MXPA96003192A - Reading reading reagent strips dire - Google Patents

Abstract

The present invention relates to a multi-layer test reagent strip measuring the concentration of an analyte in a liquid sample applied to it, the sample is guided to a number of test areas disposed along the strip, where the analyte can react with a reagent to cause a color change, each test area also includes an inhibitor for the color change reaction, the concentration of the inhibitor increases in successive test areas, thus, the number of areas that change color is a measure of the concentration of the analyte, the strip is particularly adapted to measure glucose in an intact blood sample, in a preferred embodiment, the sample is guided to the test areas along a route formed by selected areas of membrane shredding , and the non-shredded test areas of the membra

Description

DIRECT READING PROOF REAGENT STRIPS CROSS REFERENCE TO RELATED REQUEST This application is a continuation in part of the requests of E.U.A. copendientes Series No. 411,238, filed on March 27, 1995 and Series No. 442,035, filed on June 15, 1995.

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a dry test strip for measuring the concentration of an analyte in a biological fluid; very particularly, a test strip that identifies the concentration directly, without the need for a meter. 2. Description of the related art Many visual test devices have been developed to measure the concentration of certain analytes in biological fluids. These devices, for example, have measured glucose, cholesterol, proteins, ketones, phenylalanine or enzymes in blood, urine or saliva. Dry-phase reagent strips that incorporate enzyme-based compositions are used extensively in clinical laboratories, doctors' offices, hospitals and at home to test glucose concentrations with biological fluid samples. In fact, reagent strips have become a daily necessity for many of the nation's many millions of diabetics. Since diabetes can cause dangerous abnormalities in the chemistry of the blood, it can contribute to vision loss, kidney failure and other severe medical consequences. To minimize the risk of these consequences, most diabetics should periodically test themselves, then adjust their glucose concentration accordingly, for example, through diet control and / or insulin injections. . Some patients should test their blood glucose concentration as often as four or more times a day. It is especially important that diabetics who have to control their diet to regulate sugar intake and / or administer insulin injections, and that they should be guided to this screening by frequent tests of blood glucose concentration, have quick reagent strips , low cost and accurate for glucose determination. Reagent strips are known which contain an indicator that is transformed into a different shade of color, depending on the concentration of glucose in a biological fluid that has been applied to the strip. Although some of these strips use reduction chemistry, they most commonly involve a dye or pair of oxidizable dyes. 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 having peroxidative activity, which is capable of selectively catalyzing the oxidation of the oxidizable dye in the presence of hydrogen peroxide (see, for example, U.S. Patent No. 5,306,623, issued April 26, 1994). , from Kiser and others). The patent of E.U.A. No. 3,964,871, issued June 22, 1976, to Hochstrasser, discloses a disposable indicator strip for directly measuring substances, such as glucose, and biological fluids. The indicator records the concentration of the substance including both an indicator reagent, which oxidizes and changes color when it reacts with the substance, and an "antagonist" that in some way prevents the accumulation of oxidized indicator until it has been completely constructed. Palmer et al. Describe a "digital" quantitative test system for glucose and other analytes in the publication of European patent application No. 0 317 070, published on May 24, 1989 (see also US Patent No. 5,036,000, issued on July 30, 1991). That system measures the concentration of an organic compound in a biological fluid by first oxidizing the compound with an enzyme oxidase specific to the substrate to produce hydrogen peroxide. The system includes a chromogen that is a hydrogen peroxide reducer and an air-stable hydrogen peroxide reducer that has a greater reduction potential. The major reduction potential retards any color change detectable by the chromogen until the first air-stable hydrogen peroxide reductant has been consumed. In this way, no color change occurs if the hydrogen peroxide to be measured is less than a predetermined level corresponding to the concentration of the stable peroxide redoubt. As a result, the system measures the concentration quantitatively, regardless of the intensity of color change. Engle ann, patent of E.U.A. No. 4,738,823, issued April 19, 1988, discloses a test strip for analyte determination having a support member, which has an absorbent material located to remove excess sample applied to the strip. The strip may also include a cover, which includes an opening through which sample may be introduced. Burkhardt et al., Patent of E.U.A. No. 4,810,470, issued March 7, 1989, describes a device for measuring analyte concentrations in liquid samples. The device includes one or more absorbent matrices covered by a coating or film impervious to 1 liquid. The sample is deposited on a portion of an absorbent matrix and dosed in the matrix chromatographically. By wicking action, the sample travels to the test region containing a test reagent for the analyte. Daffern et al., Patent of E.U.A. No. 4,994,238, issued February 19, 1991, discloses a chemical analysis test device comprising an absorbent layer, a waterproof barrier layer and a reagent layer having a certain volume. The sample is applied to the reagent layer through aligned holes in the overlapping absorbent and barrier layers. Whether the test is conducted in the home, in the doctor's office, in the clinic or in the hospital, the accuracy and reproducibility of a glucose determination is extremely important. In the case of a color indicator reagent strip, it is desirable that the color change be pronounced and intense to variations in components of the biological fluid other than glucose. In the case of a visually readable strip of reagents, it is especially important that diabetics, who may have impaired vision, have a strip that shows a significant color change dependent on the glucose concentration, although the change in cslor, as shown A change in absorbance at a given wavelength is also important for the accuracy of meter-readable strips. Since the color change involves a series of chemical reactions, it does not happen instantaneously. In this way, the user must wait a while - typically a minute or less - for the reactions to take place. When a meter reads the strip, the time controller circuit can give a signal that the reactions have been completed. However, when a strip is read visually, without a meter, the user can underestimate the time needed, read the strip prematurely and obtain an incorrect result. Alternatively, the user may feel the need to wait an excessive amount of time before reading the strip, to ensure that the reaction is completed, which causes unnecessary delay and user dissatisfaction. Therefore, there is a need for a "chemical" time controller, that is, an element on the strip that changes the color regardless of the concentration of glucose (or other analyte of interest) in the sample, but will do so only after that sufficient time has elapsed to complete the color reactions with the sample.

BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, an elongamulti-layer reagent test strip for measuring analyte concentration in a biological fluid sample that is applied to the strip comprises a) a bottom layer with a hole passed to accept the sample; b) a membrane layer having a sample side facing the bottom layer and a test side opposite thereto, and containing a reagent that can react with the analyte to produce a color change, the reagent comprising )? n first component that interacts with the analyte to form hydrogen peroxide, ii) a second component that interacts with hydrogen peroxide to undergo a color change; and iii) a third component that inhibits the color change of the second component; c) an intermediate layer between the lower and membrane layers; and d) dosing means for distributing sample along the strip, the dosing means comprising i) a non-absorbent region in the membrane layer; and ii) a fluid transport channel formed in the intermediate layer for guiding the sample over the surface of the non-absorbent region to a plurality of discrete absorbent test areas diepueetae along the length of the membrane layer; the inhibitor concentration increased in a predetermined manner with the distance from the first end of the strip, so that a correspondingly increasing analyte concentration must be contained in a sample if a color change is to be made, so that one or more test areas may change color when a sample is applied to the strip, and the area of color change furthest from the first end indicates the concentration of analyte in the sample. During operation, a method for measuring the concentration of analyte in the biological fluid sample comprises the steps of: (a) applying the sample to a test reagent strip comprising: (i) a plurality of absorbent test areas which change each color when contacwith a fluid that contains at least a predetermined amount of analyte, greater than the amount of analyte that produces a color change of the test areas that are closer to a first end of the strip and (ii) dosing means for distributing the sample along a predetermined non-absorbent path to each one of the test areas and (b) observing a test area that changes color and is more distant from the first end of the strip. The strip is of the type that provides a visible indication of the concentration of an analyte that is contained in a biological fluid applied to the "side of the sample" of the strip. The visible indication appears on the opposite (or "test") side of the strip. The chemical chemistry of the test strip depends, of course, on the analyte / biological fluid to be measured.

The test strips can be designed to detect analytes such as glucose? Other sugars, alcohol, cholesterol, proteins, ketone, uric acid, phenylalanine or enzymes in biological fluids such as blood, urine and saliva, as well as water. For purposes of convenience and brevity, the test reagent strips described in greater detail in this specification detect blood glucose. One skilled in the art could easily adapt the information in this description to detect other analyte / biological fluid combinations. A test strip of the present invention provides a simple and rapid determination of glucose concentration in an unmeasured blood sample. The strip comprising a bottom layer with a hole through which a sample can be introduced to the sample side of a porous matrix, the opposite side of which is the test side. The matrix is generally a membrane and the terms are used interchangeably in the present specification and the appended claims. A test reagent is applied to the matrix and to a greater or lesser degree impregnawithin the pores of the matrix. For simplicity, sometimes the reagent on the matrix is called "coating", in this specification and in the appended claims, recognizing that the reagent coating penetrates the matrix. An intermediate layer is laid between the lower layer and the matrix. Cuts in the middle layer are aligned with non-absorbent areas of the membrane to guide the sa to a series of absorbent test areas that are disposed along the strip (as noted in this specification and the claim is made to annexes). , "absorbent" means precisely that it is capable of absorbing). A series of grooves in the middle layer surround the space around and above the test area to restrict the flow of sa to those areas. A fixed volume of sa - typically whole blood containing red blood cells and glucose - is therefore directed to the sa side of the membrane in each one of the series of test areas. Preferably, the lower layer has ventilation holes in alignment with the areas to facilitate the filling of the volumes uniformly. The porosity of the matrix allows the fluid to pass from the sa side to the test side, for exa, by capillary action. In this way, the test reagent can react with the blood glucose to produce a color change on or near the test side. Since strongly colored red blood cells can make it difficult to detect color change, the matrix is preferably anisotropic, with pore sizes graduated from large pores on the sa side to small pores on test sides, in order to trap red blood cells. they move away from the test side. A variety of materials can be used for the various components of the test strip and time controller of this invention. Some of these matters are described in the U.S. Patents. 5,306,623 and 5,418,142, issued April 26, 1994 and May 23, 1995, respectively, by Kiser et al., Incorporated herein by reference. The test reagent comprises a component for converting glucose to hydrogen peroxide, such as glucose oxidase; one or more component to detect the hydrogen peroxide produced from the glucose present in the sample; and an inhibitor. The components for detecting hydrogen peroxide may be a peroxidase, preferably horseradish peroxidase, together with an "indicator" which changes color in the course of the reaction. The indicator may be a dye or pair of oxidizable dyes. Peroxidase catalyses the oxidation of the indicator in the presence of hydrogen peroxide. The final element of the reagent is an inhibitor that retards the oxidation of color change of the indicator. The strip is segmented along its length in such a way that adjacent membrane segments have different concentrations of inhibitors. Each segment has an absorbent test area that only changes color if and when sufficient glucose is present to first make all the inhibitor consumed and after the indicator is oxidized and thus produces the characteristic color change. Therefore, a change of color in a particular area is an indication of a minimum glucose concentration in the original blood sample. Along the strip, in a particular direction, each successive segment has a higher inhibitor concentration per paeoe, which corresponds to a step increase in the minimum glucose concentration. The indicator concentration is the same for all segments. In principle, variable inhibitor / indicator balances are also possible. If the segments have inhibitor concentrations at the appropriate scale for a particular test sample, the adjacent test areas react with the analyte in such a way that one area is colored and one adjacent is not. That result indicates that the glucose concentration in the tooth is at least equal to the minimum concentration required to change the color of an area, but not as large as that required to change the color of the adjacent area. For blood glucose monitoring, an optional regular segment of time coating comprises the elements of the indicator strip - a porous matrix having a test reagent coated thereon - and also glucose. In the case of eeco, the chemistry of the reagent is not activated by glucose, but when the sample is applied to the strip, the time-controlling coating is hydrated and the glucose in the coating after a predetermined time causes the indicator to change. color. Preferably the glucose is present in the time controller in an amount that far exceeds that required to overcome the inhibitor. In that case, the time required is longer or shorter depending on whether this is more or less inhibitory. The color changes in the strip and in the time controller can be observed either directly with the naked eye or with an optical instrument that detects changes in reflection.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of the matrix of a direct reading test reagent strip of the present invention. Figure 2 shows a sectional plan view of the sample side of a strip of direct reading test reagents of the present invention. Figure 3 is an enlarged fragmentary perspective view of the internal part of the test strip of Figure 2, partially sectioned. Figure 4 is a cross section of the strip of Figure 2 taken along line 4-4. Figure 5 is a bottom plan view of a test strip of this invention. Figure 6 is a top plan view showing the test side of the test strip of Figure 5. Figure 7 is the strip of Figure 6 after a sample has been applied thereto.

