阅读说明：本技术 脲生物传感器和脲生物传感器在室温下的稳定化 (Urea biosensor and stabilization of urea biosensor at room temperature ) 是由 徐晓贤 P.帕米迪 H.严 于 2019-06-03 设计创作，主要内容包括：公开了在环境温度下稳定的脲生物传感器,及其制造方法。(Disclosed are urea biosensors that are stable at ambient temperatures, and methods of making the same.)

1. A biosensor, comprising:

an ammonium ion selective electrode, urease immobilized on the ion selective electrode, a diffusion barrier on the surface of the electrode, and a polysaccharide.

2. The biosensor of claim 1, wherein the ammonium ion-selective electrode comprises a metal electrode selected from the group consisting of silver, platinum, and gold.

3. The biosensor of claim 1, wherein the ammonium ion selective electrode comprises a silver/silver chloride internal reference electrode.

4. The biosensor of claim 1, wherein the ammonium ion selective electrode comprises:

a polymer matrix and

an ammonium-selective ionic support disposed on the polymer matrix.

5. The biosensor of claim 4, wherein the polymer matrix is selected from the group consisting of: polyvinyl chloride, polyurethane, poly (tetrafluoroethylene), poly (methyl methacrylate), silicone rubber, and mixtures thereof.

6. The biosensor of claim 4, wherein the polymer matrix comprises polyvinyl chloride.

7. The biosensor of claim 4, wherein the ammonium-selective ionophore is selected from the group consisting of: nonviable bacteria, mono-viable bacteria, di-viable bacteria, tri-viable bacteria, tetra-antibiotics, narasin, hexaoxaheptacycloforty-trioxane, benzocrown ethers, cyclic depsipeptides, and mixtures thereof.

8. The biosensor of claim 4, wherein the ionophore comprises a non-viable biotin.

9. The biosensor of claim 1, wherein the urease is crosslinked.

10. The biosensor of claim 1, wherein the polysaccharide is selected from the group consisting of sucrose, trehalose, raffinose, and lactitol.

11. The biosensor of claim 1, wherein the biosensor is capable of measuring urea.

12. The biosensor of claim 1, wherein the diffusion barrier comprises a polymeric compound selected from the group consisting of: polyurethanes, poly (tetrafluoroethylene) ionomers (perfluorosulfonate ionomers,) Poly (2-methylolmethacrylate), polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof.

13. The biosensor of claim 1, wherein the polysaccharide comprises 10% sucrose.

14. A method of making a stable urea biosensor, comprising:

providing an ammonium ion selective electrode;

casting urease in solution on the outer surface of the PVC ammonium ion selective electrode to form an enzyme layer;

applying a diffusion barrier on a surface of the enzyme layer;

applying a polysaccharide solution to an ammonium ion selective electrode;

the electrodes were dried to form a stable urea biosensor.

15. The method of claim 14, wherein the urease is crosslinked.

16. The method of claim 14, wherein the urease is crosslinked by a chemical selected from the group consisting of: glutaraldehyde, 1, 4-diisocyanatobutane, 1,2,7, 8-diepoxyoctane and 1,2,9, 10-diepoxydedecane, and combinations thereof.

17. The method of claim 14, wherein the ammonium ion selective electrode is selected from the group of electrodes consisting of: silver, platinum and gold.

18. The method of claim 14, wherein the ammonium ion selective electrode comprises a silver/silver chloride electrode.

19. The method of claim 14 wherein the polysaccharide is selected from the group consisting of sucrose, trehalose, raffinose, and lactitol.

20. The method of claim 14, wherein the biosensor is capable of measuring urease.

21. The method of claim 14, wherein applying the diffusion barrier comprises applying: polyurethanes, poly (tetrafluoroethylene) ionomers (perfluorosulfonate ionomers,) Poly (2-hydroxymethylmethacrylate), polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof.

22. The method of claim 14, wherein applying the polysaccharide comprises exposing the electrode to the polysaccharide solution for at least 30 minutes.

