MULTI-ENZYMATIC BIOSENSORS AND STABILIZATION OF MULTI-ENZYMATIC BIOSENSORS AT ROOM TEMPERATURE

Abstract

Disclosed are multi-enzyme biosensors that are stable at ambient temperature, and methods of making thereof.

Claims

1-34. (canceled)

35. A biosensor comprising: an electrode; a plurality of enzymes immobilized over the electrode, where an interaction among at least two of the plurality of enzymes can cause instability of one or more of the plurality of enzymes; a diffusion barrier on the plurality of enzymes; and a polysaccharide that is (i) applied to the diffusion barrier of the electrode, (ii) included with the enzymes immobilized over the electrode, or (iii) both applied to the diffusion barrier of the electrode and included with the enzymes immobilized over the electrode, the polysaccharide for maintaining stable activity of the one or more of the plurality of enzymes.

36. The biosensor of claim 35, wherein the electrode comprises platinum, gold, palladium, alloys of platinum, gold and palladium, or carbon.

37. The biosensor of claim 35, wherein the electrode comprises graphite or carbon nanotube.

38. The biosensor of claim 35, wherein the plurality of enzymes are cross-linked.

39. The biosensor of claim 35, wherein the plurality of enzymes comprise creatinase, creatininase, or sarcosine oxidase.

40. The biosensor of claim 35, wherein the polysaccharide comprises sucrose, trehalose, raffinose, or lactitol.

41. The biosensor of claim 35, wherein the biosensor is configured to measure creatine.

42. The biosensor of claim 35, wherein the biosensor is configured to measure creatinine and creatine.

43. The biosensor of claim 35, wherein the diffusion barrier comprises a polymeric compound comprising polyurethane, poly(tetrafluoroethylene) ionomers, the perfluorosulfonate ionomer NAFION®, poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures or copolymers thereof.

44. The biosensor of claim 35, wherein the polysaccharide comprises 10% sucrose.

45. A method of using a multi-enzyme biosensor, the multi-enzyme biosensor comprising: a plurality of enzymes in an enzyme solution over a surface of an electrode, where an interaction among at least two of the plurality of enzymes can cause instability of one or more of the plurality of enzymes; a diffusion barrier on a surface of the plurality of enzymes; and a polysaccharide solution (i) applied to the electrode after the diffusion barrier is applied to the surface of the plurality of enzymes, or (ii) added to the enzyme solution before the enzyme solution is applied over the electrode, or (iii) both added to the enzyme solution before the enzyme solution is applied to the electrode and applied to the electrode after the diffusion barrier is applied over the surface of the plurality of enzymes, the polysaccharide solution maintaining stable activity of the one or more of the plurality of enzymes; wherein the method comprises using the multi-enzyme biosensor to measure creatine, creatinine, or both creatine and creatinine in a body fluid sample.

46. The method of claim 45, wherein the plurality of enzymes are cross-linked.

47. The method of claim 45, wherein the plurality of enzymes are cross-linked by a chemical comprising glutaraldehyde, 1,4-diisocyanatobutane, 1,2,7,8-diepoxyoctane and 1,2,9,10-diepoxydecane, or a combination thereof.

48. The method of claim 45, wherein the electrode comprises platinum, gold, palladium, alloys of platinum, gold and palladium, or carbon based material.

49. The method of claim 45, wherein the electrode comprises graphite or carbon nanotubes.

50. The method of claim 45, wherein the polysaccharide comprises sucrose, trehalose, raffinose, or lactitol.

51. The method of claim 45, wherein the biosensor measures both creatinine and creatine.

52. The method of claim 45, wherein the biosensor measures creatine only or creatinine only.

53. The method of claim 45, wherein the diffusion barrier comprises a polyionic compound comprising polyethylenimine, poly(N-vinylimidazole), polypropyleneimine, polyallylamine, polyvinylpiridine, polyvinylpyrollidone, polylysine, protamine, or derivatives of polyionic compounds.

