MXPA99007484A - Stress for visual test of glucose in the san - Google Patents

Abstract

The present invention relates to a strip for visual blood glucose test having two membranes each incorporating a reagent that reacts with glucose in a blood sample applied to the membranes to cause a color change; one of the membranes also includes an inhibitor and a dye, a blood sample applied to the strip causes the two membranes to form two different colors, the comparison of the colors with a calibrated color table allows a user to determine the concentration of glucose in the sample of sang

Description

STRESS FOR VISUAL TEST OF GLUCOSE IN THE BLOOD BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION This invention relates to a dry test strip that measures the concentration of glucose in the blood; very particularly a strip that performs glucose measurement without requiring a meter. 2. Description of the Related Art Many visual test devices have been developed to measure the concentration of certain analytes in biological fluid. For example, these devices have measured levels of glucose, cholesterol, proteins, ketones, phenylalanine or enzymes in the blood, urine or saliva. Among the devices that have a more widespread use at present is the blood glucose monitor. In the United States alone, it is estimated that there are more than 14 million people with diabetes. To avoid serious medical problems, such as loss of vision, circulatory problems, kidney failure, etc., many of these people monitor their blood glucose on a regular basis and then take the necessary steps to maintain their glucose concentration at a level acceptable.

The test strips used in these devices contain an indicator that acquires a different shade of color, depending on the concentration of glucose in the whole blood sample that has been applied to the strip. Although some of these strips use reduction chemistry, they most commonly include an oxidizable dye or pair of oxidants. Some of the strips include an enzyme, such as glucose oxidase, which is capable of oxidizing glucose to gluconic acid and hydrogen peroxide. They also contain an oxidizable dye and a substance that has peroxidizing activity, which is capable of selectively catalyzing oxidation of the oxidizable dye in the presence of hydrogen peroxide. The patent of E.U. No. 3,298,789, issued on January 17, 1967 to R. L. Mast, describes a strip of absorbent paper that incorporates a reactive composition that changes color when blood containing glucose is applied to its surface. During use, the blood is cleaned one minute after it has been applied to the surface, and the color of the strip is compared to a color chart that has blocks of colors that represent specific levels of glucose. The patent of E.U. No. 3,630,957, issued December 28, 1971 to H. Rey et al., Describes a strip of thin sheet of plastic that is coated with a reactive composition. The method of use is similar to that described in the previous patent - the blood is applied, about one minute is expected and the resulting color is compared with a color chart.

The patent of E.U. No. 4,975,367, issued on December 4, 1990 to Albarella et al., Discloses a color matching strip that is designed to provide different shades at different analyte concentrations, using two independent indicator systems. It also describes the option of including a color retardant to delay the catalytic effect of the independent systems, changing the final tone produced by a particular concentration of analyte. The patent of E.U. No. 5,200,325, issued April 6, 1993 to J. M. Blatt et al., Describes a "self-indicating" strip, in which no comparisons with a color chart are required. The strip indicates whether or not an analyte is present at a predetermined level of concentration by conducting a subtractive reaction before the indicator reaction. With this the strip generates a predetermined level of response only if the analyte is present at a predetermined concentration or more. The patents described above include "wipe-off" strips, in which the interference between the color of the whole blood and the color developed by the indicator dye is reduced to a minimum by cleaning the excess blood sample from the surface of the strip. Other approaches include applying the blood sample to a surface of the strip and measuring the color on the opposite surface. The patents described below are examples of this. The patent of E.U. No. 4, 935,346, issued June 19, 1990 to R. Phillips et al., Describes a meter, strip and method for determining the glucose concentration in a whole blood sample. The method includes simply applying a whole blood sample to a first ("sample") surface of an inert porous matrix that is impregnated with a reagent. The sample migrates to the opposite "test" surface by interacting the glucose with the reagent to create a light absorbing reaction product. A reading of the reflectance of the test surface indicates the glucose concentration. Reflectance measurements are made at two separate wavelengths to eliminate interference. A synchronization circuit is activated by an initial decrease in reflectance caused by wetting the test surface by the sample that has passed through the matrix. The patent of E.U. No. 5,306,623, issued April 26, 1994 to Kiser et al., Discloses a strip for visual blood glucose testing which includes applying a whole blood sample containing glucose to one side of the strip and taking the glucose reading on the opposite side, after the red blood cells have separated and the sample has reacted with a reagent on the strip. It was found that an asymmetric polysulfone membrane was especially useful as a single layer matrix for the strip. (See also U.S. Patent No. 5,418,142, issued May 23, 1995 and U.S. Patent No. 5,719,034, issued February 7, 1998). The patent of E.U. No. 5,563,031, issued October 8, 1996 to Y.S. Yu, describes a pair of dyes useful in dry test strips to detect analytes, such as glucose, in biological fluids. The dye couple comprises monosodium N-sulfonylbenzenesulfonate of meta [3-methyl-2-benzothiazolinone hydrazone] combined with 8-anilino-1-naphthalenesulfonic acid-ammonium (MBTHSB-ANS) and is used as an indicator in a cascade of reactions that produces a strong oxidizing agent, such as hydrogen peroxide. An advantage of the pair is that it is soluble in aqueous solution, but becomes insoluble after oxidative coupling, thereby minimizing fading and providing a stable end point. White-Stevens and Stover, Clin. Chem. 28, 589-595 (1982), describe the interference that can be caused by ascorbic acid in diagnostic tests. Ascorbic acid causes a delay in the development of the color of tests based on the use of peroxidase and oxidation-reduction indicators. M.J. Sherwood et al., Clin. Chem. 29, 438-445 (1983) describes a visual test strip to be used in conjunction with a calibrated color scale. The strip uses two pads, each formulated for a different part of the normal scale. The technology supports the Visidex ™ test strip, which is read visually. Other commercially available visual strips include the Dextrostix® test strips and the Chem-strip bG® and SmartStrip ™ test strips.