DETAILED DESCRIPTION OF THE INVENTION The present invention is a strip of direct reading test reagents for measuring the concentration of an analyte in biological fluid. The key element of said test strip is a porous matrix that incorporates a test reagent that undergoes a change in color in response to the analyte in a sample of biological fluid that is applied to the strip. The matrix can be a uniform composition or it can be a coated substrate and can be either isotropic or anisotropic. It has a sample side to which the sample is applied and a test side where the color change is observed. Preferably, the matrix is an anisotropic membrane; most preferably, an anisotropic membrane having a wide range of pore sizes. For example, a gradient of pore sizes from about 0.1 microns to about 150 microns can be spread across the membrane. On the large pore side, the pore size is preferably in the range of about 30 microns to about 40 microns. On the side of the membrane where the pores are smaller, the hollow volume is relatively small, and the membrane material is usually very dense, within a layer that typically can constitute up to 20% of the thickness of the membrane . Within this layer, the pore size is preferably in the range of about 0.1 to about 0.8 microns, with a nominal pore size preferably of about 0.3 microns. When the biological fluid is applied to the sample side, the sample finds pores of smaller and smaller size as it penetrates the membrane. Generally, the eolids talee as red blood cells reach a position in the membrane where they can no longer penetrate. The rest of the sample, which still contains the dissolved glucose, penetrates through the side of the test. The anieotropic nature of the membrane and / or the passage of a separate component (which is described below) allows relatively rapid flow rates through the membrane, even while filtering the solids. As the mueetra passes through the matrix, the reaction with the reagent causes the light absorbing dye to form cr to decompose in the hollow volume near the test side, thereby substantially affecting the reflectance of the matrix. Polysulfones and polyamides (nylon) are examples of suitable matrix materials. Other polymers having comparable properties can also be used. The polymers can be modified to introduce other functional groups that provide charged structures, so the surfaces of the matrix can be neutral, positive or negative. A preferred method for preparing the porous material that forms the matrix and molding the polymer without a support core. Said matrix is, for example, the dielectric anisotropic polysulfone membrane of Memtec, In., Ti oniurn, MD. A matrix of less than about 200 microns in thickness is generally used, with one of about 115 to 155 microns being preferred, and a thickness of about 130 to 140 microns being most preferred., particularly when the matrix is nylon or anisotropic polysulfone. The membrane can be treated with test reactive agent by immersing it in a mixture of the components, thereby saturating the membrane. Preferably at least some of the components are applied to the membrane sequentially. The excess reactive agent can be removed by mechanical means such as, for example, an air knife, a tongue swath, or a glass rod. Deep? The membrane dries. The reactive agent tends to concentrate near the small pore (test) side of the membrane. The test reactive agent comprises (i) a component for converting glucose to hydrogen peroxide, (ii) a component for detecting hydrogen peroxide, and (iii) a component for inhibiting the component that detects hydrogen peroxide. The reactive agent can optionally also comprise a separate component that causes the solids, such as red blood cells, to be trapped in the matrix, effectively removing the solids from the biological fluid. Additional components may also be included as described hereunder and in the teachings. Preferred components for converting glucose to hydrogen peroxide include glucose oxidase, an enzyme that is usually obtained from Aspergillue niger or Penicillium. Glucose oxidase reacts with glucose and oxygen to produce gluconolactone and hydrogen peroxide. The optimal concentration of glucose oxidase depends on the composition of the indicator system. For example, if the indicator system is MBTHSB-ANS (which is described below), then the glidase oxidase in the range of about 500-10000 U./mL, is appropriate, most preferably from about 700-2- 000 U. / L, and mu preferably even of 1000 U./mL. Generally, higher concentrations of glucose oxidase cause the reaction to proceed more quickly and lower concentrations make it slower. The hydrogen peroxide thus produced reacts with the component to detect hydrogen peroxide, which comprises a peroxidase which selectively catalyzes a reaction between the hydrogen peroxide and an indicator. Peroxidase fluidizes hydrogen peroxide as an oxidant that is capable of removing hydrogen atoms from several strains. An appropriate peroxidase may contain ferriprotoporphyrin, a red hemin obtained from plantae. The peroxidases obtained from animalee, for example from the animal gland and thyroid gland, are also appropriate. Horseradish peroxidase (HRPO) is especially preferred as a constituent of the component for detecting hydrogen peroxide. The hydrogen peroxide, preferably catalyzed by peroxidase, reacts either directly or indirectly to form or decompose an indicator dye that absorbs light on a wavelength scale of predetermined wavelength. Preferably, the indicator dye strongly absorbs at a wavelength different from that to which the test reactive agent strongly absorbs. The oxidized form of the indicator can be the final product colored, dimly colored, or without color, which shows a change in the color of the test side of the matrix. That is, the test reagent can indicate the presence of analyte in a sample by a colored area that is bleached or, alternatively, by a colorless area that de-coils color. The indicators that are useful in the present invention include 3-methyl-2-benzothiazolinonhydrazone hydrochloride (MBTH) combined with 3-dimethylaminobenzoic acid (DMAB); (b) MBTH combined with 3,5-dichloro-2-hydroxybenzene sulfonic acid (CDHBS); (c) 4-aminoantipyrene (4-AAP) and 5-N- (m-tolyl) -diethanolamine (DNA); (e) 2,2'-azino-di (3-ethylbenciazolin) sulphonic acid (ABTS) (f) 4AAP and 4-methoxine tol, - (g) red of pturogalol; (h) Bro opirogalol red (BPR); (i) Acid Green 25 (AG); (j) onosodium salt of 2-benzothiazolinonhydrazone N-sulfonylbenzenesulfonate (MBTHSB), combined with the ammonium salt of 8-anilino-l-naphthalenesulfonic acid (ANS) MBTHSB-ANS is preferred. Additional information regarding MBTHSB-ANS appears in the copending US patent application, series No. 302,575, filed September 8, 1994 and incorporated herein by reference. The inhibitor component retards the reaction between the hydrogen peroxide and the indicator, for example, reduction of hydrogen peroxide or reduction of the oxidized indicator. In principle, there are several different modes of operation for an inhibitor. First, the inhibitor can compete with the indicator and therefore reduce the rate at which the color change takes place in the indicator. Second, the inhibitor may not be competitive, so that substantially all of the inhibitor is consumed before any substantial color change in the indicator is caused to occur. Other modes of operation of the indicator are also known. Preferably, the inhibitors of the present invention are non-competitive. Among the scale of appropriate inhibitors are acid 2,3,4-trihydroxybenzoic acid; propylgalate; 3,4-dihydroxy-benzaldehyde acid; Gallic acid; 5, 6-diaminofurasyl; aecorbic acid; and isoaecorbic acid; the azobic acid is the preferred one; However, the azobic acid is oxidized in solution and must be stabilized in order to allow the reactive agent to be coated. Preferred stabilizers are primary alcohols such as ethyl alcohol, methyl alcohol or propyl alcohol. Methyl alcohol is the preferred, particularly, concentrate solution; ie, solutions of 50% or more of ethanol. Although the anizotropic membrane which is the preferred matrix filters out the red blood cells and keeps them away from the test side, optionally the test reagent may also contain a separation component. The separation component must be capable of producing a relatively clear colorless fluid from the fluid containing red blood cells, for example, whole blood, sequestering red blood cells in the matrix. Separation components for use in the present invention include but are not limited to polyethylene glycol, polymethyl vinyl ether / rnaleic anhydride, polypropylene glycol, polystyrene ionic acid, polyacrylic acid, polyvinyl alcohol, and polyvinyl sulphonic acid at a pH between about 4.0- 8.0; such separation components are present in the matrix in amounts that will vary depending on their charge and molecular weight, the other components embedded in the matrix, the pH of the matrix and the pore size, and the residual unit of the matrix after of drying. Such parameters can easily be determined by one skilled in the art, for example, when propylene glycol is used as the separation component, for example, BASF's PPG-410, lyandotte, MI) is preferably present at about 2-30% by