UREA BIOSENSORS AND STABILIZATION OF UREA BIOSENSORS AT ROOM TEMPERATURE

Abstract

Disclosed is a urea biosensor that is stable at ambient temperature, and methods of making thereof.

Claims

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 said 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 said ammonium ion-selective electrode comprises 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 ionophore 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 nonactin, monactin, dinactin, trinactin, tetranactin, narasin, hexaoxaheptacyclotritrtracontane, benzocrown ethers, cyclic depsipeptides, and mixtures thereof.

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

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

10. The biosensor of claim 1 wherein the poly-saccharide 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 polyurethane, poly(tetrafluoroethylene) ionomers (the perfluorosulfonate ionomer, NAFION), poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof.

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 solution on the outer surface of the PVC ammonium ion-selective electrode to form an enzyme layer; applying a diffusion barrier on the surface of said enzyme layer; applying a polysaccharide solution to the ammonium ion-selective electrode; drying the electrode to form the stable urea biosensor.

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

16. The method of claim 14 wherein 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 said ammonium ion-selective electrode is selected from the electrode group consisting of silver, platinum, and gold.

18. The method of claim 14 wherein said 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 polyurethane, poly(tetrafluoroethylene) ionomers (the perfluorosulfonate ionomer, NAFION) poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof.

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

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

24. A disposable cartridge housing 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 calibration reagents.

26. The method of claim 14 wherein the polysaccharide solution is added to the urease enzyme solution before the urease enzyme solution is applied to the ammonium ion-selective electrode.

27. The method of claim 14 wherein the polysaccharide solution is applied 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 the enzyme solution is applied to the ammonium ion-selective electrode and the polysaccharide solution is applied to the ammonium ion-selective electrode after application of the diffusion barrier.

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

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a diagrammatic illustration of a cross-section of one embodiment of a urea biosensor according to the invention.

[0029] FIG. 2A is a graphic illustration of the effect of sucrose treatment on linearity of urea measurement in twenty-two urea sensors; the measurement of high urea concentration (70 mg/dL) in a sample versus cartridge age with drying time of 3 hours after sucrose treatment;

[0030] FIG. 2B is a graphic illustration of the impact of sucrose treatment on urea measurement (mg/dl) for combinations of two of three sucrose treatment factors (time delay (days) from sensor fabrication to sensor disaccharide treatment; length of disaccharide treatment soak time (hours); length of disaccharide treated sensor drying time hours) on the twenty-two urea sensors in FIG. 2A;

[0031] FIG. 3A is a graphic illustration of measured high urea concentration (95 mg/dL) versus cartridge age of twelve urea biosensors after short sensor shelf-life of two weeks and up to 3 week use-life; Daily urea concentration measurement in high urea sample (95 mg/dL) over 3-week use life;

[0032] FIG. 3B is a graphic illustration of measured high urea concentration (95 mg/dL) versus cartridge age of twelve urea biosensors stored at ambient temperature over 5 months and up to 3 week use-life. Daily urea concentration measurement in high urea sample (95 mg/dL) over 3-week use life.

DETAILED DESCRIPTION

[0033] The inventions described below are directed to a device and related method for enhancing enzyme stability and extending the shelf-life and use-life of urea biosensors useful in clinical analyzers for in vitro diagnostics, point-of-care applications in particular.

[0034] According to the invention, polysaccharides, for example, disaccharides, such as sucrose, are optimal compositions for preserving the stability and activity and extending the shelf-life and use-life of urea biosensors systems. Other poly-saccharides such as trehalose (-D-Glucopyranosyl--D-glucopyranoside), raffinose (O--D-Galactopyranosyl-(1.fwdarw.6)--D-glucopyranosyl -D-fructofuranoside), and lactitol (4-O--D-Galactopyranosyl-D-glucitol) (all poly-saccharides obtained from Sigma) also improve stability and activity of urease in urea biosensors extending its shelf-life and use-life.

[0035] For simplicity, 10% sucrose was used as an example polysaccharide for the studies presented below. Significant improvement in maintaining urease activity at ambient temperature was observed with sucrose stabilization. A stable shelf-life of at least 5 months was achieved when the urea sensor was stored at room temperature following sucrose-treatment enzyme stabilization.

[0036] As described below, it was determined by the inventors that a disaccharide, for example, sucrose, is one of the optimal compositions for preservation and stability of the activity of a urea biosensor. Other poly-saccharides such as trehalose, raffinose, and lactitol also have similar effect on urea sensors improving stability.

