Urea biosensor and stabilization of urea biosensor at room temperature

Abstract

Urea biosensors that are stable at ambient temperature, and methods for making the same are disclosed.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 830,191, filed on April 5, 2019, and is incorporated herein by reference in its entirety for all intents and purposes. Technical Field

[0003] The present invention relates to a urea biosensor and a method for making the same, which has enzyme stability at room temperature and an extended shelf life and service life. Background Art

[0004] Enzyme biosensors are used to detect many analytes, such as creatinine, creatine, glucose, urea, and lactate, in patient fluid samples (such as blood). As such, enzyme biosensors are particularly important in assisting point-of-care diagnosis of patient diseases.

[0005] However, one of the disadvantages of enzyme biosensors, especially in point-of-care applications, is the loss of enzyme activity during continuous use and its shelf life at ambient temperature, which is typically less than 15 days. Therefore, the short shelf life is a key factor limiting the practical application of enzyme biosensors, such as urea biosensors.

[0006] An enzyme biosensor for which shelf life is particularly problematic is the urea biosensor. Measuring urea is important for patient care because it is helpful in determining poor renal function in a patient.

[0007] Urea sensors are biosensors that contain an enzyme-modified ammonium ion selective electrode. Urea hydrolysis is a necessary step to measure urea in patient samples (such as blood, serum or plasma), which is catalyzed by urease in the urea sensor as follows:

[0008] CO(NH2)2+2H2O 脲酶 >CO2+2NH4OH

[0009] The product produced after urea hydrolysis catalyzed by urease is ammonium ions.

[0010] Urease is immobilized on the surface of the ammonium ion selective electrode by a cross-linking agent (e.g., glutaraldehyde) or by physical absorption (e.g., entrapment in a hydrogel) to form an enzyme layer on the electrode. + ) is then detected potentiometrically by an ammonium sensor, typically one of a type of sensor in a sensor array located on a substrate or card. The sensor potential is relative to the card reference electrode (Ag / Ag + ) and is proportional to the logarithm of the urea concentration based on the Nernst equation.

[0011] After forming the enzyme layer immobilized on the electrode, a permeable outer polymeric membrane is applied over the top of the enzyme layer of the electrode to protect the enzyme (urease in this case) from denaturation when in contact with the patient fluid sample and to limit the flow (flux) of urea substrate from the patient fluid sample into the enzyme layer.

[0012] For the commercialization, mass production and practical application of the above urea sensor to accurately measure urea in biological samples, a major challenge must be overcome. The challenge is to obtain long-term stability of urease at ambient temperatures ranging from 15-25°C, preferably 18-24°C, more preferably 20-24°C and 24°C for at least 5 months to one year or more of storage (shelf life).

[0013] From the design principle point of view, the sensitivity (slope) of the urea sensor measuring urea is directly related to the residual activity of the fixed enzyme mixture on the electrode of the biosensor. It is known that biologically active components (such as enzymes) are very fragile (delicate) and are unstable over a prolonged period of time at ambient temperature. The rapid decay of the sensitivity (slope loss) of the urea biosensor due to protein denaturation and the degradation of the accuracy (linearity loss) of measuring the substrate urea at high concentrations are the basis of its instability and very limited service life or shelf life.

[0014] Complete blood analyzer, such as GEM Analyzer (Instrumentation Laboratory Company, Bedford, MA), using a multi-use, single-consumable cartridge such as that 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 their entirety for all intents and purposes. The cartridge contains all key components (sensor array, reference solution, flushing fluid, 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.

[0015] Most commercially available urea sensors of similar design address the short service life and shelf life of urea sensors by refrigerating key components of the biosensor to extend its shelf life. However, this approach adds complexity to instrument operation for field operators at point-of-care locations in hospitals. PAK cartridges (Instrumentation Laboratory Company, Bedford, MA), for example, the biosensor is an integral and key feature of the cartridge. The urease sensor is an integral part of the cartridge, and it is impractical to store the entire cartridge in a refrigerator for the following reasons: cartridge size and reagent stability, for example, reagent stability of the reference solution and stabilization of the gas pO2 and pCO2 of the calibration solution.

