Test sensor reagent having cellulose polymers

The invention relates to a reagent composition used in test sensors, and in particular, a reagent composition containing from 3.6 wt. up to 6.0 wt. hydroxyethyl cellulose polymer, from 1 wt. up to 4% by weight glucose oxidase enzyme, from 15 wt. up to 20% by weight ferricyanide mediator and from 0.2 wt. up to 1.6 wt. smectite clay.

BACKGROUND OF THE INVENTION [0002] Quantification of analytes in body fluids is of great importance in the diagnosis and stabilization of certain physiological abnormalities. For example, lactate, cholesterol and bilirubin should be monitored in some people. In particular, the determination of glucose in body fluids is important for people with diabetes who, in order to regulate glucose intake in their diets, often need to check the level of glucose in their body fluids. The results of such tests can be used to determine if, if any, insulin or other light should be given. In one type of blood glucose testing system, test sensors are used to test a fluid such as a blood sample.

[0003] The test sensor contains biosensor or reagent material that will react with the analyte of interest, such as blood glucose. The test tip of the test sensor is adapted to be placed in the fluid to be tested, for example blood that has accumulated on the finger of a person after piercing the finger. Fluid is drawn into the capillary channel running in the test sensor from the end of the test to the reagent material due to the capillary effect, so that the right amount of fluid being tested is drawn into the test sensor. In some test sensors, the fluid then reacts chemically with the reagent material in the test sensor, resulting in an electrical signal indicating the glucose level in the fluid being tested.

[0004] One of the problems with commonly used test sensors is that the reagents may contain components that interfere with the stability of the sensor. In particular, some components, such as polyethylene oxide ("PEO"), may be incompatible with other components such as the enzyme and electron transfer mediator, which are important in testing sensors. A test sensor with reagents that are formulated from components that are, for example, incompatible with the enzyme and electron transfer mediator, may after some time show insufficient stability of the test sensor. Instability is especially noticeable when the total determination time is below 35 seconds. Thus, it would be beneficial if the test sensor reagent consist of components that increase the stability of the test sensor.

[0005] DE 102004003793 A1 discloses a reagent carrier layer for an electrochemical biosensor that comprises a two-layer membrane, the first of which

- the 2nd layer contains an aqueous polymer dispersion with the reagents contained, and the second layer has a thin, waterproof, permeable surface layer. Suitable polymer dispersions for the first layer of the two-layer membrane are well-known aqueous polymer dispersions. Reagents required for the detection process, i.e. usually enzymes, can be easily incorporated with the aid of the aqueous polymer dispersion phase. Additional excipients such as buffers, detergents, rheological additives and mediators may be included in this reagent formulation. Polymer solutions and preferably aqueous polymer solutions can be used to form the second layer. In the first layer based on anionic polymer dispersion, a double polyelectrolyte layer can be formed by applying a cationic polymer solution, such as, for example, a solution of polyallylamine hydrochloride (PAH), poly (diallyldimethylammonium chloride) (polyDADMAC), polyethyleneimine hydrochloride (PEI.HCl) or cellulose polymers such as diethylaminocellulose hydrochloride or chistosamine hydrochloride. Still, producing an electrochemical two-layer test sensor requires more manufacturing steps than producing a single-layer membrane. Thus, a single layer reagent composition that has components that increase the stability of the test sensor is preferred. B. Alp, et al .: "Glow-discharge-treated cellulose acetate (CA) membrane for a high linearity singlelayer glucose electrode in the food industry", Food Research International, No. 33 (2000), pp. 107-112, Toronto, Canada, discloses a study that aimed to develop a single-layer glucose enzyme electrode with extended linearity for use in the food industry to determine glucose content. An amperometric electrode with a probe type glucose enzyme was used, with a Pt working electrode and an Ag / AgCl reference electrode polarizing at + 650 mV. In this study, the selective effects of various polymeric membranes (cellulose acetate, polyurethane, polyethersulfone) and commercially available track-etched polycarbonates were tested at various pH values ​​(pH 4, 6, 7.4, 10) to estimate the amount of interference induced by different groups of electroactive compounds. Selective studies have shown that cellulose acetate membranes were the most convenient structures for forming a single-membrane recognition layer.