[0037] A typical urea biosensor 150 shown in FIG. 1, comprises a metal element 155 embedded in an electrode card 6 and a composite membrane 160 which is located between the metal element 155 and an analytical sample that flows through a channel 20 in the electrode card 6. The composite membrane 160 includes an outer diffusional membrane 165 adjacent to the patient sample channel 20, an enzymatic layer 170, an ammonium-selective polymeric membrane 175, and an inner solution layer 180 adjacent the metal element 155.

[0038] The outer 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 analytical sample in the channel 20. The enzyme layer 170 may include at least one enzyme, or a mixture of several enzymes, proteins and stabilizers that react with a particular analyte, i.e., urea in the patient sample. If the analyte diffuses through the outer diffusional membrane 165 into the enzyme layer, it can react with the enzyme in the enzyme layer 170 to produce a chemical byproduct, in the case of urea, ammonium ions. An electrical potential is generated across the composite membrane 160 that depends on the concentration of the chemical byproduct that is proportional to the concentration of urea in the analytical sample. PVC may be a constituent of the ammonium selective polymeric membrane 175.

[0039] In one embodiment of the invention, the steps for making a stable disaccharide-treated urea sensor according to the invention include:

[0040] (i) fabricating a polyvinyl chloride (PVC) based ammonium ion-selective sensor on a silver surface 155 which is covered with AgCl and inner solution layer 180 to form an internal reference electrode, the PVC ammonium ion-selective membrane 175 composition is described in US Publication No. 2004/0256227, incorporated by reference herein for all intents and purposes; followed by

[0041] (ii) immobilizing the enzyme, urease, to form an enzyme layer 170 on the outer surface of the PVC membrane 175 by applying, for example, a cross-linking agent, e.g., the cross-linker, if applied, selected from the group consisting of glutaraldehyde, diisocyanatobutane, diisocyanoto, 1, 2, 7, 8-diepoxyoctane, 1, 2, 9, 10-diepoxydecane, and combinations thereof; alternatively, immobilization of one or more enzymes on the outer surface of the PVC membrane 175 can occur by physical absorption, entrapment with a hydrogel, for example; followed by

[0042] (iii) applying a hydrophilic polyurethane layer to form the outer diffusion membrane 165, or one or more of the following polymers such as poly(tetrafluoroethylene) ionomers (the perfluorosulfonate ionomer, NAFION), poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof to the enzyme layer 170 for enzyme protection and diffusion control; followed by,

[0043] (iv) exposing the urea sensor 150 to a polysaccharide solution such as a sucrose solution or alternatively a solution of trehalose, raffinose, or lactitol in concentrations (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 leak 30 minutes to 240 minutes, at least 30 minutes to 12( )minutes, at least 30 minutes to 60 minutes, preferably at least 30 minutes; followed by

[0044] (v) air drying the polysaccharide-treated urea sensor 150; and

[0045] (vi) storing, for example, 5 months or longer, the urea sensor 150 in ambient conditions until use.

[0046] In an alternative embodiment of making a stable urea biosensor 150, the steps include;

[0047] (i) fabricating a polyvinyl chloride (PVC) based ammonium ion-selective sensor on a silver surface 155 which is covered with AgCl and an inner solution layer 180 to form an internal reference electrode, the PVC ammonium ion-selective membrane 175 composition is as described above;

[0048] (ii) preparing a urease enzyme in a polysaccharide solution such as a sucrose solution or alternatively a solution of trehalose, raffinose, or lactitol in concentrations (w/v) ranging from >0% to 2%, 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably, 10% solution

[0049] (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 membrane 175 by applying, for example, a cross-linking agent, e.g., the cross-linker, if applied, selected from the group consisting of glutaraldehyde, diisocyanatobutane, diisocyanoto, 1, 2, 7, 8-diepoxyoctane, 1, 2, 9, 10-diepoxydecane, and combinations thereof to form an enzyme layer 170; followed by,

[0050] (iv) applying a hydrophilic polyurethane layer 165 or one or more of the following polymers such as poly(tetrafluoroethylene) ionomers (the perfluorosulfonate ionomer, NAFION), poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof to the enzyme layer 170 for enzyme protection and diffusion control; followed by

[0051] (v) storing, for example, 5 months or longer, the urea sensor 150 in ambient condition until use.