54. A disposable cartridge comprising multi-enzyme sensors, where a multi-enzyme sensor among the multi-enzyme sensors comprises: an electrode; a plurality of enzymes immobilized over the electrode, where an interaction among at least two of the plurality of enzymes can cause instability of one or more of the plurality of enzymes; a diffusion barrier on the plurality of enzymes; and a polysaccharide that is (i) applied to the diffusion barrier of the electrode, (ii) included with the enzymes immobilized over the electrode, or (iii) both applied to the diffusion barrier of the electrode and included with the enzymes immobilized over the electrode, the polysaccharide for maintaining stable activity of the one or more of the plurality of enzymes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 illustrates a cross-sectional view of an enzyme sensor according to one embodiment of the invention.

[0027] FIG. 2A is a graphic illustration of creatinine sensor activity in a group of 10 cartridges, the activity of the creatinine sensor of each cartridge plotted as slope, in the unit of picoampere/milligram/deciliter (pA/mg/dL) versus age of cartridge (hours), over a three week use period after 5 month room temperature storage, for example, at 22°−25° C. Each cartridge enclosed a creatinine sensor that did not receive sucrose treatment. Testing was discontinued after one week because the slope dropped below the cut-off value of 400 picoampere/milligram/deciliter (400 pA/mg/dL). The cut-off is established to ensure analytical performance consistency from cartridge-to-cartridge (sensor to sensor);

[0028] FIG. 2B is a graphic illustration of creatinine sensor activity in another group of 5 cartridges. The activity of the creatinine sensor activity of each cartridge plotted as slope (pA/mg/dL) versus age of cartridge (hours) over a three week use period after 5 month room temperature storage. Each cartridge enclosed a creatinine sensor that received treatment with 10% sucrose;

[0029] FIG. 3 is a graphic illustration of four batches numbered 3919, 3921, 3925 and 3926, of sucrose-treated and polyethylenimine-containing creatinine sensor sensitivities over a 3 week use-life after 5 month storage at room temperature; a total of twelve creatinine sensors were tested.

[0030] FIG. 4A is a graphic illustration of creatinine sensor analytical performance in clinical samples in a group of ten cartridges, each represented by a different symbol, of creatinine and creatine sensors without polyethylenimine in the enzyme mixture. The difference (aka bias, or error) of creatinine measured between GEM PAK and a reference chemistry analyzer were plotted vs. plasma creatinine reported by the reference chemistry analyzer, the dashed lines were acceptable limits of bias at any given creatinine concentration for clinical applications. Due to sensor-to-sensor variation in performance, the bias between GEM and reference analyzers were scattered and many samples had bias exceeded the acceptable limits (data points outside dashed lines).

[0031] FIG. 4B is a graphic illustration of creatinine sensor analytical performance in clinical samples in another group of ten cartridges, each represented by a different symbol, of creatinine and creatine sensors with polyethylenimine-containing enzyme mixture. The difference of creatinine measured between GEM PAK and a reference chemistry analyzer were plotted vs. plasma creatinine reported by the reference chemistry analyzer. The dashed lines were acceptable limits of bias at any given creatinine concentration for clinical applications. With improved sensor performance, the biases were tightly distributed and most of the samples had bias within the clinical acceptable limits (data points within dashed lines).

DETAILED DESCRIPTION

[0032] The inventions described below are directed to a device and related method for enhancing enzyme stability extending the shelf-life and use-life of multi-enzyme biosensors including but not limited to creatine and creatinine biosensors used in clinical analyzers for in vitro diagnostics, point-of-care applications in particular.

[0033] 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 a multi-enzyme system such as a three-enzyme biosensor system for example, the creatinine sensor. Other poly-saccharides such as trehalose (α-D-Glucopyranosyl-α-D-glucopyranoside), raffinose (O-α-D-Galactopyranosyl-(1.fwdarw.6)-α-D-glucopyranosyl β-D-fructofuranoside), and lactitol (4-O-β-D-Galactopyranosyl-D-glucitol) (all poly-saccharides obtained from Sigma) also improve stability and activity of enzymes in multi-enzyme biosensors extending their shelf-life and use-life.