23. The method of claim 14, wherein the biosensor maintains stable urea measurement performance after dry storage for 5 months and 21 days use at ambient temperature.

24. A disposable cartridge containing a urea sensor, the urea sensor comprising: an ammonium selective electrode, urease, a diffusion barrier on the surface of the electrode, and a polysaccharide.

25. The disposable cartridge of claim 24, further comprising an array of a plurality of sensors and a calibration reagent.

26. The method of claim 14, wherein the polysaccharide solution is added to the urease solution prior to applying the urease solution to the ammonium ion selective electrode.

27. The method of claim 14, wherein the polysaccharide solution is added to the ammonium ion selective electrode after the diffusion barrier is applied to the enzyme layer.

28. The method of claim 14, wherein the polysaccharide solution is added to the enzyme solution prior to applying the enzyme solution to the ammonium ion selective electrode and the polysaccharide solution is added to the ammonium ion selective electrode after applying the diffusion barrier.

29. The method of claim 14, wherein the polysaccharide solution comprises 10% sucrose.

Technical Field

The present invention relates to a urea biosensor having enzyme stability at room temperature and extended shelf life and service life, and a method for manufacturing the same.

Background

Enzyme biosensors are used to detect many analytes, such as creatinine, creatine, glucose, urea, and lactate, in a sample of a patient's bodily fluid, such as blood. Likewise, enzyme biosensors are particularly important in assisting point-of-care (point-of-care) diagnosis of a patient's disease.

However, one of the drawbacks of enzyme biosensors, especially in point-of-care applications, is the loss of its enzymatic activity in continuous use and its shelf life at ambient temperature (typically less than 15 days). Therefore, short shelf life is a key factor limiting the practical application of enzyme biosensors (such as urea biosensors).

An enzyme biosensor whose shelf life is particularly problematic is a urea biosensor. Measuring urea is important for patient care because it is helpful in determining renal dysfunction in a patient.

The urea sensor is a biosensor comprising an enzyme-modified ammonium ion-selective electrode. Urea hydrolysis is an essential step in the measurement of urea in patient samples (such as blood, serum or plasma) and is catalyzed by urease in a urea sensor as follows:

CO(NH₂)₂+2H₂O^urease>CO₂+2NH₄OH

The product produced after urea hydrolysis catalyzed by urease is ammonium ions.

Urease is immobilized on the surface of the ammonium ion selective electrode by a cross-linking reagent (e.g., glutaraldehyde), or by physical absorption (e.g., as a hydrogel retention) to form an enzyme layer on the electrode. Product ammonium (NH)₄ ⁺) Followed potentiometrically by an ammonium sensor, typically one of the types of sensors in an array of sensors on a substrate or card. Sensor potential vs. card reference electrode (Ag/Ag)⁺) And is measured and is proportional to the logarithm of the urea concentration based on the nernst equation.

After the enzyme layer immobilized on the electrode is formed, a permeable outer polymeric membrane is applied over the top of the enzyme layer of the electrode to protect the enzyme (in this case urease) from denaturation upon contact with the patient body fluid sample and to restrict the flow (flux) of urea substrate from the patient body fluid sample into the enzyme layer.

One major challenge must be overcome for the commercialization, mass production, and practical application of the above-described urea sensors to accurately measure urea in biological samples. The challenge is to obtain long-term stability of urease at ambient temperatures ranging from 15-25 ℃, preferably 18-24 ℃, more preferably 20-24 ℃ and 24 ℃ for storage (shelf life) of at least 5 months to one year or more.

From the design principle, the sensitivity (slope) of the urea sensor to measure urea is directly related to the remaining activity of the immobilized enzyme mixture on the electrode of the biosensor. It is known that biologically active components, such as enzymes, are very fragile (delicates) and are unstable over extended periods of time at ambient temperatures. The rapid decay in sensitivity of urea biosensors due to protein denaturation (loss of slope), and the deterioration in accuracy of measuring the substrate urea at high concentrations (loss of linearity) are the basis of their instability and very limited service or shelf life.

