HK1189038A - Size self-limiting compositions and test devices for measuring analytes in biological fluids - Google Patents

Description

Self-limiting size composition and test device for measuring analytes in biological fluids

The application is a divisional application of a same-name application with the application number of 200580042703.X and the application date of 2005, 12 months and 12 days.

Technical Field

The present invention generally relates to formulations for determining the amount of an analyte in a biological fluid. In one important application, the invention is applied to measuring the glucose content of blood or other fluids.

Background

The determination of the amount of an analyte in a biological fluid, such as whole blood, is important in the diagnosis and treatment of certain diseases. For example, to determine the glucose level in the blood of individuals with diabetes, who must frequently check the glucose level in the blood to adjust their drinks and medications. The glucose content of blood can be measured by several methods. One approach is to use an electrochemical biosensor that relates the glucose content to the measured current. Another method provides a visual indication of glucose content, such as by the reaction of an indicator to develop a color. The invention is particularly useful in optical measurements, as well as in electrochemical biosensors.

There are numerous patents describing methods of using indicators that develop color or other measurable responses when they are chemically oxidized in the last step of a series of reactions. For example, methods using enzymes, such as an analyte oxidase (e.g., glucose oxidase) or an analyte dehydrogenase (e.g., glucose dehydrogenase). The procedure used is similar, but different enzymes, mediators and indicators are used.

Methods of using glucose oxidase are taught in a number of U.S. patents and patent applications. Representative are US4,211,845, US4,808,529, US5,116,729, US5,264,348, US5,620,863 and US2003/0077702a 1. These patents teach methods for the oxidation of glucose to gluconic acid with the release of hydrogen peroxide. The hydrogen peroxide is said to oxidize the indicator in the presence of peroxidase to produce a measurable color that is indicative of the glucose level in the blood sample. Some recent patents have proposed methods in which glucose is first converted to gluconic acid and then to gluconolactone with the release of hydrogen peroxide. It has also been suggested that gluconolactone is formed first and then hydrolyzed to gluconic acid. Regardless of which process is correct, glucose oxidase has been widely used in dry strips and other techniques for measuring the glucose content of blood.

Different indicators are used on glucose sensors, such as diaminobiphenyl type indicators and heterocyclic azines. For example, 3,3 ', 5, 5' -tetramethylbenzidine and syringaldazine, luminol, o-tolidine, o-dianisidine and others. Another indicator family is tetrazolesA dye precursor. Examples of patents describing such indicators include U.S. Pat. nos. 5,126,275, US5,322,680, US5,300,637, US5,290,536, US5,360,595, and US6,586,199. Tetrazoles are used in the preferred embodiments of the invention described belowAn indicator.

Of particular interest in connection with the present invention is the process described in U.S. Pat. No. 6,200,773 and its parent U.S. Pat. No. 5,902,731. In these patents, glucose dehydrogenase, NAD or PQQ or their derivatives, and tetrazole as cofactors are used for testing the glucose content in bloodA dye precursor, lipoamide dehydrogenase or the like, and nitrite. FIG. 5 of patent' 773 is by tetrazoleReduction of dye precursors to formazanAnd a graph showing the method of detecting glucose by developing color.

A prior patent that relates to the use of enzymes for determining the amount of glucose in blood is US3,630,957. Glucose oxidase and peroxidase are uniformly distributed into a water-resistant polymer membrane to react with glucose and produce color. The membrane can be supported on a substrate, such as a polymer membrane. Suggestions may additionally include chalk, titanium dioxide, colloidal silicic acid (used in the examples), etc., and may include pigments to make the film opaque. Blood is applied to the reagent-containing membrane and then removed before reading the developed color. The use of opaque fillers to reduce interference in the measurement of glucose by red blood cells is also discussed in US5,968,765.

Another interesting is US patent US4,312,834, where the use of a water-repellent film comprising insoluble particles is described, which particles provide a passage for the reagent while hindering the entry of large components. The membrane may be supported on a carrier, such as a film, foil, or the like. In view of the access of certain molecules, the patentees indicate that the amount of particles (called "film openers") should be limited. Different types of particles are suggested, such as diatomaceous earth gel, silica gel, gypsum, and the like. Titanium dioxide is suggested as a way to open the membrane and improve the membrane mitigation properties. Generally, the 200-400 μm relatively thick films of the examples were deposited on a substrate film, and in some cases, a multilayer film was applied. As in the' 957 patent, excess sample, such as blood, is removed after the reaction has occurred.

Many patents have been described with test strips as they are widely used to detect analytes in biological samples. Each test strip application has unique problems that it must address if accurate and consistent results are to be obtained. Testing whole blood requires that the red blood cells do not interfere with the developed color indicating the presence of glucose, nor do they interfere with the electrochemical measurement. In some cases a specific component is added to the test strip to allow red blood cells to be filtered from the sample. In other cases, the sample is removed after a period of time has elapsed and the developed color can be measured. Problems related to the concentration of red blood cells in the sample are encountered when testing whole blood. The amount of red blood cells in a sample is typically measured by the hematocrit value. Since the hematocrit in a blood sample may vary between 20 and 60%, the measurement of glucose may be affected. At the same time, plasma movement to carry glucose to the reagent to develop color (or electrochemical response) may be delayed or incomplete.