[0034] For simplicity, 10% sucrose was used as an example polysaccharide for the studies presented below. Significant improvement in maintaining multi-enzyme activity at ambient temperature was observed with sucrose stabilization. A stable shelf-life of at least 5 months was achieved when the multi-enzyme sensor was stored at room temperature following sucrose-treatment enzyme stabilization.

[0035] Polyelectrolytes, i.e., polyionic compounds, in addition to polysaccharides are another known class of chemicals useful as a multi-enzyme stabilizer, for example, polyethylenimine (PEI). PEI has been applied in single enzyme biosensors, e.g., lactate enzymatic sensor formulation on biosensor-containing cartridges, for example, GEM®-PAK (Instrumentation Laboratory Company; Bedford, Mass.) for that purpose. To further enhance the sucrose stabilization effect on a three-enzyme or a two enzyme biosensor system, a polyelectrolyte/sugar stabilization system was studied by introducing PEI into the three-enzyme mixture. The formulation, discussed below, has been optimized to achieve the best results, i.e., maintaining sensor slope above 400 pA/mg/dL after 5 month dry storage and three weeks of use life. PEI improves enzyme stability through its electrostatic interaction with the enzyme(s) outer shell protein structure, and may further facilitate the penetration of sucrose-water complex into the three dimensional structure of the immobilized enzyme. Thus, the immobilized enzyme system in the presence of PEI is packed with the polyhydroxyl compound, sucrose, for example, instead of free water, and maintains stable activity of the enzymes during the drying process at the sensor manufacturing step and throughout the dry storage during the shelf-life prior to use at the clinical site.

[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 two or three-enzyme system for a biosensor, for example, a creatinine sensor. Other poly-saccharides such as trehalose, raffinose, and lactitol also have similar effect on multi-enzyme sensors improving stability.

[0037] A typical enzyme biosensor, e.g., a creatine or creatinine biosensor according to the invention is illustrated in FIG. 1. The enzyme sensor 59 on sensor card 50 includes a three layer composite membrane 60 comprising, arranged from the body fluid sample flow chamber 56 to the electrode 57, an outer diffusion membrane 51 adjacent to the flow channel 56, an enzyme layer 53, located between the outer membrane 51 and an inner membrane 55 that is adjacent to the electrode 57. The enzyme sensor 59 contacts the patient sample as the sample flows along the flow channel 56 and over the outer membrane 51 of the enzyme sensor 59.

[0038] In one embodiment of the invention, the steps for making a stable multi-enzyme disaccharide-treated sensor according to the invention include:

(i) solvent casting a plurality of enzymes on an electrode, for example, an optionally cross-linked multi-enzyme mixture, e.g., a three enzyme or a two enzyme mixture, the cross-linker, if applied, selected from the group consisting of glutaraldehyde, 1,4-diisocyanatobutane, 1, 2, 7, 8-diepoxyoctane, 1, 2, 9, 10-diepoxydecane, and combinations thereof; alternatively, immobilization of one or more enzymes on the surface of the electrode can occur by physical absorption, entrapment with a hydrogel, or coated on the electrode by electro-polymerization with conductive monomers, for example. The electrode may be selected from the group consisting of platinum, gold, palladium, alloys of the foregoing, carbon, graphite, and carbon nanotubes;
(ii) applying to the solvent cast multi-enzyme electrode, a diffusion control barrier such as polyurethane, poly(tetrafluoroethylene) ionomers (the perfluorosulfonate ionomer, NAFION®), poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, or mixtures and copolymers thereof; followed by,
(iii) exposing the solvent cast electrode to a polysaccharide solution, for example, a disaccharide solution, such as, a sucrose solution, or trehalose, raffinose or lactitol in (w/v) concentrations 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, and,
(iv) air drying.

[0039] In an alternative embodiment of the above described method for making a multi-enzyme poly-saccharide-treated biosensor, as described in greater detail below, rather than exposing the electrode in step (iii) to a polysaccharide solution, polysaccharide and optionally a polyionic compound such as PEI are added directly into the enzyme mixture before solvent casting the enzyme mixture on the electrode, and then the mixture of polysaccharide, optionally, PEI, and multi-enzymes are solvent cast on the electrode.