[0016] Other methods that have been tried to maintain urease activity in urea sensors include changing the enzyme immobilization method from chemical cross-linking to physical adsorption (retention of urease with polyurethane). The retention method generally does not provide as strong immobilization of urease as chemical bonding through cross-linking, and mixing the enzyme in the polyurethane can also affect the urea sensor response time.

[0017] It has been reported in the literature that monosaccharides or polysaccharides can be used to maintain the activity of biologically active substances such as enzymes in free form, in the dry stage or in solution.

[0018] Sugars can be used as stabilizers to maintain the enzyme in free form during freeze drying (lyophilization). However, the specific sugar that best stabilizes an enzyme is based on the structure of the sugar, the hydrophilicity / hydrophobicity of the enzyme, and its interaction with water and the stabilizer.

[0019] It is speculated that in the presence of water, the polyhydroxyl groups contained in the sugar can form a complex with water, and this complex can penetrate into the enzyme structure even in the cross-linking stage. It is believed that the complex can reduce the unfolding of the enzyme structure that causes denaturation and thus maintain the activity of the enzyme.

[0020] However, sandwiching the enzyme urease between two polymer layers in a urea sensor brings about more complex interactions between the enzyme and the components in the two polymer layers (ion carriers, plasticizers, lipophilic salts, organic solvents, etc.), causing instability of the enzyme during the preparation of the sensor or during storage at ambient temperature. These interactions lead to lower than expected performance. The purpose of the invention described herein is to solve the instability of the urea sensor due to the cross-interaction of the enzyme with other chemical components, with the aim of improving the stability of the urease during storage of the urea sensor. The invention described herein improves and relates to the storage of a stable urea sensor at room temperature for at least 5 months. Summary of the invention

[0021] The present invention relates to a urea biosensor that is stable at room temperature, a method of making the same and a cartridge containing the same. The terms sensor and biosensor are used interchangeably throughout.

[0022] In one aspect, the present invention relates to a method for making a urea biosensor having a shelf life stability of at least 5 months at ambient temperature and an additional service life of three weeks. The method comprises providing an electrode, preparing a polyvinyl chloride (PVC) based ammonium ion selective membrane on a silver electrode surface covered with AgCl and an inner layer as an inner reference electrode as described in U.S. Patent Publication No. 2004 / 0256227A1 (EP1753872B1), which is incorporated herein by reference, casting a urease enzyme in a solution, i.e., an enzyme mixture, on an 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 the urea sensor, and drying the sensor to form a stable urea biosensor.

[0023] The internal reference electrode of the biosensor is selected from the group of metals such as silver, platinum or gold and is covered with an internal layer of AgCl and electrolyte. The internal dielectric layer is formed by casting an internal solution on the Ag / AgCl electrode surface, the internal solution comprising MES buffer, sodium chloride, ammonium chloride and hydroxyethyl cellulose (HEC).

[0024] Urea biosensors are capable of measuring urea in body fluid samples such as blood, plasma or serum.

[0025] In a number of embodiments, the step of applying a polysaccharide solution to the urea biosensor includes applying one or more polysaccharides, such as, but not limited to, disaccharides sucrose, trehalose and lactitol, trisaccharide raffinose, and other polysaccharides. The polysaccharide can be added to the enzyme mixture before the sensor is cast with the enzyme mixture, or the polysaccharide is added to the solution after the external diffusion membrane is applied to the sensor, or added as a combination of the above steps. After assembly, the urea biosensor can be immersed in the polysaccharide solution, dried, and immersed in the polysaccharide solution again, followed by drying each time. Optionally, immersing the urea biosensor in the polysaccharide solution can be repeated multiple times, followed by drying each time. The concentration range of the polysaccharide in the solution is greater than 0% to about 25%, and the duration of the polysaccharide treatment is thirty minutes or longer.