Preventing red blood cells from reaching reagents that react with glucose is a concern to many of those skilled in the art. In the test strip of the above-mentioned' 765 patent, a 0.002 to 0.2 inch (50.8 to 5080 μm) thick porous membrane is added with a reagent for separating red blood cells from whole blood, which includes polyacrylic acid, an indicator, and an opaque filler such as titanium dioxide, talc, etc. mixed therein. The coating solution is deposited on the surface of the porous membrane or absorbed within the membrane.

In US5,306,623, a coating capable of separating whole blood is selected from the group consisting of polymers including polyvinyl alcohol sulfonic acid, polyethylene glycol, polystyrene sulfonic acid, hydroxypropyl cellulose, polypropylene glycol, polyvinyl alcohol and polyacrylic acid. The release coating is deposited on the porous substrate together with the reagents for testing blood.

U.S. patent US5,789,255 discusses the sensitivity of blood test strips to whole blood hematocrit. The inventors found that increasing 0.1-2% w/v of a high molecular weight (>750000) acrylic acid polymer reduces the effect of varying hematocrit on glucose measurements.

An ideal test strip for measuring glucose in a whole blood sample should be insensitive to the hematocrit of the blood sample and provide rapid, accurate, and consistent results. The fast response time plus the stable endpoint (endpoint) will provide a verification that is significantly less time dependent and therefore more convenient for the user. Another important aspect for the user is that the test strip should be insensitive to the amount of blood used. Test strips with very close to ideal performance are described in more detail below.

Disclosure of Invention

The present invention relates generally to a self-limiting size (size self-limiting) reagent formulation for use in optical or electrochemical methods for measuring analytes in biological fluids and test strips containing the same. While glucose is the analyte of particular interest, other analytes in other biological fluids are also considered to be within the scope of the present invention.

The inventive formulation consists essentially of a water-soluble swellable polymer matrix containing water-insoluble particles of nominal size of about 0.05 to 20 μm, preferably about 1 to 10 μm, and an enzyme system that reacts with the analyte. The weight ratio of the water insoluble particles to the water soluble swellable polymer matrix is about 1/2 to 2/1.

In a preferred embodiment, the reagent preparation of the invention comprises as reactants an enzyme system for reaction with glucose and an indicator. The enzyme system comprises glucose dehydrogenase and cofactors of the enzyme such as NAD, tetrazoleA salt indicator, and in a particularly preferred embodiment, a lipoamide dehydrogenase as mediator. The agent is bound to a water-soluble swellable polymer matrix containing small water-insoluble particles, preferably titanium dioxide and calcium carbonate. The polymer matrix is preferably selected from the group consisting of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and copolymers thereof. The weight ratio of particles to polymer matrix is preferably from about 1/2 to 2/1. In preparing test strips, the formulations of the present invention are cast as a film having a thickness of about 6 to 16 μm, preferably about 7 to 10 μm, on a non-porous substrate. The membrane is covered with an adhesive layer and a protective top that includes the capillary channel to complete the test strip. The overlay may be transparent or opaque.

In another aspect, the invention is a method for detecting glucose in an analyte in a biological fluid, such as whole blood, by optical or electrochemical methods, which provides a fast and stable response and is insensitive to the amount of sample. The method of the invention uses a test strip in which a film is cast on a non-porous substrate, the film comprising water insoluble particles of nominal size 0.05 to 20 μm, preferably 1 to 10 μm, in a water soluble swellable polymer matrix. The film preferably has a thickness of about 6 to 16 μm, more preferably about 7 to 10 μm; and the weight ratio of reflective particles to polymer matrix is preferably from about 1/2 to 2/1.

Drawings

FIGS. 1a, b are graphs of the results of example 1.

FIGS. 2a, b are graphs of the results of example 2.

FIGS. 3a, b, c are graphs of the results of example 3.

FIGS. 4a, b, c are graphs of the results of example 4.

Detailed description of the preferred embodiments

The present invention relates generally to formulations for measuring analytes in biological fluids, including but not limited to glucose, lactate, cholesterol, triglycerides, free fatty acids, bilirubin, ascorbic acid, hydrogen peroxide, and uric acid. An important application of the present invention is the measurement of glucose content in whole blood, which is described in more detail below. The formulation may be used in place of other reagents for measuring glucose, as will be apparent to those skilled in the art.

Measuring glucose in blood

Optical method

Glucose in blood can be measured by a reagent system that oxidizes glucose using enzymes including, but not limited to, hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

In methods using glucose oxidase, these enzymes react with glucose to produce oxidized glucose and hydrogen peroxide. The hydrogen peroxide oxidizes the indicator compound in the presence of peroxidase. The oxidized indicator produces a color that is correlated to the glucose content of the blood sample.