Whole blood analyzers, e.g. GEMThe analyzer (Instrumentation Laboratory Company, Bedford, MA) uses a multi-purpose, single-consumption cartridge, such as the cartridges described in U.S. patent No.6,960,466 and U.S. patent publication No.2004/0256227a1, assigned to Instrumentation Laboratory Company (Bedford, MA), and incorporated herein by reference in its entirety for all purposes and purposes. The cartridge contains all the critical components (sensor array, reference solution, wash solution and calibration reagents), including at least one urea sensor for blood measurement of blood analytes, and requires ambient temperature storage for at least 5 months.

Most commercially available urea sensors with similar designs address the short life and shelf life of urea sensors by refrigerating key components of the biosensor to extend their shelf life. However, this approach adds complexity to the instrumentation of the field operator at the point-of-care location of the hospital. For thePAK cartridges (Instrumentation Laboratory Company, Bedford, MA), for example, biosensors are a compositional and key feature of the cartridge. Urease sensors are an integral part of the cassette and it is impractical to store the entire cassette in refrigeration for the following reasons: cassette size and reagent stability, e.g., reagent stability of reference solution and gas pO of calibration solution₂And pCO₂Stabilization of (3).

Other methods that have been tried to maintain urease activity in urea sensors include changing the enzyme immobilization method from chemical cross-linking immobilization to physical adsorption (retention of urease with polyurethane). The retention methods generally do not provide strong immobilization of the urease as by chemical bonding through cross-linking, and mixing the enzyme in the polyurethane can also affect the urea sensor response time.

The use of mono-or polysaccharides to maintain the activity of biologically active substances, such as enzymes, in the dry stage (stage) in free form or in solution has been reported in the literature.

Sugars can be used as stabilizers to maintain the enzyme in free form during lyophilization (lyophilization). However, the specific sugars that best stabilize an enzyme are based on the structure of the sugar, the hydrophilicity/hydrophobicity of the enzyme and its interaction with water and stabilizers.

It is speculated that the polyhydroxy groups contained in the saccharide may form complexes with water in the presence of water, and that the complexes are able to penetrate into the enzyme structure, even during the cross-linking stage. It is believed that the complex may reduce the unfolding of the enzyme structure leading to denaturation, and thereby maintain the activity of the enzyme.

However, sandwiching the enzyme urease between two polymer layers in a urea sensor brings about a more complex interaction between the enzyme and the components (ionophore, plasticizer, lipophilic salt, organic solvent, etc.) in the two polymer layers, causing instability of the enzyme during the manufacture of the sensor or during storage at ambient temperature. These interactions result in less than expected performance. The invention described herein is directed to addressing the instability of urea sensors due to cross-interactions of enzymes with other chemical components with the goal of improving the stability of the urease during storage of the urea sensor. The invention described herein improves and relates to the storage of stable urea sensors at room temperature for at least 5 months.

Disclosure of Invention

The present invention relates to a urea biosensor stable at room temperature, a manufacturing method and a cartridge containing the stable urea biosensor. The terms sensor and biosensor are used interchangeably throughout.

In one aspect, the invention relates to a method of making a urea biosensor having a stability of at least 5 months shelf life at ambient temperature and a useful life of an additional three weeks. The method comprises the following steps: providing an electrode, preparing a polyvinyl chloride (PVC) based ammonium ion selective membrane on the surface of a silver electrode covered with AgCl and an internal layer as an internal reference electrode as described in U.S. patent publication No.2004/0256227a1(EP1753872B1), which is incorporated herein by reference, casting urease enzyme, i.e., an enzyme mixture, in solution on the outer surface of the PVC membrane to form an enzyme layer, applying a diffusion barrier on the surface of the enzyme layer, applying a polysaccharide solution to a urea sensor, and drying the sensor to form a stable urea biosensor.

The internal reference electrode of the biosensor is selected from the group of metals consisting of, for example: silver, platinum or gold, and covered with AgCl and an electrolyte inner layer. The inner dielectric layer is formed by casting an inner solution comprising MES buffer, sodium chloride, ammonium chloride, and hydroxyethyl cellulose (HEC) on the Ag/AgCl electrode surface.