weight with respect to volume (p / v), and rnuy preferably 8-10% p / v. Other separation components can also be used in a concentration of approximately 2-30% w / v. The polymeric separation components can be impregnated or embedded in the matrix or molded into the membrane during manufacture. Some water-soluble salts can also effect blood separation. Among the salts suitable for the separation of the component from the salts are citrates, formates and sulfates as well as certain acids, such as amino acids, citric acid, phytic acid and malic acid. (see, for example, U.S. Patent 3,552,928, issued January 5, 1971 to M.C. Fetter). An advantage of including the separation component is that with solids such as red blood cells being ubiquitously recovered from the biological fluid, there is less background color at the test site to obscure a change in the coloration produced by the test reagent. Other components may be embedded within the matrix to improve the coloration and readability of reactive agents and to preserve the uniformity and integrity of the matrix. For example, the test reagent may include eals and / or pH regulators to assist in the separation of the dye in the matrix. Such pH regulators may contain, for example, citrate, present in the solution at about 0.1M to about 0.01M and preferably 0.1M. You can also use others ") 0 pH regulators. Compounds that make the matrix hydrophilic or compounds that can act as stabilizers, such as hydrolysed proteins, can also be employed. Such compounds include, but are not limited to, for example, bovine serum albumin, polypeptides, and the low molecular weight protein available as Crotein SPA (CRODA, Inc. New York, N.Y.). Such compounds are used, for example, at concentrations of about 1 mg / mL to about 100 mg / mL. In the case of Crotein, about 30 rnG / L is preferred. Other preservative stabilizers may also be included in the coating for the matrix. For example, ethylenediaminetetraacetic acid (EDTA) diethylenetriatapentaacetic acid (DTPA) and related compounds can be used, for example, at concentrations of about 0.01 mg / mL approximately 10 mg / mL. Among the purposes of the conservatives is to help stabilize the inhibitor. Some of the indicators (for example BPR) have an undesirable tendency to igrar in the matrix. When such an indicator is used, an ion-pairing agent is included to prevent such migration, for example, commercially available polyethylene glycol derivatives such as Polyquart (H) (Henkel, Inc., Ambler, PA) are particularly useful for their ability to facilitate the ionic pairing between the indicator and other matrix substituents. When the presence of an analyte is indicated by the formation of color, for example, MBTHSB-ANS) surfactants can be added to brighten the color and improve the contraete with the uncoloured contour. The organic solvents can also be used in the practice of this invention and can be included in the formulation of the test reagent for the matrix, provided, of course, that they are compatible with the matrix and the test reactive agent compositions. Suitable potential organic solvents include chloroform, acetone, alcohols, methylene chloride, diethyl and petroleum ethers, acetonitrile, and mixtures thereof. In the practice of the present invention, 70% ethanol in water is particularly preferred. The test reagent that is coated on or impregnated in the matrix is not uniform on the surface of the test strip. Instead, the reactive agent is preferably applied to the matrix in a series of parallel strips, or "segments", which extend through the narrow dimension of the strip. The composition in adjacent segments increases, in a series of steps, the inhibitor concentration. Each segment has a porous test area. It is in the test areas that the test reactive agent reacts with any blood glucose to cause a color change, provided that the glucose concentration is sufficiently large to exceed the level of inhibitor in that test area. Therefore, each subsequent test area requires, in a series of steps, a higher glucose concentration in the wheel to cause the area to change color. Optionally, one of the test areas is adapted to serve as a time controller, to indicate that sufficient time has elapsed for the reactive agent to react with glucose on each of the test areas. The time-controlling segment of the matrix is coated or impregnated with a composition consisting of the test reactive agent as well as glucose. In addition, the purpose of the test reactive agent is to change the color in response to glucose, combining the two without causing the color change to require care. An amount of inhibitor beyond that required for the time control function must be present to compensate for this effect. The speed at which the time-controlling segment dries, after the solution containing glucose is applied, is controlled. In practice, the membrane is first coated with a solution containing pH regulators, stabilizers and enzymes and the coating dried to form a first layer. Then, a second coating step applies a solution containing indicator, inhibitor and glucose. Parameters such as tape speed, furnace temperature and air flow, and the amount of deposited coating solutions will have been fixed in advance and appropriate adjustments to the inhibitor and / or glucose concentrations will be made. Instead of applying the second coating directly, an alternative, less preferred, involves making the second coating on a separate tape and then placing it on the first layer. When a sample is applied to the strip, the hydration of the composition of the time-controlling segment allows the color formation reaction to proceed. The time it takes for the time-controlling element to change the color is determined by the temperature and by the characteristics of the test reactive agent, particularly the inhibitor concentration, the amount of glucose, and the hydration and oxygen fusion rates. It can be done that e-1 time of color change of the time controller depends on the concentration of glycoea in the sample or, alternatively, that it is independent of that concentration. By incorporating a large excess of glucose in the time controller, the time is substantially independent of the glucose concentration of the sample. By incorporating less glucose in the time controller, the time can be made dependent on the glucose in the sample; that is to say; The time controller will change color faster if the glucose concentration in the sample is higher. Preferably, the glucose concentration in the time controller is about 1500 mg / dL, which makes the time controller substantially independent of the glucose concentration of the parent on the scale of about 40-400 mg / dL. The composition of the time-controlling segment includes amounts in excess of the component (such as glucose oxidase) that converts the glycemia to hydrogen peroxide, as well as glucose. The continuous composition of time should then include at least as much, or more, inhibitor that causes the resulting segment to have a higher concentration of inhibitor (which corresponds to the higher glucose reading). The time controller also serves as an important quality control function, making it evident when a test strip has been compromised by exposure to moisture. The test strip should remain dry until the moment it is to be used, since the components that convert glucose into hydrogen peroxide (usually enzyme) tend to degrade on exposure to moisture. Therefore, if the strip is exposed to moisture prematurely, it will be compromised. However, the deterioration of the test strip is not obvious to the user, who can therefore use said strip and obtain an erroneous result. However, if the strip includes a time-controlled segment, exposure to moisture causes the time controller to change color, which alerts the user to the fact that the strip has been compromised and should not be used. In addition to the matrix containing the reactive agent, the strip of the present invention includes a lower layer that is supported by the matrix. The cap > The lower is preferably a thermoplastic sheet, more preferably a polyester, and has a hole through which the metal can be applied to the side of the matrix sample. From the sample orifice, the blood sample is distributed along the length of the matrix. If the lower layer is generally opaque, then one or more transparent window sections can be placed at an appropriate distance from the sample orifice, the appearance of the sample in the window confirming that the proper width has been applied to the strip. . The distribution of the blood from the orifice of the patient to the test areas implies the intervention of an intermediate layer that is located between the lower layer and the membrane and, apcionalmente, is adhered to both. The intermediate layer is preferably a thermoplastic sheet; very preferably? n polyether. It has cuts that guide the wheelchair down the entire length of the strip through nonporous paths on the membrane and direct the sample to one of the test areas. The notches in the intermediate layer align with the test areas, so that each test area is sub-substantially enclosed by the walls of the intermediate layer. A preferred structure of the non-porous paths on the membrane