[0026] Applying a diffusion barrier comprises applying a polymeric compound to the electrode to form an outer diffusion membrane in contact with a body fluid sample introduced into a body fluid sample flow chamber (wherein urea is measured), the polymeric compound being selected from the group consisting of polyurethane, poly(tetrafluoroethylene) ionomer (perfluorosulfonate ionomer, ), poly(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof. The enzyme layer is located between the external diffusion membrane and the ammonium selective PVC membrane.

[0027] The stable urea biosensor according to the method of the present invention maintained stable urea measurement performance after at least 5 months storage and 21 days use at ambient temperature.

[0028] 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 electrode, the ammonium selective polymer membrane, the enzyme, the cross-linking agent, the polysaccharide, the diffusion barrier, and the urea biosensor is described above.

[0029] In yet another aspect, the present invention relates to a box 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 the surface of the enzyme layer adjacent to a body fluid sample flow chamber, and a polysaccharide. The stability of the urea sensor and the urea biosensor is described above, the urea sensor comprising: an electrode, an ammonium selective polymer membrane, an enzyme, a cross-linking agent, a polysaccharide, a diffusion barrier. In one embodiment according to the present invention, in addition to a card having a sensor array including an enzyme biosensor according to the present invention, the box also contains at least one urea sensor described above in a sensor array, and additionally includes: a reference solution, a fluid channel, a calibration reagent, a flushing fluid and an electronic component that is operatively docked with a clinical analyzer, and other key components. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a cross-sectional diagrammatic illustration of one embodiment of a urea biosensor according to the present invention.

[0031] Figure 2A is a graphical illustration of the effect of sucrose treatment on the linearity of urea measurement in twenty-two urea sensors; high urea concentration (70 mg / dL) in the sample is measured relative to the cell age (age) with a 3 hour drying time after sucrose treatment;

[0032] Figure 2B is Figure 2A Figure 1. Effect of sucrose treatment on urea measurements (mg / dl) for combinations of two or three sucrose treatment factors (time delay from sensor fabrication to sensor disaccharide treatment (days); length of disaccharide treatment soaking time (hours); length of disaccharide treated sensor drying time (hours)) on twenty-two urea sensors in FIG.

[0033] Figure 3A is a graphical representation of measured high urea concentrations (95 mg / dL) versus cartridge life for twelve urea biosensors after a short sensor shelf life of two weeks and up to a 3 week service life; daily urea concentration measurements in high urea samples (95 mg / dL) over a 3 week service life;

[0034] Figure 3B Figure 1 is a graph of high urea concentration (95 mg / dL) measured versus cartridge life for twelve urea biosensors over 5 months storage and up to 3 weeks of service life at ambient temperature. Daily urea concentration measurements in high urea samples (95 mg / dL) over 3 weeks of service life. DETAILED DESCRIPTION

[0035] 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.

[0036] According to the present invention, polysaccharides (e.g., disaccharides such as sucrose) are the optimal ingredients for maintaining the stability and activity of the urea biosensor system and extending its shelf life and service life. Other polysaccharides such as trehalose (α-D-pyranoglucopyranosyl-α-D-pyranoglucoside), raffinose (O-α-D-galactopyranosyl-(1→6)-α-D-pyranoglucopyranosyl β-D-fructofuranoside) and lactitol (4-O-β-D-galactopyranosyl-D-glucitol) (all polysaccharides were obtained from Sigma) also improved the stability and activity of urease in urea biosensors and extended their shelf life and service life.

[0037] For simplicity, 10% sucrose was used as an example polysaccharide for the studies presented below. With sucrose stabilization, a significant improvement in maintaining the activity of urease at ambient temperature was observed. When the urea sensor was stored at room temperature after sucrose treatment enzyme stabilization, a stable shelf life of at least 5 months was achieved.

[0038] As described below, the inventors determined that disaccharides (eg, sucrose) are one of the best components for maintaining and stabilizing the activity of urea biosensors. Other polysaccharides such as trehalose, raffinose, and lactitol also have similar effects on urea sensors, improving stability.