Urea biosensors are capable of measuring urea in a sample of bodily fluid, such as blood, plasma or serum.

In various embodiments, the step of applying the polysaccharide solution to the urea biosensor includes applying one or more polysaccharides such as, but not limited to, the disaccharides sucrose, trehalose, and lactitol, the trisaccharide raffinose, and others. The polysaccharide may be added to the enzyme mixture prior to casting the sensor with the enzyme mixture, or added to the solution after applying the outer diffusion membrane to the sensor, or as a combination of the above steps. After assembly, the urea biosensor can be immersed in the polysaccharide solution, dried, and immersed again in the polysaccharide solution, followed each time by drying. Optionally, the dipping of the urea biosensor into the polysaccharide solution can be repeated multiple times, followed by drying each time. The concentration of polysaccharide in the solution ranges from greater than 0% to about 25%, and the duration of polysaccharide treatment is thirty minutes or more.

Applying the diffusion barrier comprises applying a polymeric compound to the electrode to form an outer diffusion membrane in contact with a bodily fluid sample introduced into the bodily fluid sample flow chamber (where urea is measured), the polymeric compound selected from the group consisting of: polyurethanes, poly (tetrafluoroethylene) ionomers (perfluorosulfonate ionomers,) Poly (2-hydroxymethylmethacrylate), polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof. The enzyme layer is located between the outer diffusion membrane and the ammonium selective PVC membrane.

The stable urea biosensor according to the method of the present invention maintains stable urea measurement performance after at least 5 months of storage and 21 days of use at ambient temperature.

In another aspect, the present invention relates to a urea biosensor comprising: an ammonium-selective electrode, urease immobilized on an ammonium-selective polymer membrane as an enzyme layer, a diffusion barrier on the surface of the enzyme layer, and a polysaccharide. The stability of the electrodes, ammonium selective polymer membrane, enzyme, cross-linking agent, polysaccharide, diffusion barrier and urea biosensor is described above.

In yet another aspect, the invention relates to a cartridge containing at least one urea sensor in a sensor array, the at least one urea sensor comprising: an ammonium-selective membrane, an enzyme layer comprising urease, a diffusion barrier on a surface of the enzyme layer adjacent to the bodily fluid sample flow chamber, and a polysaccharide. The stability of a urea sensor and a urea biosensor comprising: electrodes, ammonium selective polymer membranes, enzymes, cross-linking agents, polysaccharides, diffusion barriers. In one embodiment according to the invention, the cartridge contains, in addition to the card with a sensor array comprising the enzyme biosensor according to the invention, at least one urea sensor described above in the sensor array, and additionally comprises: a reference solution, a fluid channel, calibration reagents, wash fluids and electronics that operatively interface with the clinical analyzer, and other critical components.

Drawings

FIG. 1 is a cross-sectional diagrammatic illustration of one embodiment of a urea biosensor according to the present invention.

FIG. 2A is a graphical representation of the effect of sucrose treatment on the linearity of urea measurements in twenty-two urea sensors; high urea concentration (70mg/dL) measurements in the samples versus box life (age), with a drying time of 3 hours after sucrose treatment;

FIG. 2B is a graphical representation of the effect of sucrose treatment on urea measurements (mg/dl) on the twenty-two urea sensors in FIG. 2A for a combination of two or three sucrose treatment factors (time delay (days) from sensor manufacture to sensor disaccharide treatment; length of disaccharide treatment soak time (hours); length of disaccharide treated sensor dry time hour);

FIG. 3A is a graphical representation of measured high urea concentration (95mg/dL) versus cartridge life for twelve urea biosensors after a short sensor shelf life of two weeks and a lifetime of up to 3 weeks; daily urea concentration measurements in a high urea sample (95mg/dL) over a 3 week life;

FIG. 3B is a graphical representation of measured high urea concentrations (95mg/dL) versus cartridge life for twelve urea biosensors stored at ambient temperature for 5 months and up to 3 weeks of service life. Daily urea concentration in a high urea sample (95mg/dL) over a 3 week service life was measured.