is formed by crushing the pore structure of the membrane. This can be achieved by heating, either directly or using a laser beam or ultrasound, and preferably including pressure. However, the preferred method is crushing. Therefore, the membrane is crushed to make it non-porous (although still hydrophilic) anywhere, except for the test areas. For the preferred membranes of this invention, trituration is preferably achieved by applying high pressure, at least 80,000 kPa and, optionally, heat (at least 110 ° C) to the membrane areas that are crushed. The preferred pressures and temperatures depend, of course, on the crushing mechanism and the dwell time, as well as the membrane parameters. The optimal values can be determined by routine experimentation. For precise measurements, it is important that the volume of blood provided to each test area is reproducible. If the notches completely surround the test areas, then, by adopting a liquid-tight seal between the intermediate layer and the lower layer and the crushed membrane, each test area is associated with a closed volume (cylindrical) whose walls are formed by the intermediate layer and whose ends are formed by the membrane and the lower layers. However, a distribution channel runs along the strip and feeds the sample to each of the test areas. The high precision requires that the distribution channel provide a fixed volume of filter to each test area, but afterwards it does not provide more, at least not in the time structure of the measurement, of approximately 1 or 2 minutes. Since the sample volume is variable, an absorbent layer at each end of the membrane is preferable to dislodge the excess sample from the ends of the distribution channel. Adsorbent layers at the end of the channel also improve the packing of the sample along the length of the strip. Non-woven fabrics, well known in the art, form the preferred absorbent layers. The color change caused by the glucose in the test sample appears on the test side of the membrane. It is convenient to cover that side of the membrane with an upper layer that has holes that align with the test areas. The holes make color changes visible and also allow oxygen to reach the colored sites. The upper layer can be attached to the membrane, for example, with adhesive. Any adhesive is preferably limited to the non-porous areas of the membrane, if it interferes with the glucose measurement reactions. However, if the adhesive does not interfere with glucose, its placement is less critical. Since the test areas, when they contain the preferred reactive agent, slowly support a color change when exposed to light or oxygen and since the optional time controller is sesable to moisture, the packaged eethan strips preferably in a package impervious to moisture and opaque oxygen, such as a sealed sheet metal foil. If the strips are individually packed, the strip can remain in the open wrapper during the year. The invention will now be described with reference to the figures. Figure 1 shows a matrix 10 of the present invention for measuring the amount of analytes in a biological fluid. Although shown in an arched position, the matrix 10 is flexible and is generally in a plane when used. The matrix includes a sample side 12, to which the sample of biological fluid is applied, and a test side 14 on or near which a change in color indicates the presence of the analyte. The color change results from the interaction of the analyte with the reactive agent impregnated in the pores 16. Preferably, to measure the concentration of glucose in the blood, the pore sizes are relatively large near the sample side 12 and decrease the size as it approaches the test side 14. The pore size gradient serves to trap the red blood cells near the sample side 12, so that its color does not interfere with the ability to observe the color change that indicates the presence of the analyte. Tree segments parallel to, b and c are shown schematically. Each segment has gradually more inhibitor than the previous segment. In a preferred embodiment, after the membrane reactive agent has been applied in parallel segments, as shown, the membrane is ground completely less in the test areas, where the analyte-reactive agent reactions take place. This pattern of test areas - a single area indicated in each of the parallel items - and nonabsorbent shredded areas is described in the plan view of FIG. 2 and the enlarged fragmentary perspective view of FIG. 3. FIG. 2 is a vieta on the lower floor, in partial section, on the side of the wall 12 of the membrane 10 and the absorbent layers 20 and 22, superposed with the intermediate layer 24 the bottom layer 26. The membrane 10 and the absorbent layers 20 and 22 are preferably supported by a layer top, not shown. The layer of builders 20 and 22 are preferably located at the ends of the membrane (over the dashed lines A and B) to absorb the blood sample that is in excess of the volume necessary for the measurement. That volume should be sufficient to provide a dummy for each of the test areas and, if present, also for the time controlling area. In general, a strip that has less test area does not require as much sample, but provides a smaller scale of glucoea value, less accuracy, or more things. Figure 2 shows nine absorbent areas, representing eight test areas (numbered from 1 to 8) and a time controller (T), which provides adequate scale and precision in that they do not require an unacceptably large sample volume. The intermediate layer 24 has a notch 28, which is aligned with the hole of the sample 30 in the lower layer 26. The sample is introduced through the hole of the sample 30 and is directed by capillary action along the central channel 32 of the intermediate layer 24 to each of the test areas and to the time controlling area, any excess sample being absorbed in absorbent layers 20 and 22. The appearance of the flasher through the optional deepening windows 34 and 35 confirms that? A sufficient sample has been provided for the measurement. Preferably, the intermediate layer 24 forms a seal with the side of the sample 12 of the membrane, so that the sample can not, for example, flow directly between the adjacent test areas. Figure 3 is a vieta in fragmentary elongated perspective, representing the parts of 3 test areas, 6, 7, and 8, viewed through the lower layer 26, and separated by extensions of the intermediate layer 24. Optional adhesive layers 24A the intermediate layer 24 is joined to the lower layer 26 and the membrane 10. Ventilation holes 40 in the layer 26 facilitate the flow of the sample within the strip. The pits, such as 38, in the upper layer 36 are aligned with the absorbent areas, making any color change visible in the absorption area and also admitting the oxygen necessary for the color change reaction. The optional adhesive layer 36A joins the top layer 36 to the test side of the membrane 10. Figure 4 is a transverse section taken along the line 4-4 of Figure 2, which shows the top layer 36, in FIG. addition to the layers shown in Figure 2. The ventilation holes in the lower layer 26, such as 40, are aligned with the test and controller areas of time and facilitate the filling of the sample volume surrounding each of those areas . The volumes to be filled are fastened with the membrane 10, the intermediate layer 24 and the lower layer 26. Note that the separation between the upper part of the test area 3 and the lower layer 26 is only 12 microns, but the larger one is that the scale for greater clarity. The figure & ee a vieta on the lower floor of a strip of the present invention, showing the sample hole 30 and the graphs that guide the user to introduce the sample through that hole. When the sample is seen through the clear windows 34 and 35, it confirms that adequate wall has been applied to the strip. Figure 6 is a top plan view of the upper layer 36 of a strip that has been calibrated to associate test areas with glucose concentration. Fig. 7 shows the strip of Fig. 6 after a blood sample has been applied to the opening 30 (Fig. 2), the sample has been extended along the central channel 32, and the glucose in the mueetra has reacted with the reagent in the test area. Since the lower test area has less inhibitor, that area will have changed color first. After that, the second and then the third area will change color. The upper circles do not change color because there is very little glucose in the sample. The strip can be read since sufficient time has elapsed for the time controlling area 42 to change color. In this way, the result shown in Figure 7 indicates that the concentration of glucose in the sample is at least 120 mg / dL, but less than 150 mg / dL. The reading may be taken at any time after the time controlling area 42 changes color. Note that in figure 7 the color change caused by the reaction with glycoea ee from white to colored. However, the screen may alternatively operate with an indicator dye that is destroyed by glucose-induced oxidation, with a corresponding color change from colored to white. For a better understanding of the present invention, the following examples will further illustrate various embodiments of the invention. The examples are not intended to be in any way limiting.

