVA) or any other hydrophilic polymer.

[0041] The exact size and geometry of the polymeric reference electrode is determined by the sensor into which it is incorporated. Such considerations are not a limitation of the instant invention.

EXAMPLE 1 [0042] Preparation of the reference electrode, n-butylacrylate (nBA) and methyl methacrylate (MMA) were combined in an 80:20 molar ratio. Benzoin methyl ether (BME) was added to the solution to a final concentration of 0.5%, and the mixture was stirred rapidly until it was completely dissolved. The solution was then divided into glass scintillation vials with approximately 5 ml of the solution per vial. The vials were then placed under a high intensity UV lamp for about 1 hour until fully polymerized. The polymer was then dissolved in cyclohexanone with vigorous agitation to produce copolymer solution of an appropriate viscosity. The solution was optionally mixed with a solution of PVC before use for coating the internal electrode.

[0043] The internal electrode was prepared by applying a 1-4 M solution of KCI in PVA on an Ag/AgCI contact. The aqueous phase was then dried.

[0044] The reference electrode was formed by coating the submembrane (the internal electrode) with two to three layers of the polymeric membrane of the invention. The electrode was allowed to dry completely between layers. EXAMPLE 2

[0045] Testing of the polymeric reference electrode. Comparison to Calomel reference electrode. The polymeric reference electrode was compared to a commercially available Calomel reference electrode. The mV differential from the test calibration solution to various other test solutions obtained with the polymeric reference electrode was compared to data obtained with a Calomel reference electrode. The polymeric reference electrode of the instant invention was found to be comparably stable to the Calomel reference electrode based on repeated measurements of the series of test solutions.

EXAMPLE 3

[0046] Testing of the Polymeric Reference Electrode. Practical application of the reference electrode. Polymeric reference electrodes were used in conjunction with ion selective electrodes (ISEs) to measure the concentration of various analytes in whole blood and aqueous solutions. The sensors were exposed to extensive testing over several months. The results of the ISEs that were referenced off of the polymeric reference electrode tracked well with the control ISEs that were referenced off of the standard gel electrode used in the ABL™77 analyzer (Radiometer Medical ApS, Denmark). This was true for each of the ions tested; Na⁺, Ca⁺⁺, K⁺ and H⁺ (pH) and for pCO₂. The polymeric reference electrode of the invention was found to produce stable, reproducible results over a range of concentrations of each of the ions within two standard deviations of the average value determined using a National Institute of Standards and Technology (NIST) traceable standard reference method.

[0047] Although exemplary embodiments of the invention have been described above by way of example only, it will be understood by those skilled in the field that modifications and variations may be made to the disclosed embodiments without departing from the scope and spirit of the invention, which is to be defined solely by the appended claims.

Claims

1. A polymeric reference electrode comprising: an internal electrode comprising a contact having a stable electrical potential and a membrane comprising a membrane polymer with a glass transition temperature

(T_g) of less than about 25°C, wherein the membrane polymer comprises lipophilic plasticizing groups pendant from a polymeric backbone.

2. The polymeric reference electrode according to claim 1 , wherein the T₉ of the membrane polymer is less than about O⁰C.

3. The polymeric reference electrode according to claim 2, wherein the T₉ of the membrane polymer is between about -10°C and about -100⁰C, preferably between about -10⁰C and about -60°C.

4. The polymeric reference electrode according to any of the preceding claims, wherein the lipophilic plasticizing groups are selected from C₁ to Ci₆ alkyl groups, preferably Ci to C₁₂ alkyl groups, more preferred C₃ to C₇ alkyl groups.

5. The polymeric reference electrode according to any of the preceding claims, wherein the polymeric backbone of the membrane polymer comprises a polyacrylate backbone.

6. The polymeric reference electrode according to claim 5, wherein the membrane polymer comprises segments of the following formula:

where the lipophilic plasticizing groups R₁ and R₂ are the same or different and selected from C₁ to C₁₆ alkyl groups, preferably C-i to C₁₂ alkyl groups, and R₃ and R₄ are the same or different and selected from H and CH₃.