SUMMARY OF THE INVENTION [0006] According to one embodiment, the invention relates to a reagent composition in a test sensor, wherein the reagent composition contains from 3.6 wt. up to 6.0 wt. hydroxyethyl cellulose polymer, from 1 wt. up to 4% by weight glucose oxidase enzyme, from 15 wt. up to 20% by weight ferricyanide mediator and from 0.2 wt. up to 1.6 wt. smectite clay.

[0007] According to a further embodiment, the method for determining the concentration of the analyte sample comprises activities leading to providing an electrochemical test sensor comprising a series of electrodes including, a counter electrode and a working electrode, a fluid receiving area and a reagent composition containing from 3.6 wt.%. up to 6.0 wt.

hydroxyethyl cellulose polymer, from 1 wt. up to 4% by weight glucose oxidase enzyme, from

- 15% by weight up to 20% by weight ferricyanide mediator and from 0.2 wt. up to 1.6 wt. smectite clay. The method also includes operations leading to the determination of analyte concentration with an assay time of less than 35 seconds.

[0008] According to a further embodiment of the invention, the method for determining the concentration of an analyte of a fluid sample includes additional activities leading to the collection of the fluid sample, placing the fluid sample with at least one analyte in a testing sensor, combining the fluid sample with the reagent composition and determining the concentration of the analyte in the fluid sample by using an electrical signal that is the result of a chemical reaction of the analyte in a fluid sample with a reagent material in a testing sensor.

[0009] The above summary of the invention is not intended to provide each embodiment or aspect of the invention. Additional features and advantages of the invention will be apparent from the detailed description, figures and claims set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS [0010] FIG. 1 is a perspective view of an instrument dispensing test sensors in an open position, showing the sensor pack inserted in accordance with one embodiment.

[0011] FIG. 2a is a front view of a disposable cartridge with a plurality of testing sensors in the stack, according to one embodiment.

[0012] FIG. 2b is a front view of a sensor dispensing instrument according to one embodiment that is adapted to receive the cartridge of FIG. 2a.

[0013] FIG. 3 is an exploded view of the test sensor components according to another embodiment.

[0014] FIG. 4 is a front view of the electrochemical testing sensor of FIG. 3.

[0015] FIG. 5 is a graph comparing the percentage glucose oxidase recovery for test sensors with a HEC-based reagent composition and a PEO-based reagent according to one embodiment of the invention.

[0016] FIG. 6 is a graph comparing the mediator stability based on the reagent storage time function for testing sensors with the HEC-based reagent composition and the PEO-based reagent according to one embodiment of the invention.

[0017] FIG. 7a-7b and 7c-7d are a series of graphs comparing systematic assay error and% systematic assay error based on 10-second assay and 30-second assay, respectively, for test sensors with HEC-based reagent composition and reagent-based composition PEO according to one embodiment of the invention.

DESCRIPTION OF SYRINGED EXAMPLES:

[0018] The invention relates to a reagent composition for a single-sensor instrument or a sensor dispensing instrument that includes a plurality of electrochemical or optical testing sensors. Electrochemical or optical testing sensors are used to determine blood glucose levels. Glucose may be, for example, in a whole blood sample, a blood sample, a blood plasma sample or other body fluids such as ISF (interstitial fluid) and urine.

[0019] Figures 1 to 4 and the corresponding description disclose information about test sensors that are adapted to use the reagent composition of the invention and about test sensors that can be used in the method of the invention. A number of test sensors are usually stored in a disposable cartridge or container. In one embodiment, a series of test sensors can be stored in a sensor package, where the sensors are individually packed in sensor cavities (e.g., a blister package). An example of a disposable cartridge 10 disposed in the sensor dispensing instrument 20 is shown in FIG. 1. The disposable cartridge 10 is an example of a blister pack. The cartridge 10 includes a series of testing sensors 12, which are separately stored in a corresponding recess for the sensor 14. The use of other sensor packages that keep the sensors separate is also contemplated.