Detailed Description

The invention described below relates to devices and related methods for enhancing the enzyme stability and extending the shelf-life and useful life of urea biosensors useful in clinical analyzers for in vitro diagnostics, particularly point of care applications.

According to the present invention, polysaccharides (e.g. disaccharides, such as sucrose) are the most optimal components to maintain the stability and activity of the urea biosensor system and to extend its shelf-life and service-life. Other polysaccharides such as trehalose (α -D-glucopyranosyl- α -D-glucopyranoside), raffinose (O- α -D-galactopyranosyl- (1 → 6) - α -D-glucopyranosyl β -D-fructofuranoside) and lactitol (4-O- β -D-galactopyranosyl-D-glucitol) (all polysaccharides are available from Sigma) also improve the stability and activity of urease in urea biosensors, prolonging its shelf life and service life.

For simplicity, 10% sucrose was used as an exemplary polysaccharide for the studies presented below. By virtue of sucrose stabilization, a significant improvement in maintaining urease activity at ambient temperature was observed. A stable shelf life of at least 5 months was achieved when the urea sensor was stored at room temperature after sucrose treatment enzyme stabilization.

As described below, the inventors determined that a disaccharide (e.g., sucrose) is one of the best components for maintaining and stabilizing the activity of a urea biosensor. Other polysaccharides such as trehalose, raffinose and lactitol also have similar effects on the urea sensor, improving stability.

The exemplary urea biosensor 150 shown in fig. 1 comprises a metal element 155 embedded in an electrode card 6 and a composite membrane 160, the composite membrane 160 being located between the metal element 155 and the analyte sample flowing through the channel 20 in the electrode card 6. The composite film 160 includes: an outer diffusion membrane 165 adjacent the patient sample channel 20, an enzyme layer 170, an ammonium selective polymer membrane 175, and an inner solution layer 180 adjacent the metal element 155.

Outer diffusion layer 165 controls diffusion of the analyte into enzyme layer 170 and protects other components of urea biosensor 150 from direct contact with the analyte sample in channel 20. Enzyme layer 170 may include at least one enzyme or a mixture of enzymes, proteins, and stabilizers that react with a particular analyte (i.e., urea in a patient sample). If the analyte diffuses through the outer diffusion membrane 165 into the enzyme layer, it may react with the enzyme in the enzyme layer 170 to produce a chemical by-product, ammonium ions in the case of urea. An electrical potential is generated across the composite membrane 160 that depends on the concentration of chemical byproducts that is proportional to the concentration of urea in the analyte sample. PVC may be a constituent of the ammonium selective polymer membrane 175.

In one embodiment of the present invention, the step of manufacturing a stabilized disaccharide treated urea sensor according to the present invention comprises:

(i) a polyvinyl chloride (PVC) based ammonium ion selective sensor was fabricated on a silver surface 155 covered with AgCl and an internal solution layer 180 to form an internal reference electrode, the PVC ammonium ion selective membrane 175 composition being described in U.S. patent publication No.2004/0256227, which is incorporated herein by reference for all purposes and purposes; then the

(ii) Immobilizing the enzyme urease to form enzyme layer 170 on the outer surface of PVC film 175, for example by applying a cross-linking agent, if applied, for example selected from: glutaraldehyde, diisocyanatobutane, diisocyanato, 1,2,7, 8-diepoxyoctane, 1,2,9, 10-diepoxydedecane, and combinations thereof; alternatively, immobilization of one or more enzymes on the outer surface of PVC membrane 175 may be achieved by physical absorption (e.g., retention with hydrogel); then the

(iii) A hydrophilic polyurethane layer is applied to the enzyme layer 170 to form an outer diffusion membrane 165 for enzyme protection and diffusion control, or one or more of the following polymers, such as: poly (tetrafluoroethylene) ionomers (perfluorosulfonate ionomers,) Poly (2-hydroxymethylmethacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof; then, the user can use the device to perform the operation,