[0052] In yet another embodiment of making a stable urea biosensor 150, the steps include:

[0053] (i) fabricating a polyvinyl chloride (PVC) based ammonium ion-selective sensor on a silver surface 155 which is covered with AgCl and an inner solution layer 180 to form an internal reference electrode, the PVC ammonium ion-selective membrane 175 composition is as described above;

[0054] (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 concentrations (w/v) ranging from >0% 2% to 25%, 2% to 20%, 5% to 15%, 10% to 15%, preferably, 10% solution;

[0055] (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 membrane 175 by applying, for example, a cross-linking agent, e.g., the cross-linker, if applied, selected from the group consisting of glutaraldehyde, diisocyanatobutane, diisocyanoto, 1, 2, 7, 8-diepoxyoctane, 1, 2, 9, 10-diepoxydecane, and combinations thereof to form an enzyme layer 170; followed by

[0056] (iv) applying a hydrophilic polyurethane layer 165 or one or more of the following polymers such as poly(tetrafluoroethylene) ionomers (the perfluorosulfonate ionomer, NAFION), poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof to the enzyme layer 170 for enzyme protection and diffusion control; followed by

[0057] (v) exposing the urea sensor 150 to a polysaccharide solution such as a sucrose solution or alternatively a solution of trehalose, raffinose, or lactitol in concentrations (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; followed by

[0058] air drying the polysaccharide-treated urea sensor 150; optionally repeating the step exposing the sensor to a polysaccharide solution once, twice, 3-4, 5-8, and 9-10 times, followed by

[0059] storing, for example, five months or longer, the urea sensor 150 in ambient condition until use.

EXEMPLIFICATION OF THE INVENTION

Example 1

[0060] Example 1 described below provides one embodiment of the method for making the urea sensor with sucrose stabilization.

[0061] 1. According to one method of the invention, a solution for an ammonium selective polymer membrane is prepared in THF and contains, for example, 25-35% PVC, 60-70% DOS, 1-5% nonactin, and 0-1% KTpClPB by weight.

[0062] Nonactin is an ionophore with specific selectivity towards ammonium. Other reagents, for example, monactin, dinactin, trinactin, tetranactin, narasin, benzocrown ethers, cyclic depsipeptides, and mixtures of the preceding can also be used for this purpose.

[0063] DOS (bis(2-ethylhexyl) sebacate) is a plasticizer. o-nitrophenyl octyl ether (NPOE) is another commonly used plasticizer.

[0064] KTpC1PB (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.

[0065] Other than PVC, the polymer membrane can also include, for example, polyurethane, poly(tetrafluoroethylene), poly(methyl methacrylate), silicone rubber, and mixtures of above.

[0066] THF (tetrahydrofuran) is a solvent. Cyclohexanone can also be used for this purpose.

[0067] 2. The ammonium sensor is planar and formed by casting the ammonium selective polymer membrane solution made as described above on a Ag/AgCl metal electrode and an inner electrolyte layer to form an internal reference electrode which is embedded in a solid substrate, for example, but not limited to PVC. The inner electrolyte layer is formed by casting the inner solution on the Ag/AgCl electrode surface. The inner solution contains, for example, a 65% MFS buffer, 1% sodium chloride, 1% ammonium chloride, and 33% HEC (percentages by weight) in water.

[0068] 3. The enzymatic layer is formed by casting an enzyme solution on the outer surface of the ammonium-selective PVC membrane. The enzyme solution includes an enzyme urease, enzyme stabilizer glutathione, inert proteins bovine serum albumin, cross-linking agent glutaraldehyde and solvents. Glutathione is used together with one or more inert proteins, e.g., bovine serum albumin, to stabilize the urease in the enzyme layer. Cross-linking also secures the enzymatic layer to the underlying ion-selective polymeric layer. During fabrication of the enzymatic layer, the enzyme stabilizers are generally added to the solution containing the enzyme prior to the addition of the cross-linking agent to ensure the stabilizers are cross-linked together with the enzyme. A typical enzyme solution was prepared containing 50 mg/mL urease, 20 mg/mL glutathione. 10 mg/mL bovine serum albumin, and 0.12% glutaraldehyde in 0.1 M phosphate buffer at a pH of 7.2, and the solution was applied to the top of the ammonium ion-selective PVC membrane.