[0039] Figure 1 The typical urea biosensor 150 shown in the figure 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 analysis sample flowing through the channel 20 in the electrode card 6. The composite membrane 160 includes: an external diffusion membrane 165 adjacent to the patient sample channel 20, an enzyme layer 170, an ammonium selective polymer membrane 175, and an internal solution layer 180 adjacent to the metal element 155.

[0040] The external diffusion layer 165 controls the diffusion of the analyte into the enzyme layer 170 and protects the other components of the urea biosensor 150 from direct contact with the analysis sample in the channel 20. The enzyme layer 170 may include at least one enzyme or a mixture of multiple enzymes, proteins, and stabilizers that react with a specific analyte (i.e., urea in a patient sample). If the analyte diffuses through the external diffusion membrane 165 into the enzyme layer, it may react with the enzyme in the enzyme layer 170 to produce a chemical byproduct, which is ammonium ions in the case of urea. A potential is generated across the composite membrane 160, which depends on the concentration of the chemical byproduct, which is proportional to the concentration of urea in the analysis sample. PVC may be a constituent of the ammonium selective polymer membrane 175.

[0041] In one embodiment of the present invention, the steps of making a stable disaccharide-treated urea sensor according to the present invention include:

[0042] (i) fabricating a polyvinyl chloride (PVC) based ammonium ion selective sensor 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 intents and purposes; and then

[0043] (ii) immobilizing the enzyme urease to form an enzyme layer 170 on the outer surface of the PVC film 175, for example by applying a cross-linking agent, if applied, the cross-linking agent is selected from the group consisting of glutaraldehyde, diisocyanatobutylene, diisocyanate, 1,2,7,8-diepoxyoctane, 1,2,9,10-diepoxydecane, and combinations thereof; alternatively, immobilizing one or more enzymes on the outer surface of the PVC film 175 can be achieved by physical absorption (e.g., retention with a hydrogel); then

[0044] (iii) applying a hydrophilic polyurethane layer to the enzyme layer 170 to form an external diffusion membrane 165 for enzyme protection and diffusion control, or applying one or more of the following polymers, such as: poly(tetrafluoroethylene) ionomer (perfluorosulfonate ionomer, ), poly(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof; then,

[0045] (iv) exposing the urea sensor 150 to a polysaccharide solution, such as a sucrose solution or alternatively a solution of trehalose, raffinose or lactitol, at a concentration (w / v) ranging from >0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably a 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; and then

[0046] (v) air drying the polysaccharide treated urea sensor 150; and

[0047] (vi) Storing the urea sensor 150 under ambient conditions until use, for example, 5 months or longer.

[0048] In an alternative embodiment of making a stable urea biosensor 150, the steps include;

[0049] (i) fabricating a polyvinyl chloride (PVC) based ammonium ion selective sensor 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 being composed as described above;

[0050] (ii) preparing urease in a polysaccharide solution, such as a sucrose solution or alternatively a solution of trehalose, raffinose, or lactitol, in a concentration (w / v) range of >0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably a 10% solution

[0051] (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, the cross-linking agent is selected from the group consisting of glutaraldehyde, diisocyanatobutylene, diisocyanate, 1,2,7,8-diepoxyoctane, 1,2,9,10-diepoxydecane, and combinations thereof; and then

[0052] (iv) applying a hydrophilic polyurethane layer 165 or one or more of the following polymers to the enzyme layer 170 for enzyme protection and diffusion control, such as poly(tetrafluoroethylene) ionomer (perfluorosulfonate ionomer, ), poly(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof; then

[0053] (v) The urea sensor 150 is stored under ambient conditions until use, for example, 5 months or longer.