In other embodiments of the invention, glucose dehydrogenase is combined with a cofactor such as NAD, FAD or PQQ, a mediator such as lipoamide dehydrogenase, and an indicator such as tetrazoleDye precursors are used together to produce a visible response proportional to the glucose content of the sample. This reaction is generally described by the following sequence of reactions:

glucose + GDH-cofactor_{Oxidation by oxygen}→ gluconolactone + GDH-cofactor_{Reduction of}

GDH-cofactor_{Reduction of}+ medium_{Oxidation by oxygen}→ GDH-cofactor_{Oxidation by oxygen}+ medium_{Reduction of}

Medium_{Reduction of}+ tetrazoleIndicator → medium oxidation + A

According to this sequence of reactions, glucose is converted to gluconolactone while the dehydrogenase-cofactor is reduced and then reoxidized by the mediator for further reaction with available glucose.

Dehydrogenase enzyme

The dehydrogenase specifically reacting with glucose is called glucose dehydrogenase. They are from Toyobo, Kyowa, Amano, Genzyme, Biozyme and other commercial enzymes and either natural enzymes or recombinant enzymes produced by typical fermentation and/or recombinant methods. To be effective, this dehydrogenase requires cofactors such as NAD (nicotinamide adenine dinucleotide) and its derivatives, FAD (flavin adenine dinucleotide) and its derivatives, and PQQ (pyrroloquinoline quinone) and its derivatives.

Medium

Mediators are typically used to reoxidize the reduced dehydrogenase-cofactor after the glucose reaction to form the corresponding lactone. Examples of the medium (referred to as a hydride extractor in U.S. Pat. No. 6,200,773) include lipoamide dehydrogenase, PMS (phenazine dimethyl sulfate), PES (phenazine ethosulfate), DCIP (2, 6-difluorophenolinophonol) and ferrocene. A commonly used medium in electrochemical sensors is ferricyanide.

Tetrazole derivatives Indicator agent

Tetrazoles are widely described in U.S. patent No. US5,360,595 and other patents mentioned aboveAn indicator. Tetrazoles identified in U.S. Pat. No. 6,200,773Dye precursors are listed as being particularly useful for combined reactions with dehydrogenase-cofactors. One of which is tetrazole named WST-4Compounds, which are used in the examples below.

Supporting matrix

The reagent layer of the present invention is typically disposed on a substantially non-porous support substrate, typically a polymeric tape or handle such as polyester, polycarbonate, and the like. Such a strip has dimensions suitable for use with an instrument for reading the appearing color. For example, the tape of the examples is about 0.060 by 0.160 inches (1.5 by 4.1 mm) and about 0.002 inches (51 μm) thick. Although not critical to the invention, the substrate is preferably optically clear and may contain one or more coatings that are used to aid in adhesion to other surfaces during processing, such as reagent bearing layers.

Electrochemical process

In an electrochemical sensor, a voltage is applied to an electrode in contact with a blood (or other) sample to generate a current, which is measured and correlated to the amount of analyte (e.g., glucose) in the sample. The electrode is in contact with a solid layer containing an enzymatic reagent that oxidizes the analyte in the sample and a mediator that reoxidizes the reduced enzyme. The reactants may be described by the following steps:

glucose + enzyme_{Oxidation by oxygen}→ enzymes_{Reduction of}+ oxidized glucose (gluconolactone)

Enzyme_{Reduction of}+ n medium_{Oxidation by oxygen}Dielectric substance → n_{Reduction of}+ enzyme_{Oxidation by oxygen}

n medium_{Oxidation by oxygen}→ medium_{Oxidation by oxygen}＋ne-

Wherein the enzyme_{Oxidation by oxygen}And enzymes_{Reduction of}Is an oxidized and reduced form of the redox center of the enzyme, and the mediator_{Oxidation by oxygen}And a medium_{Reduction of}Are the oxidized and reduced forms of the mediator.

In general, the reactive agent used in electrochemical sensors is similar to that used in optical methods, except that no indicator is needed in the electrochemical sensor.

Reagent layer preparation

The reagent layer of the present invention is a single, very thin layer containing reagents that react with glucose in the blood sample and produce a rapid and uniform response. The reagent layer is characterized by a self-defined size, in which movement of large particles, such as red blood cells, is impeded and movement of desired components is allowed. It can be generally described as a water-soluble swellable polymeric matrix containing water-insoluble particles, an enzyme that reacts with glucose, such as glucose dehydrogenase and its co-factor, a mediator, and an indicator using optical detection. Additional components may include detergents, surfactants, and the like.

The polymer may include polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and copolymers thereof. Typically such polymers are characterized as having a relatively low molecular weight, for example 100 to 100000 daltons, including low molecular weight oligomers. Such polymers have high solubility in aqueous solutions and typically have low viscosity. They are typically dissolved in a buffer solution that maintains the desired pH, e.g., about 7.5. The polymer will expand rapidly upon rehydration in a neutral pH solution such as whole blood. It is believed that such polymers provide rapid access of the analyte (e.g., glucose) to the reagent.

The particles may include titanium dioxide, calcium carbonate, bentonite, silica, barium sulfate, powdered metals, latex, and the like. Suitable particles are substantially water insoluble and have a nominal size of about 0.05 to 20 μm, but preferably in the range of 1 to 10 μm. The particles should not have pores that allow undesired movement of the reagent or sample components.

Detergents and surfactants may include proprietary materials such as Silwet L-7600 (dimethyl silicone methyl ethoxylate), Gerepon T-77 (sodium N-oleyl-N-methyl taurate), Zwittergentt3-12 (N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate), and others.