BRIEF DESCRIPTION OF THE INVENTION According to the present invention, a strip for visual blood glucose test comprises: a) a dispersion layer for accepting a blood sample on a main surface and passing the sample to a second opposing main surface; b) an intermediate layer comprising first and second membranes, substantially collateral, each having a major upper surface adjacent to the second major surface of the dispersion layer to receive a part of the blood sample, and each containing a reagent that it can react with the glucose in the sample, while passing through the membrane, to cause a color change in the reagent, the second membrane also comprises an inhibitor and a dye, and c) a support layer for supporting the other layers and to allow any color change in the membranes to be visible through it. As used in this description and the appended claims, the term "reactive" refers to the components that may be on, or in, both membranes, unlike the inhibitor and dye, which are only on, or in, the second membrane. The dye is inert, in the sense that its color is substantially independent of the glucose concentration. Its purpose is to improve the difference in color between the two membranes. Among the advantages of the test strip of this invention are that it does not require cleaning, it can use a small blood sample and it provides results quickly.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a top plan view of a test strip of this invention. Figure 2 is a bottom plan view of the test strip of Figure 1. Figure 3 is an exploded perspective view of another embodiment of a test strip of this invention. Figure 4 is an exploded perspective view of yet another embodiment of a test strip of this invention. Figure 5 is a sweep diagram of glucose values measured by one embodiment of the present invention, plotted against values measured on a standard meter.