[0054] In another embodiment of making the stable urea biosensor 150, the steps include:

[0055] (i) fabricating a polyvinyl chloride (PVC) based ammonium ion selective sensor 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 being composed as described above;

[0056] (ii) preparing a urease enzyme solution in a polysaccharide solution, such as a sucrose solution in water or alternatively a solution of trehalose, raffinose or lactitol, in a concentration (w / v) range of >0% 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably a 10% solution;

[0057] (iii) applying the urease enzyme-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, the cross-linking agent is selected from the group consisting of glutaraldehyde, diisocyanatobutylene, diisocyanate, 1,2,7,8-diepoxyoctane, 1,2,9,10-diepoxydecane, and combinations thereof; and then

[0058] (iv) applying a hydrophilic polyurethane layer 165 or one or more of the following polymers to the enzyme layer 170 for enzyme protection and diffusion control, such as poly(tetrafluoroethylene) ionomer (perfluorosulfonate ionomer, ), poly(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof; then

[0059] (v) exposing the urea sensor 150 to a polysaccharide solution, such as a sucrose solution or alternatively a solution of trehalose, raffinose or lactitol, at a concentration (w / v) ranging from >0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably a 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; and then

[0060] air drying the 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

[0061] The urea sensor 150 is stored under ambient conditions until use, for example, five months or longer.

[0062] Example

[0063] Example 1

[0064] Example 1 described below provides one embodiment of a method for making a sucrose-stabilized urea sensor.

[0065] 1. According to a method of the present 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% inactive antibiotics, and 0-1% KTpClPB.

[0066] Anabactin is an ionophore with a specific selectivity for ammonium. Other agents, for example, monoactin, diactin, triactin, tetraactin, narasin, benzocrown ether, cyclic depsipeptide and mixtures of the foregoing, can also be used for this purpose.

[0067] DOS (di(2-ethylhexyl) sebacate) is a plasticizer. O-nitrophenyl octyl ether (NPOE) is another commonly used plasticizer.

[0068] KTpClPB (potassium tetrakis[4-chlorophenyl]borate) is a lipophilic salt. Potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTTFPB) can also be used for this purpose.

[0069] In addition to PVC, the polymer film may include, for example, polyurethane, poly(tetrafluoroethylene), poly(methyl methacrylate), silicone rubber, and mixtures thereof.

[0070] THF (tetrahydrofuran) is the solvent. Cyclohexanone can also be used for this purpose.

[0071] 2. The ammonium sensor is planar and is formed by casting an ammonium selective polymer membrane solution prepared as described above on an Ag / AgCl metal electrode and an inner electrolyte layer, and the inner electrolyte layer forms an inner reference electrode embedded in a solid substrate (for example, but not limited to PVC). The inner electrolyte layer is formed by casting an inner solution on the surface of the Ag / AgCl electrode. The inner solution contains, for example, 65% MES buffer, 1% sodium chloride, 1% ammonium chloride, and 33% HEC (weight percentage) in water.

[0072] 3. The enzyme layer is formed by casting an enzyme solution on the outer surface of the ammonium selective PVC membrane. The enzyme solution includes urease, enzyme stabilizer glutathione, inert protein bovine serum albumin, cross-linking agent glutaraldehyde and solvent. Glutathione is used together with one or more inert proteins (such as bovine serum albumin) to stabilize the urease in the enzyme layer. Cross-linking also binds the enzyme layer to the ion-selective polymer layer below. During the manufacture of the enzyme layer, an enzyme stabilizer is generally added to the solution containing the enzyme before adding the cross-linking agent to ensure that the stabilizer is cross-linked with the enzyme. A typical enzyme solution containing 50 mg / mL urease, 20 mg / mL glutathione, 10 mg / mL bovine serum albumin and 0.12% glutaraldehyde is prepared in 0.1 M phosphate buffer at pH 7.2, and the solution is applied to the top of the ammonium ion selective PVC membrane.