Other additives may include emulsifiers, wetting agents, thickeners, pigments, and similar additives.

The weight ratio of reflective particles to polymer matrix after the coating is applied and dried is typically between about 1/2 and 2/1. The coating may be applied at a thickness of about 6 to 16 μm, preferably about 7 to 10 μm. Such coatings are much thinner than those typically used in the prior art. It has surprisingly been found that such a film provides a fast and stable response, as illustrated in the examples below.

Preparation of test strips for optical methods

The reagent layer is prepared by mixing the above components into a uniform aqueous coating, applying it to a substrate such as a gravure or Mayer-rod coating using techniques well known to those skilled in the art, and then drying. Other coating methods may be used because the method of applying the coating is not considered a successful element.

After the coating is applied to the substrate, it is covered with a protective top (protective top), preferably a hydrophilic polyester coating, which may be transparent or opaque. Additional opaque layers may also be added. An adhesive layer is placed between the reaction layer and the protective top which connects the other two layers and also forms a capillary channel for the introduction of the blood sample. As seen in the examples, the test strips of the present invention are insensitive to capillary dimensions. In other words, the test strip does not require the use of a defined amount (or other biological fluid).

In each of the following examples, the listed components were combined by mixing the polymer, buffer, particles, and additives into distilled water, with low shear followed by high shear for a period of time until uniformly mixed to prepare the base. The diagnostic reagent is then mixed into the base material and the mixed components are applied to a strip of substantially non-porous polycarbonate or polyester support material, such as a 10 μm thick coating. A spacer-adhesive layer 3M F9460 having a thickness of 50 or 80 μm was added. Finally a protective top of hydrophilic polyester coating (3M 9971) was applied by using a transfer adhesive. A piece of opaque material is applied to the exterior of the polyester top to provide an additional small amount of opacity.

Electrochemical sensor

A reagent layer is applied to the electrodes in addition to the optical indicator to produce an electrochemical sensor, such as those exemplified in US patent application US 2001/0042683.

Example 1

The test strip was prepared by mixing the components listed in Table A to prepare reagent layers and applying them to the substrate as previously described. The performance of the test strip is determined by adding a small sample of whole blood (about 600 nL) containing a known amount of glucose to the reagent layer, placing the test strip in a small reading area (e.g., 0.75mm diameter) diffuse reflectance measuring instrument and reading the color developed over a period of about 60 seconds. The results are reported as K/S, Kubelka-Munk function (1-R) 2/2R where R is the measurement coefficient. As seen in fig. 1a, the measured color intensity remained unchanged over each glucose concentration test time. The glucose content corresponding to the response of the glucose meter is given as shown in fig. 1 b. It is clear that the response time is fast and that the results are very similar for the 12 th and 52 th seconds. Thus, it can be concluded that the test strip of the present invention provides a fast response and is not overly sensitive to read time.

TABLE A

(1)Rheox,Bentone EW

(2)Sigma-Aldrich,T-8141

(3) Polysciences Ltd, 18611

(4) Pragmatics Ltd

(5)OSi Specialties

(6) Pragmatics Ltd

(7) Dojindo laboratory

(8)Calbiochem

(9)Unitika,Diaphorase I

(10) Amano Enzyme Inc., Amano2

Example 2

Preparation of the test strips was completed by mixing the components listed in Table B to prepare another reagent layer and applying them to the substrate as previously described. A series of tests were performed on whole blood samples containing known amounts of glucose as described in example 1. The results of the test are shown in FIGS. 2 a-b. It was observed that the developed color was substantially unchanged over a period of 60 seconds, except that some additional color was developed at higher concentrations.

TABLE B

(1)Rheox,Bentone EW

(2)Sigma-Aldrich,T-8141

(3) Polysciences Ltd

(4)Dow,UCAR Latex455

(5)Calbiochem

(6) Dojindo laboratory

(7)Sigma-Aldrich

(9)Unitika,Diaphorase I

(10) Amano Enzyme Inc., Amano2

Example 3

The test strips were prepared by mixing the components listed in Table C to prepare reagent layers and applying them to the substrate as previously described. The reagent layer was tested as described in examples 1 and 2. The results are shown in FIGS. 3a, b, c. The results reported in FIGS. 3a and 3b are similar to those of examples 1 and 2. Additional color development at the higher glucose concentrations recorded in fig. 2a and b did not occur in this example.

Additional results are shown in figure 3 c. Whole blood samples with 3 different hematocrits (20%, 40%, 60%) were measured and the results plotted. It is clear that the test strips of the present invention are not significantly affected by the concentration of red blood cells in the blood.

Watch C

(1)Research Organics

(2)Rheox,Bentone EW

(3)Sigma-Aldrich,T-8141

(4) Polysciences Ltd

(5) Pragmatics Ltd

(6) Pragmatics Ltd

(7) Pragmatics Ltd

(8)OSi Specialties

(9) Dojindo laboratory

(10)Sigma-Aldrich,#N-6522

(11)Unitika,Diaphorase I

(12) Toyobo Co., Ltd. # GLD311

Example 4

The test strips were prepared by mixing the components listed in Table D to prepare reagent layers and applying them to the substrate as previously described. The resulting test strips were tested as described in the previous examples. The results are shown in FIGS. 4a, b, and c. The measured color appeared even more intense than seen in the previous examples. That is, it can be inferred that the color rendering is sufficiently completed within the first few seconds.