[0040] In yet another alternative embodiment for making a multi-enzyme poly-saccharide-treated biosensor, as described in greater detail below, step (iii) above is combined with the step of adding polysaccharide and, optionally adding a polyionic compound such as PEI directly into the enzyme mixture and then the enzyme mixture is solvent cast on the electrode.

EXEMPLIFICATION OF THE INVENTION

[0041] An exemplification of the utility and method of making multi-enzyme biosensors with extended biosensor activity shelf-life and use-life is presented below from studies that were conducted for proof of principle.

[0042] Creatinine sensors were fabricated by solvent casting a platinum electrode with a three enzyme mixture. The three enzyme mixture was made with 30% creatininase, 30% creatinase, 30% sarcosine oxidase and 10% glutaraldehyde (percentages by weight) in water. The two enzyme creatine electrode was manufactured by solvent casting a platinum electrode with a two enzyme mixture of 45% creatinase, 45% sarcosine oxidase and 10% glutaraldehyde in water (percentages by weight). The enzyme mixtures are cast on the platinum electrode by dispensing the aqueous enzyme solutions onto the surface of the electrode. The solvent cast enzyme layer on the electrode has a thickness in the range of 1 to 10 microns, preferably 2-5 microns. The enzyme cast electrode was next covered with polyurethane to form the outer membrane. The outer membrane of the electrode is exposed to the patient's body fluid sample that flows in the flow channel of the sensor card.

[0043] The outer membrane comprises a blend of polyurethanes having different water uptake levels. A typical composition for one embodiment of the outer membrane is 77% aliphatic, polyether-based polyurethane with 20% water uptake, 17% aliphatic, polyether-based polyurethane with 60% water uptake, and 6% aliphatic, polyether-based polyurethane with 3% water uptake. Alternative polyurethane blends in diffusion barrier outer membranes are also possible such as those described in U.S. Pat. No. 6,960,466 and Outer Membrane Compositions for Creatinine/Creatine Sensors (Attorney Docket no. INL-169), filed on even-date, each incorporated by reference herein in their entirety for all intents and purposes.

[0044] In one embodiment, the outer membrane of the sensor with this composition can be produced, for example, by dispensing a solution of 3.0 mL cyclohexanone solvent, 17.0 mL tetrahydrofuran solvent, 1.08 g of 20% water uptake polyurethane, 0.24 g of 60% water uptake polyurethane and 0.08 g of 3% water uptake polyurethane is layered onto and in direct contact with the enzyme layer of the composite membrane. Other than polyurethane, other suitable polymers include, but are not limited to, poly(tetrafluoroethylene) ionomers (the perfluorosulfonate ionomer, NAFION®), poly-(2-hydroxymethyl methacrylate), polyvinyl chloride, cellulose acetate, and mixtures and copolymers thereof.

[0045] Referring to FIG. 2A, in the conducted studies, some creatinine biosensors (controls) were not sucrose-treated. These creatinine sensors, like the sucrose-treated creatinine biosensors were stored at room temperature.

[0046] Referring to FIG. 2B, other creatinine biosensors were immersed in a 10% sucrose solution buffered at biological pH of 7.4 after application of the diffusion barrier, in this case polyurethane, for 30 minutes followed by drying in air at ambient temperature.

