MXPA97009995A - Reactive test strip that can be read visual - Google Patents

Abstract

A multi-layered, reactive test strip measures the analyte concentration in a sample of liquid applied to it, the sample is guided to several test areas arranged along the strip, where the analyte can react with a reagent to cause a color change, each analysis area also includes an inhibitor for the color change reaction, the concentration of the inhibitor increases in successive analysis areas, so the number of areas of color change is a measurement of the concentration of analyte the test strip is particularly adapted to measure glucose in a whole blood sample, in a preferred embodiment, the screen is guided to the analysis areas along a path formed by crushing selected areas of a membrane, and the areas of non-crushed sonar analysis of the membra

Description

J and REACTIVE TEST STRAP THAT CAN BE READ VISUALHENT REFERENCE TO RELATED REQUESTS This application is a continuation in part of the pending US application serial number 08 / 743,432, filed on November 1, 1996, which is a continuation of serial number 528,511, filed on August 3, 1995, abandoned, Which is a continuation in part of serial number 411,238, filed on March 27, 1995 and serial number 442,035, filed May 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, - more particularly, to a test strip that directly measures the concentration, 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, which incorporate enzyme-based compositions, are widely used in clinical laboratories, doctors' offices, homes and hospitals to test glucose concentration in biological fluid samples. In fact, test strips have become a daily necessity for many of the several million existing diabetics. Since diabetes can cause dangerous abnormalities in blood chemistry, it can contribute to vision loss, kidney failure and other medical consequences. To minimize the risk of these consequences, most diabetics should be tested periodically, then adjust their glucose concentration accordingly, for example, by dietary control and / or insulin injections. Some patients should test their concentration of blood glucose up to four times a day or more. This is especially important for diabetics who must control their diet in order to regulate the ingestion of sugar and / or administer insulin injections, and those who should be guided in this direction by frequent tests of blood glucose concentration, have strips fast, cheap and accurate reagents for the determination of glucose. It is known that the test strips contain an indicator that changes to a different shade of color, depending on the concentration of glucose in a biological fluid, which has been applied to the strip. Although strips sometimes use reduction chemicals, they most commonly involve an oxidizable dye or a pair of dyes. Some of the strips include an enzyme, for example, glucose oxidase, which is capable of oxidizing glucose to gluconic acid and hydrogen peroxide. They also contain an oxidizable dye and a substance having peroxidating activity, which is capable of selectively catalyzing 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 to Kiser and co-inventors). U.S. Patent No. 3,964,871, issued June 22, 1976 to Hochstrasser, discloses a disposable indicator strip for measuring substances directly, such as glucose, in biological fluids. The indicator records the concentration of the substance including both an indicator reagent, which is oxidized and changes color when it reacts with the substance, as an "antagonist" that, in some way, prevents the accumulation of oxidized indicator until it has been completely consumed . Palmer and co-inventors describe a "digital" quantitative analysis system for glucose and other analytes in European Patent Application Publication No. 0 317 070, published May 24, 1989 (see also US Patent No. 5)., 036,000, issued July 30, 1991). That system identifies the concentration of an organic compound in a biological fluid by first oxidizing the compound to a specific oxidase enzyme for the substrate, in order to produce hydrogen peroxide. The system includes a chromogen that is a hydrogen peroxide reducer and an air-stable hydrogen peroxide reducer, which has a greater reduction potential. The potential for further reduction retards any color change detectable by the cryogen, until the first hydrogen peroxide reductant, stable to air, has been consumed. In this way, a color change is not obtained as a result if the hydrogen peroxide to be measured is less than a predetermined level, which corresponds to the concentration of the peroxide reducer stable to the air. As a result, the system measures the concentration quantitatively, independent of the intensity in the color change. Englenann, in U.S. Patent No. 4,738,823, issued April 19, 1988, discloses a test strip for the determination of analyte, which has a support member having an absorbent material placed to remove the excess sample applied to it. strip. The strip may also include a cover that includes an opening through which the sample may be introduced. Burkhardt and co-inventors, in U.S. Patent No. 4,810,470, issued March 7, 1989, describe a device for measuring analyte concentrations in liquid samples. The device includes one or more rna + bibulous rices covered by a coating or film impervious to liquid. The sample is deposited in a portion of a bibulous matrix and dosed in the matrix chromatographically. Through a capillary action, the sample moves to a region of analysis that contains a test reagent for the analyte. Daffern and co-inventors, in US Patent No. 4,994,238, issued February 19, 1991, discloses a test device for chemical analysis comprising an absorbing layer + e, an impermeable barrier layer and a reactive layer having a volume determined. The sample is applied to the reactive layer by means of aligned holes in the absorbent and barrier layers that remain on top. Whether the test is carried out at home, at the doctor's office, at the clinic or at a hospital, the precision and reproduction of a glucose determination is extremely important. In the case of an indicator strip for color, it is advisable that the color change be pronounced and insensitive to variations in the components of the biological fluid other than glucose. In the case of a reactive strip that is read visually, it is especially important that diabetics, who may have damaged vision, have a strip that exhibits an important color change that depends on the concentration of the glucose, although the Color change that is exhibited in a change in absorbance at a given wavelength, is also important for the accuracy of the measuring and reading strips. As + or q? E the change of color implies a series of chemical reactions, this does not happen instantaneously. Thus, the user must wait for a certain time, 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 indicating that the reactions are complete. However, when a strip is read visually, without a meter, the user can underestimate the time required, read the strip prematurely and obtain an incorrect result. Alternatively, the user may feel the need to wait for an excessive amount of time before reading the strip, to ensure that this completes the reaction, which causes unnecessary delay and user dissatisfaction. Thus, there is a need for a "chemical" time controller, that is, an element in the strip that changes color independently of the concentration of the glucose (or other analyte of interest) present in the sample., but only after sufficient time has elapsed to complete the color forming reactions with the sample.