[0020] In an alternative embodiment, a series of test sensors can be stacked in a disposable cartridge such as shown in FIG. 2a. With reference to FIG. 2a, the disposable cartridge 50 includes a housing 52 and a series of test sensors in stack 54, which by means of a spring 56 move in the direction of arrow A. The cartridge 50 also includes a series of seals 58 a, b, which protect the test sensors 54 in the stack from moisture. The testing sensors 54, one after the other, leave the cartridge 50 through the opening 60. The disposable cartridge 50 may be stored in the sensor dispensing instrument 70 of FIG. 2b. The use of other cartridges than cartridges 10, 50 in the invention is contemplated.

[0021] Cartridges 10, 50 of FIG. 1 and 2a or a container with individual test sensors may differ in the number of test sensors contained to meet the needs of different users. Typically, the cartridges or containers contain from 10 to 100 test sensors and in particular contain from 25 to 50 test sensors. Due to the shelf life and use of the testing sensors, it is anticipated that the user who rarely tests will require a cartridge or container with fewer sensors as opposed to the user who tests more often.

[0022] In some embodiments, test sensors for use in cartridges or containers are usually provided with a capillary channel that extends from the front or back of the test test sensor to the biosensor or reagent material placed in the test sensor. When the testing end of the testing sensor is placed in fluid (e.g. blood that has accumulated on the finger of a person after piercing the finger), some of the fluid is drawn into the capillary channel due to the capillary phenomenon. The fluid then reacts with the reagent in the testing sensor so that an electrical signal that indicates the level of analyte (e.g. glucose) in the fluid being tested is delivered and then passed to the electrical assembly.

[0023] The reagent composition of the invention may be applied to the substrate by a screen printing process. The screen printing process allows a thin layer of reagent to be applied to a small, flat testing sensor, such as the testing sensor shown in FIG. 4. The process usually uses a screen made of expanded stainless steel or polyester mesh and a coating of photosensitive emulsion with appropriate pattern. The reagent ink is usually applied to the screen and the squeegee blade is used to squeeze the reagent through the screen in the desired pattern. The desired pattern may include one part of the screen with a photosensitive emulsion and the other part of the screen without a photosensitive emulsion. In one embodiment, a portion of the screen without the photosensitive emulsion may come into contact with the reagent ink.

[0024] The reagent composition that is applied to the test sensor may affect such elements as the time needed to perform the test to determine the analyte concentration (i.e. assay time), stability of the test sensor and ease of application of the reagent in the screen printing process. The reagent composition of the invention includes components that provide desirable test sensor features, such as increased test sensor stability, reduced assay time, and better reagent adhesion to the substrate. A reagent composition of the invention that provides such desirable features includes a reagent composition with cellulose polymers. Cellulose polymers serve as a binder for reagent layer components and allow the viscosity of the reagent composition to be increased. It has also been found that the use of cellulose polymers in the reagent composition increases the stability of the test sensor. The cellulose polymer includes a hydroxyethyl cellulose polymer ("HEC"). HEC is beneficial because of its stabilizing properties. Particularly when HEC is used in place of other polymeric materials, glucose oxidase degradation decreases as well as the occurrence of mediator reduction. Limiting these reactions leads to increased stability of the test sensor by reducing the background intensity of the test sensor.

[0025] According to the invention, the reagent composition generally comprises from 3 wt. up to 6 wt. cellulose polymers with molecular weights between 300,000 and 1,000,000. Cellulose polymers are commercially available from various suppliers. For example, Natrasol^® and HEC polymer available from Hercules Inc. from Wilmington, Delaware.

[0026] According to another embodiment of the invention, in addition to the cellulose polymers described above, the reagent composition includes additional components such as an enzyme, electron transfer mediator and rheological additives.