[0073] 4. Polyurethane (PU) is one of the polymers with excellent biocompatibility in many successful in vivo and in vitro applications in medical devices. The optimized specific hydrophilic medical grade polyurethane type selected for this application is Tecophlic from Lubrizol (Wickliffe, Ohio). TM and 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), etc. Within these polyurethane categories, there are available materials of different grades with combinations of multiple hardness and water absorption levels for different applications. In various embodiments, the external diffusion membrane of the urea biosensor in contact with the patient sample comprises different layers of one or more identical or different polymers and / or identical or different copolymers. A typical external diffusion membrane solution is prepared in THF containing 0.12 g / ml polyurethane (Lubrizol, Wickliffe, Ohio). The external diffusion membrane solution is applied to the enzyme layer to form the external diffusion membrane. In addition to polyurethane, one or more of the following polymers may also be candidates for the external diffusion membrane: (poly(tetrafluoroethylene) ionomer), poly(2-hydroxymethyl methacrylate), polyvinyl chloride, polycarbonate, and cellulose acetate.

[0074] 5. In one embodiment of the present invention, the urea sensor having a composite membrane of inner layer-PVC layer-enzyme layer-polyurethane layer is then immersed in a disaccharide solution, such as a sucrose solution, with a concentration (w / v) ranging from >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 is buffered at a biological pH of 7.4.

[0075] 6. The sensor is dried at room temperature for 0.5 to 3 hours. Optionally, the sucrose treatment is repeated multiple times, followed by air drying each time. The sensor is then stored at ambient temperature until use.

[0076] Example 2.

[0077] Example 2 is a study evaluating the effect of sucrose treatment on the stabilization of the above urea sensors at ambient temperature using a multi-factorial experimental design with three main factors (each factor under two conditions) to study - (i) the delay time between sensor fabrication and exposure of the fabricated sensors to sucrose solution (2 or 7 days), (ii) the soaking time (2 or 16 hours), which is the duration of sensor exposure to sucrose solution, and the drying time after the sucrose exposure is completed (0.5 or 3 hours). Subgroups of the urea sensors were assembled into cartridges and for lifetime stability, the urea sensors were tested at room temperature. Urea concentrations were measured in a Premier clinical analyzer. Aqueous samples with high concentrations of urea (70 mg / dL) were measured daily on the urea sensor in the cartridge to evaluate the linearity of the urea sensor measurement.

[0078] Reference Figure 2A , provides the service life stability of each tested urea sensor under various applied experimental conditions when the drying time is fixed at 3 hours. Figure 2B The influence of experimental conditions on urea measurements is illustrated.

[0079] Figure 2A The results shown in are easy to summarize. The condition applied with a shorter delay time (2 days) and a longer soak time in the 10% sucrose solution (16 hours) provided the most stable performance and the most consistent sensor-to-sensor performance over the useful life of the sensor. The condition of a longer delay time (7 days) combined with a shorter soak time in the 10% sucrose solution (2 hours) produced 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 showed inconsistency from sensor to sensor (7 days, 16 hours), or performance degradation over the useful life (2 days, 2 hours). Figure 2B Urea measurements and favorable directions for the three sucrose treatment condition factors are illustrated: shorter delay time, longer soaking time, and longer drying time.

[0080] These results confirm that urease enzyme activity in disaccharide-treated urea biosensors continues to decay, even when urease is immobilized on the sensor. The faster the fabricated sensor is exposed to sucrose treatment, the better the urease activity is maintained. These studies also show that removing all moisture (e.g., by drying) is critical to long-term enzyme stability.

[0081] Example 3

[0082] Example 3 provides stability data at ambient temperature for urea sensors made according to the present invention from multiple production batches in a three-week service life stability and a five-month shelf life stability study.

[0083] Urea was measured daily over a 3 week lifespan of these cartridges in aqueous samples with high concentrations of urea (95 mg / dL) to evaluate the sensor urea measurement linearity over time.

[0084] Figure 3A The urea measured in twelve urea sensors selected from three batches of sucrose treated sensors and over a short shelf life storage of two weeks at ambient temperature is illustrated graphically. Daily measurements of high urea concentration (95 mg / dL) samples over a three week shelf life with short sensor shelf life showed stable shelf life performance with a consistent measured urea concentration of about 95 mg / dl.