FIG. 4c illustrates another advantage of the test strip of the present invention. Two test strip designs were compared. The first one has a capillary gap of 50 μm through which the blood sample is entered, while the second one has a capillary gap of 80 μm. Figure 4c shows that the difference in capillary gap height and the resulting change in sample volume has no significant effect on the results. The reason for this is unclear, but can be attributed to the inability of glucose to diffuse from the blood sample into the reagent layer after it has been initially rehydrated by the blood sample. In any event, this represents a significant advantage of the inventive test strips, as it means that they are not sensitive to the amount of blood administered.

Table D

(1) Polysciences Ltd

(2) Polysciences Ltd

(3)DuPont,R-706

(4) Huber Inc., 4Optifil □

(5) Pragmatics Ltd

(6) Air Products and Chemicals Co., Ltd

(7)OSi Specialties

(8) Dojindo laboratory

(9)Sigma-Aldrich,#N-6522

(10)Unitika,Diaphorase I

(11) Toyobo Co., Ltd. # GLD311

Alternative embodiment A

A reactive formulation for measuring the amount of an analyte in a biological fluid, comprising:

(a) a water-soluble swellable polymer matrix;

(b) water insoluble particles having a nominal size of about 0.05 to 20 μm;

(c) an enzyme system that reacts with the analyte; and

wherein the weight ratio of the water insoluble particles to the water soluble swellable polymer matrix is about 1/2 to 2/1.

Alternative embodiment B

The reaction formulation of alternative embodiment a, wherein the enzyme system is reacted with one of glucose, lactate, cholesterol, triglycerides, free fatty acids, bilirubin, ascorbic acid, hydrogen peroxide, and uric acid.

Alternative embodiment C

The reaction formulation of alternative embodiment a wherein the analyte is glucose and the enzyme system comprises one of hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

Alternative embodiment D

The reaction formulation of alternative embodiment a wherein the particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

Alternative embodiment E

The reaction formulation of alternative embodiment a, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

Alternative embodiment F

The reaction formulation of alternative embodiment a, further comprising at least one surfactant, detergent, or thickener.

Alternative embodiment G

The reaction formulation of alternative embodiment a, for use as a coating having a thickness of about 6 to 16 μm.

Alternative embodiment H

The reaction formulation of alternative embodiment G, wherein the coating thickness is about 7 to 10 μm.

Alternative embodiment I

The reaction formulation of alternative embodiment a, wherein the molecular weight of the water-soluble swellable polymer is less than about 100000.

Alternative embodiment J

The reaction formulation of alternative embodiment a, wherein said water insoluble particles have a nominal size of about 1 to 10 μm.

Alternative Process K

A method of measuring the amount of an analyte in a biological fluid by applying the biological fluid sample to a test strip or electrochemical sensor to obtain a fast and stable response insensitive to the sample amount, the method comprising:

(a) applying the biological fluid sample to the test strip or electrochemical sensor, the test strip or electrochemical sensor comprising a non-porous substrate on which is deposited a thin film of an enzyme system for reacting with the analyte contained in a formulation, wherein the formulation comprises a water-soluble swellable polymer matrix and water-insoluble particles having a nominal size of about 0.05 to 20 μm; and

(b) measuring the response of the sample to the enzyme system by optical or electrochemical means and determining the amount of the analyte present in the biological fluid.

Alternative Process L

The method of alternative Process K, wherein the analyte is glucose and the biological fluid is whole blood.

Alternative Process M

A method of alternative treatment L, wherein the enzyme system comprises glucose oxidase or glucose dehydrogenase.

Alternative Process N

The method of alternative Process K, wherein the film thickness is about 6 to 16 μm.

Alternative Process O

The method of alternative process N, wherein the film thickness is about 7 to 10 μm.

Alternative Process P

The method of alternative Process K, wherein the weight ratio of the particles to the polymer matrix is about 1/2 to 2/1.

Alternative Process Q

The method of alternative Process K, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

Alternative Process R

The method of alternative Process K, wherein the particles comprise at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

Alternative Process S

The method of alternative Process K, wherein the swellable polymer has a molecular weight of less than about 100000.

Alternative Process T

The method of alternative process K, wherein the insoluble particles have a nominal size of about 1 to 10 μm.

Alternative embodiment U

A reactive preparation for measuring glucose content in whole blood, comprising:

(a) a water-soluble swellable polymer matrix;

(b) water insoluble particles having a nominal size of about 0.05 to 20 μm;

(c) an enzyme system for oxidizing said glucose;

(d) an indicator;

wherein the weight ratio of reflective particles to polymer matrix in (b) is from about 1/2 to 2/1.

Alternative embodiment V

The reaction formulation of alternative embodiment U, wherein the enzyme system comprises one of hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

Alternative embodiment W

The reaction formulation of alternative embodiment U wherein the enzyme system comprises a glucose dehydrogenase, a cofactor for said glucose dehydrogenase, a tetrazoleA salt indicator and a medium.