[0027] The enzyme glucose oxidase is used to test blood glucose levels. The glucose oxidase enzyme reacts with glucose in a blood sample and produces an electrical signal that indicates glucose concentration. Enzyme activity can be measured in terms of an activity unit (U), which is defined as the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute under standard conditions. The reagent composition contains from 1.0 wt. up to 4.0 wt. glucose oxidase enzyme.

[0028] Glucose oxidase enzyme can be obtained commercially from companies such as Biozyme Laboratories International Ltd. of San Diego, California, Genzyme Corporation of Cambridge, Massachusetts and Amano Enzyme Inc. from Elgin, Illinois.

[0029] As described above, the reagent composition also includes an electron transfer mediator. The mediator used in the invention contains ferricyanide in addition to other mediators. The electron transfer mediator is a compound of mixed valence, capable of forming redox pairs. The reagent composition generally contains from 15 wt. up to 20% by weight electron transfer mediator. In the invention, the electron transfer mediator is a ferricyanide mediator. Ferricyanide mediators, as well as other electron transfer mediators, are commercially available from various suppliers such as Sigma-Aldrich Co. The invention contemplates the use of other electron transfer mediators in addition to ferricyanide mediators.

[0030] Rheological additives that are included in the reagent composition include smectite clays such as montmorillonite, hectorite or bentonite clays or other suitable natural or synthetic materials. Hectorite consists of clay minerals and is commercially available as Bentone® from Elementis Specialties Inc. from Hightstown, New Jersey or as OPTIGEL® SH Synthetic Hectorite which is commercially available from SudChemie Inc. from Louisville, Kentucky.

[0031] The rheological additives that can be used in the reagent composition of the invention are preferably thixotropic or viscosity modifying materials. Such materials improve the screen printing properties of the reagent composition. Particularly, the thixotropic additives of the invention include materials that after some time exhibit reduced viscosity. In addition, the viscosity of the thixotropic additives of the invention also decreases the longer the additives are subjected to shear. Rheological additives in the reagent composition can also serve as a binder or filler material.

[0032] In one embodiment, the reagent composition contains from 0.2 wt. up to 1.6 wt. smectite clay. The amount and type of rheological additive used may vary depending on the polymer used in the reagent composition as well as whether the reagent is based on water or an organic compound.

[0033] In yet further embodiments of the invention, the reagent composition may contain additional components such as buffer and wetting agent. Examples of buffers that can be used include citric acid and other suitable buffers such as phosphate buffers. The reagent composition may contain from 10 mmol to 500 mmol buffer and preferably from 25 mmol to 200 mmol. Other suitable buffers include sodium acetate, Hepes buffer etc. The buffer used in the reagent composition can be selected based on the electron transfer mediator used. For example, if the ferricyanide mediator is in the reagent composition, a buffer that maintains a lower pH level and which will not react with the ferricyanide mediator is preferred.

[0034] Suitable wetting agents may include fluorocarbon-based or hydrocarbon-based surfactants. Some examples of surfactants that can be used with the invention include Triton surfactants from The Dow Chemical Company in Midland, Michigan and Surfynol® additives from Air Products and Chemicals, Inc. in Allentown, Pennsylvania. The reagent composition may comprise from 0.01 wt. up to 0.3 wt. surfactant based on fluorocarbons and preferably from 0.02 wt. up to 0.06 wt. Additionally or alternatively, the reagent composition may contain from 0.1 wt. up to 5.0 wt. hydrocarbon-based surfactant and preferably from 1.0 wt. up to 3.0 wt.

[0035] The remainder of the formulation may contain water or other suitable solvents, which may vary depending on the enzyme chosen and the electron transfer mediator. The solvent should be inert towards the enzyme and electron transfer mediator.