(iv) exposing the urea sensor 150 to a polysaccharide solution, such as a sucrose solution or alternatively a trehalose, raffinose or lactitol solution, at a concentration (w/v) in the range > 0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably 10% solution for at least 30 minutes to 24 hours, at least 30 minutes to 240 minutes, at least 30 minutes to 120 minutes, at least 30 minutes to 60 minutes, preferably at least 30 minutes; then the

(v) An air-dried polysaccharide-treated urea sensor 150; and

(vi) the urea sensor 150 is stored at ambient conditions until use, for example, for 5 months or longer.

In an alternative embodiment of making a stable urea biosensor 150, the steps include;

(i) a polyvinyl chloride (PVC) based ammonium ion selective sensor was fabricated on a silver surface 155 covered with AgCl and an internal solution layer 180 to form an internal reference electrode, PVC ammonium ion selective membrane 175 being composed as described above;

(ii) preparing urease in a polysaccharide solution, such as a sucrose solution or alternatively a solution of trehalose, raffinose, or lactitol, at a concentration (w/v) in the range > 0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably 10% solution

(iii) (iii) applying the urease-polysaccharide solution described in step (ii) and immobilizing the enzyme urease in the polysaccharide solution on the outer surface of the PVC film 175 to form the enzyme layer 170, for example by applying a cross-linking agent, if applied, for example selected from: glutaraldehyde, diisocyanatobutane, diisocyanato, 1,2,7, 8-diepoxyoctane, 1,2,9, 10-diepoxydedecane, and combinations thereof; then the

(iv) To enzyme layer 170 is applied a hydrophilic polyurethane layer 165 or one or more of the following polymers for enzyme protection and diffusion control, such as: poly (tetrafluoroethylene) ionomers (perfluorosulfonate ionomers,) Poly (2-hydroxymethylmethacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof; then the

(v) The urea sensor 150 is stored at ambient conditions until use, for example, for 5 months or longer.

In another embodiment of making a stable urea biosensor 150, the steps comprise:

(i) a polyvinyl chloride (PVC) based ammonium ion selective sensor was fabricated on a silver surface 155 covered with AgCl and an internal solution layer 180 to form an internal reference electrode, PVC ammonium ion selective membrane 175 being composed as described above;

(ii) preparing a urease enzyme solution in a polysaccharide solution, such as a sucrose solution or alternatively a solution of trehalose, raffinose or lactitol in water, at a concentration (w/v) in the range > 0% 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably 10%;

(iii) (iii) applying the urease-polysaccharide solution described in step (ii) and immobilizing the enzyme urease in the polysaccharide solution on the outer surface of the PVC film 175 to form the enzyme layer 170, for example by applying a cross-linking agent, if applied, for example selected from: glutaraldehyde, diisocyanatobutane, diisocyanato, 1,2,7, 8-diepoxyoctane, 1,2,9, 10-diepoxydedecane, and combinations thereof; then the

(iv) To enzyme layer 170 is applied a hydrophilic polyurethane layer 165 or one or more of the following polymers for enzyme protection and diffusion control, such as: poly (tetrafluoroethylene) ionomers (perfluorosulfonate ionomers,) Poly (2-hydroxymethylmethacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof; then the

(v) Exposing the urea sensor 150 to a polysaccharide solution, such as a sucrose solution or alternatively a trehalose, raffinose or lactitol solution, at a concentration (w/v) in the range > 0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably 10% solution for at least 30 minutes to 24 hours, at least 30 minutes to 240 minutes, at least 30 minutes to 120 minutes, at least 30 minutes to 60 minutes, preferably at least 30 minutes; then the

An air-dried polysaccharide-treated urea sensor 150; optionally repeating the step of exposing the sensor to the polysaccharide solution once, twice, 3-4 times, 5-8 times, and 9-10 times, and then

The urea sensor 150 is stored at ambient conditions until use, for example, five months or longer.