Alternative embodiment X

The reaction formulation of alternative embodiment U wherein the particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

Alternative embodiment Y

The reaction formulation of alternative embodiment U, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

Alternative embodiment Z

The reaction formulation of alternative embodiment U, wherein the water-soluble swellable polymer is dissolved in a solution buffered to maintain a desired pH.

Alternative embodiment AA

The reaction formulation of alternative embodiment U, further comprising at least one surfactant, detergent, or thickener.

Alternative embodiment BB

The reaction formulation of alternative embodiment U, for use as a coating having a thickness of about 6 to 16 μm.

Alternative embodiment CC

The reaction formulation of alternative embodiment BB, wherein the coating thickness is about 7 to 10 μm.

Alternative embodiment DD

The reaction formulation of alternative embodiment U, wherein the water soluble swellable polymer has a molecular weight of less than about 100000.

Alternative embodiment EE

The reaction formulation of alternative embodiment U, wherein said insoluble particles have a nominal size of about 1 to 10 μm.

Alternative embodiment FF

A test strip for measuring the amount of glucose in a whole blood sample, comprising:

(a) a substantially non-porous substrate;

(b) a reagent layer disposed on the substrate, the reagent layer comprising:

(1) a water-soluble swellable polymer matrix having a polymer core,

(2) water insoluble particles having a nominal size of about 0.05 to 20 μm,

(3) an enzyme system for oxidizing said glucose, and

(4) an indicator;

(c) a protective layer for the reagent layer in (b);

(d) an adhesive layer between the reagent layer and the protective layer, the adhesive layer having capillary channels to receive the blood sample.

Alternative embodiment GG

The test strip of alternative embodiment FF, wherein the enzyme system comprises one of hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

Alternative embodiment HH

The test strip of alternative embodiment FF, wherein the enzyme system comprises glucose dehydrogenase, a cofactor for the glucose dehydrogenase, tetrazoliumA salt indicator and a medium.

Alternative embodiment II

The test strip of alternative embodiment FF, wherein the water soluble swellable polymer matrix is at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

Alternative embodiment JJ

The test strip of alternative embodiment FF, wherein the water-insoluble particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

Alternative embodiment KK

The test strip of alternative embodiment FF, wherein the weight ratio of the reflective particles in b (2) to the polymer matrix in b (1) is about 1/2 to 2/1.

Alternative embodiment LL

The test strip of alternative embodiment FF, wherein the polymer is dissolved in a solution buffered to maintain a desired pH.

Alternative embodiment MM

The test strip of alternative embodiment FF, wherein the reagent layer (b) further comprises at least one surfactant, detergent, or thickener.

Alternative embodiment NN

The test strip of alternative embodiment MM, wherein the reagent layer is about 6 to 16 μm thick.

Alternative embodiment OO

The test strip of alternative embodiment NN wherein the reagent layer is about 7 to 10 μm thick.

Alternative embodiment PP

The test strip of alternative embodiment FF, wherein the swellable polymer has a molecular weight of less than about 100000.

Alternative embodiment QQ

The test strip of alternative embodiment FF, wherein the particles have a nominal size of about 1 to 10 μ ι η.

Alternative processing RR

A method of measuring the amount of glucose in a whole blood sample by applying the blood sample to a test strip to obtain a response that is fast, stable, and insensitive to the amount of the blood sample, the method comprising:

(a) applying said blood sample to said test strip, said test strip comprising a non-porous substrate having deposited thereon a film of an enzyme system for reacting with said glucose contained in a formulation, wherein the formulation comprises a water-soluble swellable polymer matrix, water-insoluble particles having a nominal size of about 0.05 to 20 μm, and an indicator; and

(b) measuring the response of the indicator and determining the amount of the glucose in the blood sample.

Alternative processing SS

The method of alternatively treating RR, wherein the enzyme system comprises glucose oxidase or glucose dehydrogenase.

Alternative treatment TT

The method of alternative processing RR, wherein the film thickness is about 6 to 16 μm.

Alternative processing UU

The method of alternative treatment of TT, wherein the film thickness is about 7 to 10 μm.

Alternative treatment VV

The method of alternatively treating TT, wherein the weight ratio of the particles to the matrix is from about 1/2 to 2/1.

Alternative Process WW

The method of alternative processing RR, wherein the particles are about 1 to 10 μm in nominal size.

Alternative Process XX

The method of alternative treatment of RR, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

Alternative treatment YY

The method of alternative processing of RR, wherein the particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

Alternative treatment ZZ

The method of alternative treatment of RR, wherein the water soluble swellable polymer has a molecular weight of less than about 100000.

The invention also comprises the following contents:

1. a reactive formulation for measuring the amount of an analyte in a biological fluid, comprising:

(a) a water-soluble swellable polymer matrix;

(b) water insoluble particles having a nominal size of about 0.05 to 20 μm;

(c) an enzyme system that reacts with the analyte; and

wherein the weight ratio of the water insoluble particles to the water soluble swellable polymer matrix is about 1/2 to 2/1.

2. The reactive formulation of item 1, wherein the enzyme system is formulated to react with a member of the group consisting of glucose, lactate, cholesterol, triglycerides, free fatty acids, bilirubin, ascorbic acid, hydrogen peroxide, and uric acid.