[0036] FIG. 3 and 4 show another embodiment of a test sensor that is adapted to use the reagent described above. FIG. 3 is an exploded view of the test sensor. The testing sensor 110 includes an insulating base 112 on which it is printed in order (usually by screen printing techniques), an electrical conductor pattern including the first and second conductors 114a, 114b (low resistance contacts), an electrode pattern including a working electrode 116, a counter electrode 118, an insulating layer (dielectric) 120 including opening and channel 125 and reaction layer 124.

[0037] Reaction layer 124 includes a reagent composition that converts the analyte of interest (e.g., glucose) into a chemical form that is electrochemically measurable with respect to the electric current that it produces, using electrode formula components 116, 118. Reaction layer 124 is located above the opening 122 and the channel 125 in the insulating layer 120. Thus, part of the reaction layer 124 exposed to the pattern of the electrode 116, 118 is defined by the opening 122 and the channel 125 in the insulating layer 120. The working electrode 116 is electrically coupled to the first conductor 114a and the counter electrode 118 is electrically coupled to the second conductor 114b. The trigger counter electrode subunit 119 is electrically coupled to the counter electrode 118 and serves as a detection electrode in a two-electrode system.

[0038] The test sensor 110 includes a lid 130 with a concave portion 132 that forms a capillary channel when cooperating with an insulating layer 120 to displace a fluid sample from the inlet 134 to the test sensor 110. The distal end of the capillary channel has one or more holes 136 for venting the capillary channel - the fluid sample flows through the inlet 134 to the test sensor 110 toward the opening 136. In use, the test sensor 110 collects a fluid sample (e.g., a blood sample from a patient's finger) by contacting the capillary channel inlet 134 with the fluid sample.

[0039] The reagent composition of the invention described herein can be used in various testing sensors. Some examples of testing sensors that can use the reagent formulation are Ascensia ™^andAutodisc ™ and⁰Glucodisc Blood Glucose Test Strips that are designed for use with Ascensia ™⁰ BREEZE Blood Glucose Meter and Ascensia ™⁰DEX® 2 / DEX® Blood Glucose Meter from Bayer Healthcare LLC from Tarrytown, New York.

[0040] As mentioned above, the stability of the test sensor is increased by the use of a reagent composition containing a hydroxyethyl cellulose polymer. This applies in particular to analytical tests lasting less than 35 seconds and particularly preferably to analytical tests lasting less than 25 seconds. The improved stability of the test sensor leads to a longer life and use of the test sensor.

EXAMPLES [0041] To compare the stability of the test sensor, the change of reagent background as a function of time and temperature, and thermal stability, a group of test sensors was provided with the HEC based reagent composition as described below in Example 1. Another group of sensors with the PEO based reagent composition provided as described below in Example 2. The test results are described in Examples 3, 4 and 5 and are indicated in FIG. 5, 6 and 7a-7d.

Example according to the invention 1: HEC based reagent [0042]

<td>REAGENT COMPONENT</td><td>% BY WEIGHT *</td>

<td>Purified Water</td><td> 60-80</td>

<td>Smectite clay</td><td> 0,2-1,6</td>

<td>Citrate buffer</td><td>25-200 mMol</td>

<td>A fluorine hydrocarbon surfactant</td><td> 0,02-0,1</td>

<td>hydroxyethyl cellulose</td><td> 3,6-6,0</td>

<td>Potassium ferrocyanide</td><td> 15-20</td>

<td>Glucose oxidase enzyme</td><td> 1,0-4,0</td>

<td colspan="2">* Unless other units are indicated</td>

Comparative Example 2: Reagent based on PEO [0043]