EXAMPLE 1-BPR INDICATOR The following solution was prepared: Enzyme solution Distilled water 83.5 9 Aconite acid 0.2M 27.0 g EDTA Na2 1% (p /?) 23.8 9 Glucose oxidase 165,000 U Aconitic acid 6.0 9 HRPO 340,000 U NaOH (solid) 2.2 9 Crotein SPA 4.2 g Imidazole 0.6 9 Mannitol 3.0 9 Surfactol 01 at 5% (w / w) 3.0 9 Adjust pH to 4.80 Ethyl alcohol 40.0 9 PPG-410 5.6 9 Enzyme solution 28.0 9 Metec membrane BTSH 55 was coated by immersion in this solution and the excess was removed with glass bars. The coated membrane was dried in a flotation dryer at 82 ° C under moderate airflow so that the belt dried substantially within 20 seconds. The tape was wound in preparation for the second revelation, described below. The following sol? Cionee were prepared: Ascorbate supply solution Diluent (Inhibitor) Distilled water 190 g 370 g EDTA Na2 1% 55 g 107 g BPR 0.36 g 0.71 g PoliOuartR H 6 g 11.8 g PPG-410 14.2 g 27.8 g Azobic acid 1.37 g Ethyl alcohol 243 g 477 g Time controlling solution Diluent (above formula) 120 g Azobic acid 0.885 g Glucose solution * 17.25 g * The glucose solution is a solution of 16.0 g / dL of glucose in water, left in rnutarrotation for 24 hours, stored in refrigeration. The following dilutions of the supply solution were made: 0.0405: 1, 0.108: 1, 0.236: 1, 0.369: 1, 0.569: 1, 1.260: 1. This stepwise increase in inhibitor concentration corresponds to the glucose concentration stepwise larger than the report of the test area. These solutions, together with the time controlling solution, were coated side by side on the large pore side of the membrane loaded with the enzyme so as to deposit approximately 1.2 x 10-μm per square millimeter of membrane. The membrane was wetted approximately 15 seconds before experiencing the same drying conditions as those described above for the passage of the enzymatic coating. The results showed that the time controller reacted in approximately 70 seconds with approximately 95% of the results falling between 64 and 79 seconds.