3. The reaction formulation of item 1, wherein the analyte is glucose and the enzyme system comprises a member of the group consisting of hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

4. The reaction formulation of item 1, wherein the particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

5. The reactive formulation of clause 1, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and copolymers thereof.

6. The reactive formulation of item 1, further comprising at least one surfactant, detergent, or thickener.

7. The reactive formulation of item 1, for use as a coating having a thickness of about 6 to 16 μm.

8. The reaction formulation of clause 7, wherein the coating thickness is about 7 to 10 μm.

9. The reaction formulation of item 1, wherein the water-soluble swellable polymer has a molecular weight of less than about 100000.

10. The reactive formulation of clause 1, wherein the water insoluble particles have a nominal size of about 1 to 10 μm.

11. A method of measuring the amount of an analyte in a biological fluid by applying a sample of the biological fluid to a test strip or electrochemical sensor and obtaining a response that is fast, stable, and insensitive to the amount of the sample, the method comprising:

(a) applying the biological fluid sample to the test strip or electrochemical sensor, the test strip or electrochemical sensor comprising a non-porous substrate on which is deposited a thin film of an enzyme system for reacting with the analyte contained in a formulation comprising a water-soluble swellable polymer matrix and water-insoluble particles having a nominal size of about 0.05 to 20 μm; and

(b) measuring the response of the sample to the enzyme system by optical or electrochemical means and determining the amount of the analyte present in the biological fluid.

12. The method of claim 11, wherein the analyte is glucose and the biological fluid is whole blood.

13. The method of item 12, wherein the enzyme system comprises glucose oxidase or glucose dehydrogenase.

14. The method of item 11, wherein the film thickness is about 6 to 16 μ ι η.

15. The method of item 14, wherein the film thickness is about 7 to 10 μ ι η.

16. The method of item 11, wherein the weight ratio of the particles to the polymer matrix is about 1/2 to 2/1.

17. The method of clause 11, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

18. The method of item 11, wherein the particles comprise at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

19. The method of item 11, wherein the swellable polymer has a molecular weight of less than about 100000.

20. The method of item 11, wherein the insoluble particles have a nominal size of about 1 to 10 μm.

21. A reactive preparation for measuring glucose content in whole blood, comprising:

(a) a water-soluble swellable polymer matrix;

(b) water insoluble particles having a nominal size of about 0.05 to 20 μm;

(c) an enzyme system for oxidizing said glucose;

(d) an indicator;

wherein the weight ratio of reflective particles to polymer matrix in (b) is about 1/2 to 2/1.

22. The reaction formulation of item 21, wherein the enzyme system comprises one of hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

23. The reaction preparation of item 21, wherein the enzyme system comprises glucose dehydrogenase, a cofactor of said glucose dehydrogenase, and tetrazoleA salt indicator and a medium.

24. The reactive formulation of clause 21, wherein said particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

25. The reactive formulation of clause 21, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

26. The reaction formulation of clause 21, wherein the water-soluble swellable polymer is dissolved in a solution buffered to maintain a desired pH.

27. The reactive formulation of clause 21, further comprising at least one surfactant, detergent, or thickener.

28. The reactive formulation of clause 21, for use as a coating having a thickness of about 6 to 16 μm.

29. The reaction formulation of clause 28, wherein the coating thickness is about 7 to 10 μm.

30. The reaction formulation of item 21, wherein the water-soluble swellable polymer has a molecular weight of less than about 100000.

31. The reactive formulation of clause 21, wherein the insoluble particles have a nominal size of about 1 to 10 μm.

32. A test strip for measuring the glucose content of a whole blood sample, comprising:

(a) a substantially non-porous substrate;

(b) a reagent layer disposed on the substrate, the reagent layer comprising:

(1) a water-soluble swellable polymer matrix having a polymer core,

(2) water insoluble particles having a nominal size of about 0.05 to 20 μm,

(3) an enzyme system for oxidizing said glucose, and

(4) an indicator;

(c) a protective layer for the reagent layer in (b);

(d) an adhesive layer between the reagent layer and the protective layer, the adhesive layer having capillary channels to receive the blood sample.

33. The test strip of item 32, wherein the enzyme system comprises a member of the group consisting of hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

34. The test strip of item 32, wherein the enzyme system comprises glucose dehydrogenase, a cofactor for the glucose dehydrogenase, tetrazoliumA salt indicator and a medium.

35. The test strip of item 32, wherein the water-soluble swellable polymer matrix is at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and copolymers thereof.

36. The test strip of item 32, wherein the water insoluble particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

37. The test strip of item 32, wherein the weight ratio of reflective particles in b (2) to polymer matrix in b (1) is about 1/2 to 2/1.

38. The test strip of clause 32, wherein the polymer is dissolved in a solution buffered to maintain a desired pH.

39. The test strip of item 32, wherein the reagent layer (b) further comprises at least one surfactant, detergent, or thickener.