7. The polymeric reference electrode according to claim 6, wherein the lipophilic plasticizing groups R-i and R₂ are the same and selected from C-i to C₇ alkyl groups, preferably C₁ to C₄ alkyl groups, and R₃ and R₄ are the same and selected from H and CH₃.

8. The polymeric reference electrode according to any of claims 6 or 7, wherein the membrane polymer comprises a homopolymer of a monomer selected from the group consisting of methacrylate, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate and heptyi acrylate.

9. The polymeric reference electrode according to claim 6, wherein the lipophilic plasticizing groups Ri are selected from C₁ to C₃ alkyl groups, the lipophilic plasticizing groups R₂ are selected from C₄ to Ci₂ alkyl groups, preferably C₄ to C₇ alkyl groups, and R₃ and R₄ are the same or different and selected from H and CH₃.

10. The polymeric reference electrode according to any of claims 6 or 9, wherein the membrane polymer comprises a copolymer of at least two monomers selected from the group consisting of methacrylate, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate and heptyi acrylate.

11. The polymeric reference electrode according to claim 10, wherein the membrane polymer is a copolymer of butyl acrylate and methyl methacrylate.

12. The polymeric reference electrode according to claim 11 , wherein the molar ratio of the butyl acrylate to the methyl methacrylate is in a range of about 50:50 to about

95:5, preferably about 80:20.

13. The polymeric reference electrode according to any of the preceding claims, wherein the membrane further comprises at least one lipophilic polymer or polymer substituent.

14. The polymeric reference electrode according to claim 13, wherein the lipophilic polymer or polymer substituent is selected from the group consisting of silicone rubber, polyvinyl chloride (PVC), polyurethane, polyvinylchloride carboxylated copolymer, carboxylated polyvinylchloride, polyvinyl chloride-co-vinyl acetate-co- vinyl alcohol and combinations thereof.

15. The polymeric reference electrode according to any of the preceding claims, wherein the membrane further comprises at least one lipophilic additive.

16. The polymeric reference electrode according to any of the preceding claims, wherein the membrane is covered by at least one polymeric cover layer.

17. The polymeric reference electrode according to claim 16, wherein the at least one polymeric cover layer is selected from the group consisting of hydrophilic polyurethane (PU) and cellulose acetate.

18. The polymeric reference electrode according to any of the preceding claims, wherein the contact of the internal electrode comprises a metal, a metal alloy, a metal oxide, a metal salt or a combination thereof.

19. The polymeric reference electrode according to claim 18, wherein the contact comprises silver/silver chloride.

20. The polymeric reference electrode according to claim 18, wherein the contact comprises sodium vanadium bronze.

21. The polymeric reference electrode according to any of the preceding claims, wherein the internal electrode further comprises an internal electrolyte in electrolytic correspondence with the contact.

22. The polymeric reference electrode according to claim 21 , wherein the internal electrolyte comprises a salt, wherein the cationic and anionic components of the salt are of similar size.

23. The polymeric reference electrode according to any of claims 21 or 22, wherein the internal electrolyte is entrapped in at least one hydrophilic polymer.

24. The polymeric reference electrode according to claim 23, wherein the at least one hydrophilic polymer is selected from the group consisting of polyhydroxyethylmethacrylate (pHEMA), polyvinylpyrrollidone (PVP) and polyvinylacrylate (PVA).

25. The polymeric reference electrode according to any of claims 21 - 24, wherein the internal electrolyte further comprises at least one hygroscopic compound.

26. The polymeric reference electrode according to any of claims 21 - 25, wherein the internal electrolyte coats at least a portion of the contact.

27. The polymeric reference electrode according to any of claims 21 - 26, wherein the membrane is doped with the internal electrolyte.

28. A membrane for use in a polymeric reference electrode comprising a membrane polymer with a glass transition temperature (T₉) of less than about 25°C, wherein the membrane polymer comprises lipophilic plasticizing groups pendant from a polymeric backbone.