DETAILED DESCRIPTION OF THE INVENTION This invention provides a strip for measuring blood glucose that does not need to be cleaned and that allows rapid determination of blood glucose using a small sample of blood and without the need for a meter. Figure 1 is a top plan view of the strip of the invention in partial open cut and Figure 2 is a bottom plan view. As shown there, the strip 10 has three components: an upper dispersion layer 12, an intermediate layer 14 containing a reagent and a support 16. The intermediate layer 14 consists of two collateral membranes 14a and 14b which are seen through openings 16a and 16b,. respectively, in support 16. Membrane 14a contains a reagent that reacts with glucose to cause a visible color change. The membrane 14b contains a reagent that also reacts with the glucose to cause a color change, for a color formed is different (as will be described below) from that formed in the membrane 14a. During operation, a blood sample that does not have to be greater than about 10 μL is applied to the dispersion top layer 12. As the sample 12 penetrates the layer 12, it disperses, whereby the sample is distributed substantially uniformly to the membranes 14a and 14b. The glucose in the blood sample reacts with the reagents in the membranes as they pass to the support layer 16 to form colors. If the support layer 16 is not transparent, the colors are viewed through optional openings 16a and 16b and compared with a color calibration table to determine the concentration of glucose in the blood. A variety of materials are suitable for the dispersion layer; for example, paper, glass fibers, polymer fibers, sintered plastics, woven and non-woven fabrics, and membranes. The materials that are preferred require minimum sample sizes, absorb the sample quickly, distribute it evenly to the membranes and do not interfere with the chemistry of the reagent in the membrane. Of course, the cost is a practical consideration too. A preferred material for the dispersion layer is a hydrophilic polyester woven mesh available from Tetko, Inc., Deprew, N.Y. A minimal sample size and rapid absorption suggest a thin layer. However, proper contact with the membranes is important and that is difficult if the layer is so thin that it wrinkles easily. The commercially available Tetko 7-280 / 44 mesh is among the thickest commercially available meshes -225 microns- and does not wrinkle easily. The Tetko mesh offers fast absorption and a low minimum sample size; however, it may not have the ability to absorb a large sample of blood (undesirably) leaving excess sample on the surface of the dispersion layer. Another preferred dispersion layer material is heat-set plastic, such as polyethylene or polypropylene, which has been made hydrophilic by pre-or post-treatment with a blood-compatible surfactant. One of these materials is a porous polyethylene treated with sodium methyloleoyltaurate and available from Porex Corp. of Fairbum, Georgia. An advantage of this material is that it has an unusually strong absorption, which causes the fluid to be drawn away from the surface, where it could otherwise be transferred to objects or people with whom it made contact. The pores of this material vary from about 20 microns to about 350 microns, preferably about 150 microns or an average of about 100 microns. A mesh measuring approximately 0.5 to 0.6 mm thick, approximately 5 to 6 mm wide and approximately 30 mm long is suitable. Figure 3 illustrates an exploded view of a strip that allows a larger sample. The strip shown has, in addition to the elements shown in Figures 1 and 2, an optional cover layer 18 with a through hole 20 for applying a sample to the dispersion layer. Figure 4 illustrates an exploded view of another strip of this invention. In that embodiment, the optional absorbent layer 22 is attached to the dispersion layer 12 to absorb the excess sample. When the dispersion layer 12 is of the thicker concrete polyethylene type (Porex), the absorbent layer 22 is generally not necessary. However, when the dispersion layer is of the woven mesh type (Tetko), which has a capacity of of lower fluid retention, the absorbent layer 22 is preferably present.

Optionally, the elements of the strip are joined together with an adhesive, it being convenient to use a tape with adhesive on both sides.

The embodiment of Figure 4 includes an adhesive layer 24 (with passage openings 24a and 24b) for attaching the support layer 16 to the intermediate layer 14; adhesive layers 26 for joining the intermediate layer 14 to the dispersion layer 12; and adhesive layer 28 (with passage opening 28a) for attaching the cover layer 18 to the dispersion layer 12 and the absorbent layer 22. The adhesive layers 26 are shown by splicing windows 14a and 14b; however, relative placement is not critical. In this way, there may be a gap between the membrane and the adhesive layer or they may overlap slightly. The most suitable membrane materials and reagents for use in the strips of this invention provide fast reaction times, use minimum sample sizes and produce a uniform color with stable endpoints. In addition, the color of the blood should be substantially hidden from the observation side, so that it does not interfere with the evaluation. Based on these criteria, the membrane material referred to is an asymmetric polysulfone membrane whose pore size gradually increases from one surface to the opposite surface (see U.S. Patent No. 4,629,563, issued December 16, 1986 to Wrasidlo ). This type of membrane is available from the Memtec division of U.S. Filter Co., San Diego, CA. These membranes provided uniform color development and stable endpoints with samples of 10 μL or less, less than 45 seconds. The ability of the membranes to hide the blood color of the observation side depends on their pore size. Large pores do not hide well the color of the blood, but membranes with pores of less than about 0.10 μL, have a tendency to crack after being coated with reagent. The pore size designated for an asymmetric membrane is the size of the smallest pores. Preferably, membranes with pore sizes of less than about 1.0 μm are used, most preferably around 0.15 to 0.35 and give adequate blood hiding on the hematocrit scale of 30-55%.

Said membranes include memtec BTS membranes 30, 45, 55 and 65. Preferably, both the membranes 14a and 14b are of the same material. Approximately 10 μL of blood are typically required to saturate both test pads, but excess blood is absorbed up to a volume determined by the porosity and dimensions of the dispersion layer material. The reagent composition incorporated in both membranes reacts with glucose in the blood sample to cause a detectable color change. Among suitable compositions are those described in patents 4, 935,346 and 5,563,031 described above, which are incorporated herein by reference. Generally, these compositions comprise a component for converting glucose to hydrogen peroxide, such as glucose oxidase, and one or more components for detecting hydrogen peroxide produced from the glucose present in the sample. The components for detecting hydrogen peroxide may be a peroxidase, preferably bitter horseradish peroxidase, together with an "indicator" that changes color during the course of the reaction. The indicator can be an oxidizable dye or a couple of dyes. Peroxidase catalyses the oxidation of the indicator in the presence of hydrogen peroxide. The indicator can be 3-methyl-2-benzothiazolinone hydrasone hydrochloride combined with 3,3-dimethylaminobenzoic acid (MBTH-DMAB) or 8-anilino-1-naphthalenesulfonic-ammonium acid (MBTH-ANS).