EXAMPLE 2-INDICATOR OF MBTHSB-ANS The following solution was prepared: Water HPLC 1500 mL Citrus acid 16.92 g Sodium citrate 20.88 g Mannitol 15 g Disodium EDTA 1.26 g Gantrez S95 6.75 g Crotein SPA 36 g Glucoea oxidaea 1.69 MU HRPO 1.5 MU Carbopol 910 * 75 rnL Citrate disodium * 225 mL * Acetone solution 11% ** 0.1M, pH 5.0 Memtec BTS 35 membrane was coated in a cuvette so that the large pore surface made contact with the coating solution; the excess of eolución was eliminated with glass bars as antee. The membrane was dried and wound as in Example 1. The following solutions were made: Solution A (indicator) Solution B (Wetting agent) Ethanol 70% (v / v) 2819 mi Maphos "60A 41 g MBTHSB 2.98 g Ethanol 70% (v / v) 205 rnL (NHA) ANS 25.83 g Solution B 205 rnl DTPA 2% 51.25 rnl Solution C Solution D (supply of ascorbate) (time controller) Water 115 mi Water 53 mi Ascorbic acid 4.58 g Ascorbic acid 8.75 g Ethanol 267 ml Ethanol 123 ml Volume is brought to 175 rnL with 70% EtOH. Glucose solution 40.5 ml For each inhibitor solution, the volume of solution A ee fixed at 263 ml. For the different test areas, the proportion of EtOH 70%: Solution C was varied from 58.9 to 0.200 for the volume of EtOH 70% + Sol. C added to solution A to be 87.5 ml for all the inhibitor solutions. This effectively altered only the inhibitor concentration in each solution. The solutions containing the increasingly increased concentration of the inhibitor and the time controlling solution (solution D) were coated side by side on the large pore side of the membrane. The rate of accumulation was adjusted to reach approximately 8 X 10-5 mL of inhibitor per square millimeter of membrane. The membrane was dried as before, except that the delay between coating and drying was approximately 1.6 minutes. The results showed that the time controller reacts in approximately 60 seconds with little effect because of hematocrit of the blood, from 30 to 55%, or glucose from 78 to 420 mg / dL. Those skilled in the art will understand that the foregoing description and the examples are illustrative of the practice of the present invention, but are in no way limiting. Variations can be made to the details that are presented herein without departing from the scope and spirit of the present invention.