40. The test strip of clause 39, wherein the reagent layer is about 6 to 16 μm thick.

41. The test strip of clause 40, wherein the reagent layer is about 7 to 10 μm thick.

42. The test strip of item 32, wherein the swellable polymer has a molecular weight of less than about 100000.

43. The test strip of item 32, wherein the particles have a nominal size of about 1 to 10 μm.

44. A method of measuring the amount of glucose in a whole blood sample by applying the blood sample to a test strip and obtaining a fast, stable response that is insensitive to the amount of the blood sample, the method comprising:

(a) applying said blood sample to said test strip, said test strip comprising a non-porous substrate having deposited thereon a film of an enzyme system for reacting with said glucose contained in a formulation comprising a water-soluble swellable polymer matrix, water-insoluble particles having a nominal size of about 0.05 to 20 μm, and an indicator; and

(b) measuring the response of the indicator and determining the amount of the glucose in the blood sample.

45. The method of item 44, wherein the enzyme system comprises glucose oxidase or glucose dehydrogenase.

46. The method of item 44, wherein the film thickness is about 6 to 16 μm.

47. The method of item 46, wherein the film thickness is about 7 to 10 μm.

48. The method of item 46, wherein the weight ratio of the particles to the matrix is about 1/2 to 2/1.

49. The method of item 44, wherein the particles have a nominal size of about 1 to 10 μm.

50. The method of item 44, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

51. The method of clause 44, wherein the particles are at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

52. The method of item 44, wherein the water soluble swellable polymer has a molecular weight of less than about 100000.

Claims (23)

1. A method of determining the amount of an analyte in a liquid sample, the method comprising the steps of:

(a) applying the sample to a test strip, the test strip comprising a substrate having a film deposited thereon, the film comprising a water-soluble swellable polymer matrix, and an enzyme;

(b) determining the response of said sample to said enzyme, and

(c) determining the amount or concentration of said analyte present in the sample based on the determined response of said enzyme of said sample, said determined response of the sample being substantially independent of the time of detection.

2. The method of claim 1, wherein the step of determining comprises performing an optical or electrochemical assay.

3. The method of claim 1, wherein the step of determining the response is completed within the first 10 seconds of the determination.

4. The method of claim 1, wherein the step of determining the response is completed within the first 5 seconds of the determination.

5. The method of claim 1, wherein the water insoluble particles are 0.5 to 20 μm in size.

6. The method of claim 1, wherein the film thickness is 6 to 16 μm.

7. The method of claim 1, wherein the measured response of the sample is substantially independent of the size of the sample.

8. A formulation for measuring the amount of an analyte in a biological fluid, comprising:

(a) a water-soluble swellable polymer matrix;

(b) water-insoluble particles;

(c) an enzyme system that reacts with the analyte; and

wherein the weight ratio of the water-insoluble particles to the water-soluble swellable polymer matrix is from 1/2 to 2.1/1, said formulation being used as a coating having a dry film thickness of from 6 to 16 μm.

9. The formulation of claim 8, wherein the analyte is glucose, lactate, cholesterol, triglycerides, free fatty acids, bilirubin, ascorbic acid, hydrogen peroxide, or uric acid.

10. The formulation of claim 9, wherein the analyte is glucose and the enzyme system comprises a member of the group consisting of hexokinase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase-PQQ, and glucose oxidase.

11. The formulation of claim 8, wherein the water insoluble particles comprise titanium dioxide, calcium carbonate, silicon dioxide, barium sulfate, powdered metal, or latex.

12. The formulation of claim 8, wherein the water-soluble swellable polymer matrix comprises at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and copolymers thereof.

13. The formulation of claim 8, further comprising at least one surfactant, detergent, or thickener.

14. The formulation of claim 8, wherein the molecular weight of the water-soluble swellable polymer matrix is less than 100000.

15. A method of determining the amount of an analyte in a liquid sample, the method comprising:

(a) applying the biological fluid sample to a test strip, the test strip comprising a non-porous substrate having deposited thereon a monolayer film of an enzyme system for reacting with the analyte contained in a formulation comprising a water-soluble swellable polymer matrix and water-insoluble particles;

(b) measuring the response of the sample to the enzyme system by optical or electrochemical means, the response of the sample determined being substantially independent of the size of the liquid sample; and

(c) determining the amount or concentration of the analyte present in the biological fluid.

16. The method of claim 15, wherein the analyte is glucose and the biological fluid is whole blood.

17. The method of claim 16, wherein the enzyme system comprises glucose oxidase or glucose dehydrogenase.

18. The method of claim 15, wherein the film has a thickness of 6 to 16 μm.

19. The method of claim 15, wherein the weight ratio of the particles to the polymer matrix is 1/2 to 2.1/1.

20. The method of claim 15, wherein the water-soluble swellable polymer matrix is a polymer matrix comprising at least one of polyacrylic acid, polyvinyl alcohol, sodium polystyrene sulfonate, polyacrylic acid latex, polyethylene glycol, styrene acrylate, and interpolymers thereof.

21. The method of claim 15, wherein the particles are particles comprising at least one of titanium dioxide, calcium carbonate, silica, barium sulfate, powdered metal, and latex.

22. The method according to claim 15, wherein the molecular weight of the swellable polymer matrix is lower than 100000.

23. The method of claim 15, wherein the response of the sample is substantially independent of detection time.