[0069] 4. Polyurethane (KJ) is one of the polymers that has superior biocompatibility in many successful in vivo and in vitro applications in medical devices. The specific hydrophilic medical grade polyurethane families selected to be optimized for this application are Tecophlic and Tecoflex from Lubrizol (Wickliffe, Ohio). These commercially available polymeric resins are aliphatic, polyether-based polyurethane which can be dissolved in organic solvents or mixture of solvents such as dimethylacetamide (DMA), tetrahydrofuran (THE), etc. Within these polyurethane families, there are different grades of materials available with the combination of various hardness and water uptake levels for different applications. The outer diffusional membrane of the urea biosensor in contact with the patient sample in various embodiments includes one or more distinct layers of identical or different polymers and/or identical or different co-polymers. A typical outer diffusional membrane solution was prepared containing 0.12 g/ml polyurethane (Lubrizol, Wickliffe, Ohio) in THF. The outer diffusional membrane solution was applied over the enzyme layer to form the outer diffusional membrane. Other than polyurethane, one or more of the following polymers can also be candidates for the outer diffusional membrane, NAFION (the poly(tetrafluoroethylene) ionomers), poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, polycarbonate, and cellulose acetate.

[0070] 5. In one embodiment of the invention, the urea sensor with composite membrane of inner layerPVC layerEnzyme layerpolyurethane layer is then immersed in a disaccharide solution, for example, a sucrose solution, in concentrations ranging from 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 biological pH of 7.4.

[0071] 6. The sensor is dried at room temperature for 0.5 hours to 3 hours. Optionally, sucrose treatment is repeated multiple times followed by air drying each time. The sensor is then stored at ambient temperature until use.

Example 2

[0072] Example 2 is a study to assess the effect of sucrose treatment on stabilization of urea sensor described above at ambient temperature using a multi-factorial experimental design that was carried out with three main factors (each at two conditions) to investigate(i) the delay time (2 or 7 days) between the sensor fabrication and exposing the fabricated sensor to the sucrose solution, (ii) the soak time (2 or 16 hours) during which the sensor is exposed to the sucrose solution, and the drying time (0.5 or 3 hours) after the sucrose exposure is completed. A subgroup of urea sensors were assembled into cartridges and urea concentration was measured in GEM Premier clinical analyzers for use-life stability. An aqueous sample having a high concentration of urea (70 mg/dL) was tested daily on the urea sensors in the cartridges to assess the linearity of urea sensor measurement.

[0073] Referring to FIG. 2A, the use-life stability of each of the tested urea sensors at various applied experimental conditions when the drying time is fixed at 3 hours is provided. FIG. 2B illustrates the effect of the experimental conditions on urea measurement.

[0074] The results shown in FIG. 2A are readily summarized. The condition applied with shorter delay time (2 days) and longer soak time (16 hours) in 10% sucrose solution provided the most stable performance over the use-life of the sensor and most consistent sensor-to-sensor performance, The poorest stabilization performance occurred with the condition of longer delay time (7 days) combined with shorter soak time (2 hours) in 10% sucrose solution. The other two conditions provided better performance compared to the poorest performance of 7 day delay time/2 hour soak time but also exhibited inconsistency from sensor-to-sensor (7 days, 16 hours), or performance deterioration over the-use-life (2 days, 2 hours). FIG. 2B illustrates urea measurement and the favorable direction for all three sucrose treatment condition factors: shorter delay time, longer soak time and longer drying time.

[0075] These results confirmed that urease enzyme activity in a urea biosensor that is disaccharide treated is continuously undergoing decay even when urease is immobilized on the sensor. The quicker the fabricated sensor is exposed to sucrose treatment, the better urease activity is preserved. These studies also indicated that removal of all moisture, by drying, is critical for long term enzyme stability.

Example 3

[0076] Example 3 provides stability data at ambient temperature for urea sensors obtained from multiple production batches made according to the invention in a three week use-life stability and 5-month shelf life stability studies.

[0077] Urea was measured daily in an aqueous sample with a high concentration of urea. (95 mg/dL) over the 3 week use-life on these cartridges to assess the sensor urea measurement linearity over time.

[0078] FIG. 3A graphically illustrates urea measured in twelve urea sensors selected from three batches of sucrose treated sensors and short shelf-life storage at ambient temperature of two weeks. Daily measurement of the high urea concentration (95 mg/dL) sample over the three week use-life at short sensor shelf-life showed stable use-life performance with consistent measured urea concentration of about 95 mg/dl.

[0079] FIG. 3B graphically illustrates the results from another twelve urea sensors from three different batches of sucrose treated urea sensors. Daily measurement of the high urea concentration (95 mg/dL) sample over three-week use-life at 5-month ambient storage shelf life showed same stable use-life performance with consistent measured urea concentration of about 95 mg/dl.

[0080] By applying polysaccharide treatment, sucrose used as an exemplary polysaccharide in these studies, urea sensor activity and stability at ambient temperature storage shelf-life for 5 months was maintained.

[0081] The invention described above is applicable to urea biosensors developed on multiple urea sensor platforms.