Alternatively, the indicator may be monosodium N-sulfonylbenzenesulfonate of meta [3-methyl-2-benzothiazolinone hydrasone] combined with 8-anyl-1-naphthalenesulfonic acid-ammonium (MBTHSB-ANS). This last combination is preferred. The membrane 14b also includes an inhibitor, to delay the color-forming reaction, and a dye. The membrane 14a, with the composition of the color-forming reagent alone, makes it possible to distinguish several levels in the lower glucose scale; e.g., 25, 50 and 80 mg / dL. For a higher glucose concentration, the color is too dark to allow reliable discrimination between glucose levels. The color development in membrane 14b is inhibited, so that, depending on the concentration of inhibitor used, color development can initiate at higher glucose levels. A number of different inhibitors are suitable, including 2,3,4-trihydroxybenzoic acid, propylgalate and ascorbic acid (see U.S. Patent No. 5, 306,623 mentioned above). Ascorbic acid is preferred, and preferably the concentration of ascorbic acid is selected to retard color development until the glucose levels reach approximately 120 mg / dL. The inclusion of an inert dye with the inhibitor produces a different color to provide even better glucose level discrimination. Suitable inert dyes include mordant yellow, direct yellow of primulin, primaquine diphosphate, thiazole yellow G, bright yellow and O-anhydrous auramine. By varying the type and concentration of the dye, several shades of green, yellow, purple or blue may occur.

O-anhydrous auramine is preferred at a concentration of 0.1%, because in combination with MBTHSB-ANS it gives a bright green color, maximum visual resolution between glucose levels and excellent coating uniformity. In combination, the reagent compositions that are preferred in membranes 14a and 14b allow a user to discriminate between 8 levels of glucose concentration in the range of 25 to 600 mg / dL. To further increase the blood-hiding effect and thereby allow measurements on a larger scale of sample hematocrit, several water-soluble, charged polymers, including polyvinylpyrrolidone (PVP), GAFQUAT, polyacrylic acid, and acid may be added to the reagent. polystyrene sulphonic. Empirically, PVP is preferred. The following examples demonstrate the present invention in its different modalities, but are not designed to be limiting in any way.

EXAMPLE 1 A test strip was prepared using an asymmetric polysulfone hydrophilic membrane having an adequate size of 0.2 μm (Memtec BTS-55). The membrane was coated allowing the opaque side (larger pore size) to make contact with solution 1 (tangential coating). The excess solution was removed by cleaning the membrane strip with a glass rod. The membrane was dried with air in a forced air oven at 56 ° C for 10 minutes. The composition of the coating was: SOLUTION 1 Water 25 mL Citric acid 0.282 g Trisodium citrate 0.348 g Mannitol 0.25 g EDTA 0.021 g Gantrez 0.1 125 g Crotein 0.36 g Glucose oxidase 0.36 g (125 Units / mg) Horseradish peroxide 0.071 g (501 Units / mg) Carbopol solution * 1.25 mL "(0.1375 g in 1.25 mL of MeCN) Citrate pH regulator * 3.75 mL * (0.13 g of disodium citrate in 5.0 mL of water) For the low scale membrane (14a), a membrane coated with solution 1 was then tangentially coated with dye solution 2 and dried in the same prior oven for 5 minutes. The composition of dye solution 2 was: SOLUTION 2 Solvent * 38.16 mL MBTHSB 0.1552 g ANS 0.2142 g Maphos 20% (solvent) 1.84 g ("Solvent: 50% water, 30% EtOH and 20% MeOH ) For the high-scale membrane (14b), the membrane coated with solution 1 was then tangentially coated with dye solution 3 and dried in the same previous oven for 5 minutes. The composition of dye solution 3 was: SOLUTION 3 Solvent * 38.16 mL MBTHSB 0.1552 g ANS 0.2142 g Maphos ai 20% (solvent) 1.84 g Auramine O 0.0316 g Ascorbic acid 0.13 g ("Solvent: 50% water, 30% of EtOH and 20% MeOH) The resulting membranes were cut into strips 0.6 cm wide and used to make the strips illustrated in Figures 1 and 2.