Claims (25)

NOVELTY OF THE INVENTION CLAIMS

1. - An elongated multi-layer test reagent strip for measuring the concentration of analyte in a biological fluid sample that is applied to the strip, comprising a) a lower layer with a continuous hole to accept the sample; b)? a membrane layer, having a rrrrrrrr side facing the bottom layer and a test side facing it, and containing a reagent that can react with the analyte to produce a color change, the reagent comprising )? n first component that interacts with the analyte to form hydrogen peroxide; ii) a second component that interacts with hydrogen peroxide to undergo a color change; and iii) a third component q and inhibits the color change of the second component; c) an intermediate layer between the lower and membrane layers; and d) measuring means for distributing the sample along the strip, the measuring means comprising i) a non-absorbent region in the membrane layer; and ii) a fluid transport channel formed in the intermediate layer for guiding the sample over the surface of the non-absorbent region to a multitude of discrete absorbent test areas disposed along the length of the membrane layer; the concentration of the inhibitor increasing with distance in a predetermined manner from a first end of the strip, so that a correspondingly increasing analyte concentration must be contained in the sample if a color change is made, whereby a or more test areas may change color when a unit is applied to the strip, and. the area of color change more distant from the first end indicates the concentration of analyte in the sample.

2. The strip according to claim 1, wherein the analyte is glucose.

3. The strip according to claim 1, wherein the biological fluid is blood.

4. The strip according to claim 1, wherein the lower layer comprises a thermoplastic sheet.

5. The strip according to claim 4, wherein the lower layer comprises polyester.

6. The strip according to claim 1, wherein the lower layer further comprises a plurality of continuous holes in alignment with the test areas.

7. The strip according to claim 1, wherein the lower layer has a transparent section located at a predetermined distance from the hole that accepts the sample to ensure an adequate sample size.

8. The strip according to claim 1, wherein the membrane layer comprises an anisotropic porous membrane having pores that are larger near the sample side and smaller near the test side.

9. The strip according to claim 8, in which the size of the pores is selected so that the red blood cells of an intact blood sample are trapped in the membrane.

10. The strip according to claim 8, wherein the membrane comprises polysulfone.

11. The strip according to claim 1, wherein the first component comprises glucose oxidase.

12. The strip according to claim 1, wherein the second component comprises a peroxidase and an indicator dye or pair of dyes that change color when oxidized.

13. The strip according to claim 12, wherein the peroxidase is horseradish peroxidase.

14. The strip according to claim 12, wherein the dye, indicator or pair of dyes is MBTHSB-ANS.

15. The strip according to claim 1, wherein the third component comprises ascorbic acid.

16. The strip according to claim 1, wherein the reagent further comprises a separation component selected from the group consisting of polyethylene glycol, polymethylvinyl ether / anhydridomalieic ether, polypropylene glycol, polystyrene sulfonic acid, polyacrylic acid, polyvinyl alcohol, and polyvinyl sulphonic acid.

17. The strip according to claim 1, wherein the intermediate layer comprises a thermoplastic sheet.

18. The strip according to claim 1, wherein the intermediate layer comprises polyester.

19. The strip according to claim 1, wherein the absorbent and non-absorbent regions comprise crushed and non-crushed regions of the membrane layer, respectively.

20. The strip according to claim 1, further characterized in that it comprises a top layer q? E is contiguous with the upper surface of the membrane layer and has continuous holes that align with the test areas.

21. The strip according to claim 20, wherein the membrane layer is adhered to the upper layer.

22. The strip according to claim 21, wherein the membrane layer is adhered to the upper layer with an adhesive that is restricted to the non-aromatic regions of the membrane layer.

23. The strip according to claim 1, further characterized in that it comprises absorbent layers that make contact with each end of the membrane.

24. The strip according to claim 1, further characterized in that it comprises a time controlling element, which comprises a test area that includes, in addition to the reagent, an amount of glucose that causes the area to change color to a predetermined time after the sample is applied to the strip.

25. A method for measuring the concentration of analyte in a biological fluid sample, comprising the steps of: a) applying the sample to the test test strip comprising: i) a multitude of absorbent test areas that each one changes color when it makes contact with fluid containing at least a predetermined amount of analyte, greater than the amount of analyte that causes a change in color of the test areas that are closest to a first end of the strip and ii) measurement means for the distribution of the sample along a predetermined non-absorbent path to each of the test areas and b) observing the test area that changes color and that is more distant from the first end of the test. strip.