Examples

Example 1

Example 1, described below, provides one embodiment of a method of making a sucrose-stabilized urea sensor.

1. According to one method of the invention, for example, a solution for an ammonium selective polymer membrane is prepared in THF and contains by weight: 25-35% PVC, 60-70% DOS, 1-5% non-viable bacteria, and 0-1% KTpClPB.

The non-viable bacteria are ionophores with a specific selectivity for ammonium. Other agents, such as, for example, mono-, di-, tri-, tetra-, narasin, benzocrown, cyclic depsipeptides, and mixtures of the foregoing, may also be used for this purpose.

DOS (di (2-ethylhexyl) sebacate) is a plasticizer. O-nitrophenyloctyl ether (NPOE) is another commonly used plasticizer.

KTpClPB (potassium tetrakis [ 4-chlorophenyl ] borate) is a lipophilic salt. Potassium tetrakis [3, 5-bis (trifluoromethyl) phenyl ] borate (KTTFPB) may also be used for this purpose.

In addition to PVC, the polymer film may include, for example: polyurethanes, poly (tetrafluoroethylene), poly (methyl methacrylate), silicone rubbers, and mixtures thereof.

THF (tetrahydrofuran) is the solvent. Cyclohexanone can also be used for this purpose.

2. The ammonium sensor is planar and is formed by casting an ammonium selective polymer membrane solution prepared as described above over an Ag/AgCl metal electrode and an internal electrolyte layer, and the internal electrolyte layer forms an internal reference electrode embedded in a solid substrate (e.g., without limitation, PVC). The internal electrolyte layer is formed by casting an internal solution on the surface of the Ag/AgCl electrode. The internal solution contains, for example, 65% MES buffer, 1% sodium chloride, 1% ammonium chloride and 33% HEC (weight percent) in water.

3. The enzyme layer is formed by casting an enzyme solution on the outer surface of the ammonium selective PVC membrane. The enzyme solution comprises urease, an enzyme stabilizer glutathione, an inert protein bovine serum albumin, a cross-linking reagent glutaraldehyde and a solvent. Glutathione is used with one or more inert proteins (e.g., bovine serum albumin) to stabilize the urease in the enzyme layer. Crosslinking also binds the enzyme layer to the underlying ion-selective polymer layer. During the manufacture of the enzyme layer, an enzyme stabilizer is typically added to the enzyme-containing solution prior to the addition of the crosslinking reagent to ensure that the stabilizer is crosslinked with the enzyme. A typical enzyme solution containing 50mg/mL urease, 20mg/mL glutathione, 10mg/mL bovine serum albumin and 0.12% glutaraldehyde was prepared in 0.1M phosphate buffer at pH 7.2 and applied to the top of an ammonium ion selective PVC membrane.

4. Polyurethane (PU) is one of the polymers with excellent biocompatibility in many successful in vivo and in vitro applications in medical devices. The particular hydrophilic medical grade polyurethane class optimized for this application was selected as Tecophlic from Lubrizol (Wickliffe, Ohio)^TMAnd Tecoflex^TM. These commercially available polymer resins are aliphatic, polyether-based polyurethanes that are soluble in organic solvents or mixtures of solvents such as Dimethylacetamide (DMA), Tetrahydrofuran (THF), and the like. Within these polyurethane classes, there are different grades of materials available with a combination of various hardness and water absorption levels for different applications. In various embodiments, the outer diffusion membrane of a urea biosensor that is in contact with a patient sample comprises one or more distinct layers of the same or different polymers and/or the same or different copolymers. A typical external diffusion membrane solution was prepared in THF, containing 0.12g/ml polyurethane (Lubrizol, Wickliffe, Ohio). Applying an outer diffusion membrane solution on the enzyme layer to form an outer diffusion membrane. In addition to polyurethanes, one or more of the following polymers may also be candidates for an external diffusion membrane:(poly (tetrafluoroethylene) ionomer), poly (2-hydroxymethyl methacrylate), polyvinyl chloride, polycarbonate, and cellulose acetate.