29. The membrane according to claim 28, wherein the T₉ of the membrane polymer is less than about O⁰C.

30. The membrane according to claim 29, wherein the T₉ of the membrane polymer is between about -10⁰C and about -100⁰C, preferably between about -10⁰C and about -60°C.

31. The membrane according to any of claims 28 - 30, wherein the lipophilic plasticizing groups are selected from C₁ to C₁₆ alkyl groups, preferably C₁ to C₁₂ alkyl groups, more preferred C₃ to C₇ alkyl groups

32. The membrane according to any of claims 28 - 31 , wherein the polymeric backbone of the membrane polymer comprises a polyacrylate backbone.

33. The membrane according to claim 32, wherein the membrane polymer comprises segments of the following formula:

where the lipophilic plasticizing groups R₁ and R₂ are the same or different and selected from C₁ to C₁₆ alkyl groups, preferably C₁ to C₁₂ alkyl groups, and R₃ and R₄ are the same or different and selected from H and CH₃.

34. The polymeric reference electrode according to claim 33, wherein the lipophilic plasticizing groups R₁ and R₂ are the same and selected from C₁ to C₇ alkyl groups, preferably C₁ to C₄ alkyl groups, and R₃ and R₄ are the same and selected from H and CH₃.

35. The membrane according to any of claims 33 or 34, wherein the membrane polymer comprises a homopolymer of a monomer from the group consisting of methacrylate, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate and heptyl acrylate.

36. The polymeric reference electrode according to claim 33, wherein the lipophilic plasticizing groups R₁ are selected from C₁ to C₃ alkyl groups, the lipophilic plasticizing groups R₂ are selected from C₄ to C₁₂ alkyl groups, preferably C₄ to C₇ alkyl groups, and R₃ and R₄ are the same or different and selected from H and CH₃.

37. The membrane according to any of claims 33 or 36, wherein the membrane polymer comprises a copolymer of at least two monomers selected from the group consisting of methacrylate, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate and heptyl acrylate.

38. The membrane according to claim 37, wherein the membrane polymer is a copolymer of butyl acrylate and methyl methacrylate.

39. The membrane according to claim 38, wherein the molar ratio of the butyl acrylate to the methyl methacrylate is in a range of about 50:50 to about 95:5, preferably about 80:20.

40. The membrane according to any of claims 28 - 39, wherein the membrane further comprises at least one lipophilic polymer or polymer substituent.

41. The membrane according to claim 40, wherein the at least one lipophilic polymer or polymer substituent is selected from the group consisting of silicone rubber, polyvinyl chloride (PVC), polyurethane, polyvinylchloride carboxylated copolymer, carboxylated polyvinylchloride, polyvinyl chloride-co-vinyl acetate-co-vinyl alcohol and combinations thereof.

42. The membrane according to any of claims 28 - 41 , wherein the membrane further comprises at least one lipophilic additive.

.

43. The membrane according to any of claims 28 - 42, wherein the membrane is doped with an internal electrolyte.

44. The membrane according to any of claims 28 - 43, wherein the membrane is covered by at least one polymeric cover layer.

45. The membrane according to claim 44, wherein the at least one polymeric cover layer is selected from the group consisting of hydrophilic polyurethane (PU) and cellulose acetate.

46. Use of the polymeric reference electrode according to any of claims 1 - 27 or the membrane for use in a polymeric reference electrode according to any of claims 28 - 45 in potentiometric measurements of one or more parameters in a physiological fluid, such as blood.

47. A sensor board comprising one or more measuring sensors and a polymeric reference electrode according to any of claims 1 - 27 disposed on an inert support material.

48. The sensor board according to claim 47, wherein the one or more measuring sensors are selective towards one or more parameters selected from the group consisting of pH, pCO₂, pθ₂, electrolytes such as Li⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, Cl^", HCO₃ ^" and NH₄ ⁺, haemoglobin, haemoglobin derivatives, hematocrit (Hct), and metabolites, such as bilirubin, glucose, lactate, urea, blood urea nitrogen (BUN), creatine or creatinine.