[0085] Figure 3B The results for the other twelve urea sensors from three different batches of sucrose treated urea sensors are illustrated graphically. The high urea concentration (95 mg / dL) samples measured daily over a three week shelf life at 5 months ambient temperature showed the same stable shelf life performance, with the measured urea concentration being consistently about 95 mg / dL.

[0086] By applying polysaccharide treatment (sucrose was used as an exemplary polysaccharide in these studies), urea sensor activity and stability were maintained over a storage shelf life of 5 months at ambient temperature.

[0087] The present invention described above is applicable to urea biosensors developed on a variety of urea sensor platforms.

Claims

1. A biosensor comprising: an ammonium ion selective electrode, urease immobilized on the ion selective electrode, a diffusion barrier on the electrode surface, and a polysaccharide, The biosensor is obtained by a method comprising the following steps: providing an ammonium ion selective electrode; casting urease in a solution on the outer surface of a PVC ammonium ion selective electrode to form an enzyme layer; applying a diffusion barrier to the surface of the enzyme layer; applying the polysaccharide solution to an ammonium ion selective electrode; The electrodes are dried to form the biosensor, wherein the polysaccharide is selected from sucrose, and The sucrose solution contains 10% to 15% sucrose.

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: The polymer matrix and Ammonium selective ionophores on a polymer matrix.

5. The biosensor of claim 4, wherein the polymer matrix is ​​selected from the group consisting of polyvinyl chloride, polyurethane, polytetrafluoroethylene, polymethyl 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: anactivin, monoactivin, diactivin, triactivin, tetraactivin, narasin, hexaoxacyclotricarboxane, benzocrown ether, cyclic depsipeptide, and mixtures thereof.

8. The biosensor of claim 4, wherein the ionophore comprises inactivin.

9. The biosensor of claim 1, wherein the urease is cross-linked.

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

11. The biosensor of claim 1, wherein the diffusion barrier comprises a polymeric compound selected from the group consisting of polyurethane, polytetrafluoroethylene ionomer, poly-2-hydroxymethyl methacrylate, polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof.

12. The biosensor of claim 11, wherein the polytetrafluoroethylene ionomer is a perfluorosulfonate ionomer.

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

14. A method for making a stable urea biosensor, comprising: providing an ammonium ion selective electrode; casting urease in a solution on the outer surface of a PVC ammonium ion selective electrode to form an enzyme layer; applying a diffusion barrier to the surface of the enzyme layer; applying the polysaccharide solution to an ammonium ion selective electrode; The electrode is dried to form a stable urea biosensor. Wherein the polysaccharide is selected from sucrose, The sucrose solution contains 10% to 15% sucrose.

15. The method of claim 14, wherein the urease is cross-linked.

16. The method of claim 14, wherein the urease is cross-linked by a chemical selected from the group consisting of glutaraldehyde, 1,4-diisocyanatobutane, 1,2,7,8-diepoxyoctane and 1,2,9,10-diepoxydecane, 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 biosensor is capable of measuring urease.

20. The method of claim 14, wherein applying the diffusion barrier comprises applying: polyurethane, polytetrafluoroethylene ionomer, poly-2-hydroxymethyl methacrylate, polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof.

21. The method of claim 20, wherein the polytetrafluoroethylene ionomer is a perfluorosulfonate ionomer 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 5 months of dry storage at ambient temperature and 21 days of use.

24. A disposable cartridge containing a urea sensor, the urea sensor comprising: an ammonium selective electrode, urease, a diffusion barrier on the electrode surface, and a polysaccharide, The sensor is obtained by a method comprising the following steps: providing an ammonium ion selective electrode; casting urease in a solution on the outer surface of a PVC ammonium ion selective electrode to form an enzyme layer; applying a diffusion barrier to the surface of the enzyme layer; applying the polysaccharide solution to an ammonium ion selective electrode; The electrode is dried to form a urea sensor, wherein the polysaccharide is selected from sucrose, and The sucrose solution contains 10% to 15% sucrose.

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

26. The method of claim 14, wherein the polysaccharide solution is added to the urease enzyme solution prior to applying the urease enzyme 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 before 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.

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