The strips were tested by visual comparison with a color chart (table 1) that was constructed with Munsell color fragments and calibrated at the glucose value scale of 0-600 mg / dL. The randomized test of 228 venous blood samples that reached their maximum level gave an excellent correlation (R2 = 0.965) to the determinations made in a normal blood glucose meter -Yellow Springs Instruments (YSI) Model 2300 STAT Glucose Analyzer- corrected for plasma glucose values (figure 5).

EXAMPLE 2 The procedure of Example 1 was followed, except that solutions 1 and 2 (or 3) were applied on the small pore side of the membrane and a coating of a blood masking polymer was added on the dye solution coating. A number of different polymers were tested. The aqueous solutions of each of the following were effective to hide the color of the blood: Polyvinylpyrrolidone - MW 160,000, 2%. GAFQUAT 734 (quaternized vinyl pyrrolidone copolymer and dimethylaminoethyl methacrylate) - MW 60,000-100,000, 7%. Sodium salt of polystyrene sulphonic acid-MW 70,000, 7%.

EXAMPLE 2 Munsell Annotation * for color chart Glucose levels (mg / dL) Pad # 1 Pad # 2 0 10 P 7/2 10 YR 7/6 25 5 PB 6/2 2.5 Y 6/8 50 10 B 6 / 4 2.5 Y 6/8 80 10 B 5/4 2.5 Y 6/8 120 2.5 PB 5/6 7.5 AND 6/6 180 2.5 PB 4/8 5 GY 5/6 240 5 PB 4/8 10 GY 5 / 6 400 7.5 PB 3/10 5 G 4/6 600 5 PB 3/8 10 G 3/4 * see The Munsell Book of Color, Macbeth div. of Kollmorgen Instruments Corp., New Windsor, NY.

Claims (15)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A strip for visual test of blood glucose, comprising: a) a dispersion layer to accept a blood sample on a main surface and pass the sample to a second opposite main surface; b) an intermediate layer comprising first and second membranes, substantially collateral, each having a major upper surface adjacent to the second major surface of the dispersion layer to receive a part of the blood sample, and each containing a reagent that it can react with the glucose in the sample, while passing through the membrane, to cause a color change in the reagent, the second membrane also comprises an inhibitor and a dye, and c) a support layer for supporting the other layers and to allow any color change in the membranes to be visible through it.
2. 2. The strip according to claim 1, wherein the dispersion layer comprises a hydrophilic polyester woven fabric.
3. 3. The strip according to claim 1, wherein the dispersion layer comprises a specific polyethylene.
4. 4. The strip according to claim 1, wherein each of the membranes of the intermediate layer is an asymmetric polysulfone membrane. 5. - The strip according to claim 1, wherein the reagent in each membrane is the same. 6. The strip according to claim 5, wherein the reagent comprises 3-methyl-2-benzothiazolinone hydrazone hydrochloride combined with 3,3-dimethylaminobenzoic acid (MBTH-DMAB) 7.- The strip in accordance with Claim 5, wherein the reagent comprises 3-methyl-2-benzothiazolinone hydrazone hydrochloride combined with 8-anilino-1-naphthalenesulfonic acid-ammonium (MBTH-ANS). 8. The strip according to claim 5, wherein the reagent comprises monosodium N-sulfonylbenzenesulfonate of meta [3-methyl-2-benzothiazolinone hydrazone] combined with 8-anilino-1-naphthalenesulfonic acid-ammonium (MBTHSB-ANS) ). 9. The strip according to claim 5, wherein the reagent further comprises a soluble polymer. 10. The strip according to claim 9, wherein the soluble polymer is polyvinylpyrrolidone. 11. The strip according to claim 1, wherein the inhibitor comprises ascorbic acid. 12. The strip according to claim 1, wherein the dye comprises O-anhydrous Auramine. 13. The strip according to claim 1, in which any change of color in the membrane is allowed to be visible through holes in the support layer. 14. The strip according to claim 1, further comprising a cover layer on the dispersion layer, with a through hole for applying a sample to the dispersion layer. 15. The strip according to claim 1, further comprising an absorbent layer adjacent to the dispersion layer to absorb excess sample.