5. In one embodiment of the invention, the urea sensor with the composite membrane of inner layer-PVC layer-enzyme layer-polyurethane layer is subsequently immersed in a disaccharide solution, for example a sucrose solution, at a concentration (w/v) in the range > 0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably 10% solution for at least 30 minutes to 24 hours, at least 30 minutes to 240 minutes, at least 30 minutes to 120 minutes, at least 30 minutes to 60 minutes, preferably at least 30 minutes. The sucrose solution was buffered at a biological pH of 7.4.

6. The sensor was dried at room temperature for 0.5 to 3 hours. Optionally, the sucrose treatment is repeated multiple times and then air dried each time. The sensor is then stored at ambient temperature until use.

Example 2.

Example 2 is a study to evaluate the effect of sucrose treatment on the stabilization of the above-described urea sensor at ambient temperature using a multi-factor experimental design that was conducted with three main factors, each under two conditions, to study (i) the delay time between sensor fabrication and exposure of the fabricated sensor to a sucrose solution (2 or 7 days), (ii) the soak time (2 or 16 hours), which is the duration of exposure of the sensor to the sucrose solution, and the dry time after sucrose exposure was completed (0.5 or 3 hours). Subgroups of urea sensors are assembled into a cartridge and for life stability, inUrea concentration was measured in a Premier clinical analyzer. Aqueous samples with high concentrations of urea (70mg/dL) were measured daily on a urea sensor in a cartridge to assess the linearity of the urea sensor measurements.

Referring to FIG. 2A, the life stability of each of the tested urea sensors under various applied experimental conditions when the drying time was fixed at 3 hours is provided. Figure 2B illustrates the effect of experimental conditions on urea measurements.

The results shown in fig. 2A are easy to summarize. The conditions applied with a shorter delay time (2 days) and a longer soak time in a 10% sucrose solution (16 hours) provided the most stable performance and the most consistent sensor-to-sensor performance over the lifetime of the sensor. The longer delay time (7 days) condition combined with a shorter soaking time (2 hours) in a 10% sucrose solution resulted in the worst stabilization performance. The other two conditions provided better performance compared to the worst performance of 7 day delay time/2 hour soak time, but also exhibited sensor-to-sensor inconsistencies (7 days, 16 hours), or performance degradation over the lifetime (2 days, 2 hours). Figure 2B illustrates urea measurements and the favorable direction for three sucrose treatment condition factors: shorter delay times, longer soaking times and longer drying times.

These results demonstrate that urease enzyme activity in the disaccharide treated urea biosensor continued to decay with manager, even when urease was immobilized on the sensor. The faster the fabricated sensor was exposed to sucrose treatment, the better the urease activity was maintained. These studies also show that removal of all water (e.g., by drying) is critical for long-term enzyme stability.

Example 3

Example 3 stability data at ambient temperature for urea sensors made according to the present invention from multiple production lots is provided in a three week life stability and 5 month shelf life stability study.

The cartridges were tested daily for urea in aqueous samples with high concentrations of urea (95mg/dL) over a 3 week life to evaluate sensor urea measurement linearity over time.

Figure 3A graphically illustrates urea measured in twelve urea sensors selected from three batches of sucrose-treated sensors and in a short shelf-life storage at ambient temperature of two weeks. The high urea concentration (95mg/dL) samples measured daily over a three week service life with a short sensor shelf life exhibited stable service life performance, with the measured urea concentration consistently being about 95 mg/dL.

Figure 3B graphically illustrates the results of the other twelve urea sensors from three different batches of sucrose-treated urea sensors. Shelf life when stored at ambient temperature of 5 months the high urea concentration (95mg/dL) samples measured daily over a three week service life exhibited the same stable life performance, with the measured urea concentration consistently being about 95 mg/dL.

By applying the polysaccharide treatment (sucrose was used as an exemplary polysaccharide in these studies), urea sensor activity and stability were maintained over a storage shelf life at ambient temperature of 5 months.

The invention described above is applicable to urea biosensors developed on a variety of urea sensor platforms.