<td>REAGENT COMPONENT</td><td>% BY WEIGHT *</td>

<td>Purified Water</td><td> 60-80</td>

<td>smectite clay</td><td> 0,2-2,2</td>

<td>Citrate buffer</td><td>25-200 mMol</td>

<td>A fluorine hydrocarbon surfactant</td><td> 0,02-0,1</td>

<td>Hydrocarbon surfactant</td><td> 0,2-3,0</td>

<td>Polyethylene oxide</td><td> 3,0-9,0</td>

<td>Potassium ferrocyanide</td><td> 15-20</td>

<td>Glucose oxidase enzyme</td><td> 1,0-4,0</td>

<td colspan="2">* Unless other units are indicated</td>

Example 3 [0044] To assess sensor stability, a test batch was performed on two batches of test sensors to determine the percentage of glucose oxidase recovery after storage of test sensors at -20 degrees C and 50 degrees C for two and four weeks. One lot of HEC-based reagent sensors was compared with the other lot of testing sensors with a PEO-based reagent. At the end of the storage period of the testing sensors, the testing sensors were extracted with buffer, and the glucose oxidase activity in the test sensor extracts was analyzed using standard methods of enzymatic activity analysis.

[0045] The test results are shown in FIG. 5. Although both batches of test sensors were formulated using the same amount of glucose oxidase in the reagent, the recovery of glucose oxidase activity for the HEC-based reagent was greater than the recovery of glucose oxidase activity for the PEO-based reagent. This was especially true for both batches of test sensors that were stored at -20 degrees C and 50 degrees C for periods of two and four weeks. The results from the table indicated that the test sensors containing the PEO-based reagent contained more non-reactive glucose oxidase. These results indicated that glucose oxidase activity was more stable at various temperatures and for extended periods of time, in some cases close to 100% recovery for testing sensors with reagents that contained HEC. This translated into a more stable sensor during storage of the test sensor.

Example 4 [0046] FIG. 6 illustrates the results of the study to assess the change in reagent background in HEC-based reagent testing sensors and reagent-based testing sensors

- 10 PEO as a function of storage time at 5 degrees C for 6 weeks. The increase in reagent background as a function of reagent storage time is the result of non-glucose conversion of ferricyanide to ferriocyanide. At each stability checkpoint, the reagent background was analyzed by an injection flow analysis system to quantify the relative amount of ferrocyanide formed during storage. The percentage of reagent background increase was calculated by comparing the reagent background intensity at each control point to the reagent background intensity at the starting point. As seen in FIG. 6, the HEC-based reagent showed less background increase after 6 weeks compared to the PEO-based reagent.

Example 5 [0047] FIG. 7a, 7b, 7c and 7d show a comparison of the systematic error of determination for stressed test sensors formulated with HEC and PEO based reagents. To assess the thermal stability of the test sensors, the test sensors were stored at -20 degrees C and 50 degrees C for two and four weeks. At the end of the storage periods of the test sensors, the test sensors were evaluated using whole blood with 40% hematocrit at 50, 100, and 400 mg / dL glucose levels. 20 replicates per sample were collected using 30 and 10 second assay protocols. The difference in glucose results between the test sensors under stress at 50 degrees C and the test sensors under stress at -20 degrees C was calculated. For samples with 50 mg / dL glucose, the difference in the results of the determinations was expressed as "systematic error of the determination" (see FIGS. 7a and 7c) and for examples with 100 mg / dL the difference in the results of the determination was expressed as "% of the systematic error of determination "(see FIGS. 7b and 7d).

[0048] Improved stability of the test sensors was most noticeable at lower glucose levels due to less background drift of the test sensor. The results showed that the differences in assay systematic error and% assay systematic error were more noticeable when the total assay time was changed from 30 seconds to 10 seconds. The HEC-based reagent dramatically reduced the systematic error of the assay between the test sensors at 50 degrees C and -20 degrees C. This was noted for both 30-second and 10-second determinations.

[0049] Although the inventive test reagent composition has been described for use primarily in an electrochemical test sensor, it is contemplated that the inventive test sensor reagent composition may also be adapted for use with other test sensors, such as optical test sensors.

[0050] Although the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown as an example in the drawings and are described in detail herein. It should be understood, however, that this is not intended to limit the invention to the specific forms disclosed but, on the contrary, the intention is to include

- 11 all modifications, equivalents and alternatives falling within the scope of the invention as defined in the appended claims.

Prepared and verified

Mirosława Ważyńska Patent attorney