[0047] Referring to FIG. 3, another group of creatinine sensors were studied by adding polyethylenimine into the three enzyme mixture. These creatinine biosensors were prepared from a mixture of 5% creatininase by weight, 55% creatinase by weight, 30% sarcosine oxidase by weight, 5% PEI by weight and 5% glutaraldehyde by weight, for example. The weight fractions of creatininase, creatinase and sarcosine oxidase in the creatinine electrode and the weight fraction of creatinase and sarcosine oxidase in the creatine electrode can vary and are not limited to the above mentioned percentages by weight. The weight percent of polyethylenimine in creatinine and creatine electrodes can vary, for example, from 1% to 20%, and the weight percent of glutaraldehyde in the creatinine and creatine electrodes can also vary, for example, from 1% to 10%. Polyionic stabilizers, other than polyethylenimine, can also be used for stabilizing the enzyme mixture. Examples of polyionic compounds include but are not limited to poly(N-vinylimidazole), polypropyleneimine, polyallylamine, polyvinylpiridine, polyvinylpyrollidone, polylysine, protamine, and their derivatives. As described in the previous example, a polyurethane outer membrane was then directly applied over the enzyme layer. Then the sensor was exposed to 10% sucrose for at least 30 minutes and followed by drying in the air.

[0048] The application of a polysaccharide such as sucrose to extend multi-enzyme biosensor stability is not limited to the sucrose concentrations with or without PEI disclosed in the aforementioned studies. In another embodiment of the invention, following application of the sucrose-containing enzyme mixture onto the electrode, further sucrose treatment, for example, by immersing the electrode with the sucrose containing enzyme mixture already applied to the surface of the electrode, into a solution having various concentrations of sucrose. For example, a three enzyme mixture was made with 6% creatininase, 50% creatinase, 15% sarcosine oxidase, 1% glutaraldehyde, and 28% sucrose (percentages by weight) in water. The creatinine sensor with this sucrose containing enzyme mixture may subsequently undergo additional sucrose treatment again for multiple sucrose treatment after applying the outer membrane as described in the aforementioned examples.

[0049] The creatinine biosensors made according to the method described above were stored at room temperature for 5 months. The sensitivity of the two groups of sensors, control creatinine sensors that were not sucrose-treated (FIG. 2A) and sucrose-treated creatinine sensors (FIG. 2B), were studied over a three week period after 5 months of storage at room temperature. The minimum slope (pA/mg/dL) required by the end of the three week use-life was 400 pA/mg/dL.

[0050] Referring again to FIG. 2A, studies of the control sensors that were not treated with sucrose were discontinued after one week because the slope of the sensors dropped below the minimum slope threshold (400 pA/mg/dL) required.

[0051] Referring again to FIG. 2B, the slope started much higher for the creatinine sensors treated with sucrose and remained above the 400 pA/mg/dL threshold for the duration of the three week study period.

[0052] Referring again to FIG. 3, twelve creatinine sensors were selected from four batches (indicated as batch nos. 3919, 3921, 3925, and 3926) three sensors per batch of production sucrose-treated (10% solution) creatinine sensors. These sensors also contained PEI in their enzyme mixture. The sensitivities of the twelve creatinine sensors were studied after 5 months storage and three week use-life. The study demonstrated the consistency of performance among multiple batches of PEI and sucrose-treated creatinine sensors over the three week use-life of the creatinine sensors.

[0053] Referring to FIG. 4A, studies of sucrose-treated creatinine sensor analytical performance in measuring creatinine in clinical samples without polyethylenimine in the enzyme mixture were conducted. The difference of creatinine measured between GEM PAK and a reference chemistry analyzer were plotted vs. plasma creatinine reported by the reference chemistry analyzer, the dashed lines were acceptable limits of bias at any given creatinine concentration for clinical applications. Due to sensor-to-sensor variation in performance, the biases were scattered across wide space and many samples had bias exceeded the acceptable limits (data points outside dashed lines).

[0054] Referring to FIG. 4B, studies of sucrose-treated creatinine sensor analytical performance in clinical samples with polyethylenimine-containing enzyme mixture are illustrated. The difference of creatinine measured between GEM PAK and a reference chemistry analyzer were plotted vs. plasma creatinine reported by the reference chemistry analyzer, the dashed lines were acceptable limits of bias at any given creatinine concentration for clinical applications. With modified sensor formulation by the addition of polyethylenimine, the improved sensor performance was demonstrated (data illustrated in FIG. 4B vs. 4A), biases were tightly distributed and most of the samples measured with sensors modified with polyethylenimine had biases within the clinical acceptable limits (data points within dashed lines).