BRIEF DESCRIPTION OF THE INVENTION According to the present invention, an elongated multi-layered reagent test strip for measuring the analyte concentration in a biological fluid sample, which is applied to the strip, comprises: (a)? with a through hole to accept the sample; (b) a membrane layer having a sample side that will face the lower layer and a test side opposite to it, and having a plurality of biblical analysis areas disposed along its length; slabs, discrete, separated by a non-bibulous region, the membrane containing a reagent that can react * with the analyte to produce a color change; the reagent comprising: (i) a first component that interacts with the analyte to form hydrogen peroxide; (11) a second component that interacts with hydrogen peroxide to undergo a color change; and (m) a third component that inhibits the color change in the second component; (c) an intermediate layer between the lower and membrane layers; and (d) dosing means for distributing the sample along the strip; the dosing means comprising a fluid transport channel formed in the intermediate layer to guide the sample over the membrane surface towards the bibulous analysis areas; increasing the concentration of inhibitor in a predetermined manner with the distance from a first end of the strip, so that a correspondingly increasing concentration of analyte should be contained in a sample if it is to effect a color change; with which one or more analysis areas may change color when a sample is applied to the strip, and the color change area distant from the first end indicates the concentration of analyte in the sample. In the operation, a method for measuring the concentration of the analyte in a biological fluid sample, comprises the steps of: (a) applying the sample to a reagent test strip comprising: (i) a bottom layer with a through hole to accept the sample; (11) A membrane layer having a sample side facing the bottom layer and comprising a plurality of bibulous analysis areas, each of which changes color when it comes in contact with the fluid it contains when a predetermined amount of analyte, greater than the amount of analyte that causes a color change in the analysis areas that are closest to the first end of the strip; and (ni) dosing means for distributing the sample from the through hole along a predetermined, non-biblical path to each of the analysis areas; and (b) determining the analyte concentration by observing the analysis area that changes color and that 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 a "sample side" of the strip. The visible indication appears on the opposite (or "test") side of the strip. The chemical composition of the strip depends, of course, on the analyte / biological fluid that is to be measured. Test strips can be designed to detect analytes such as glucose or other sugars, alcohol, cholesterol, proteins, ketones, uric acid, phenylalanine or enzymes in biological fluids such as blood, urine and saliva, as well as in water. For the sake of convenience and brevity, the reactive test strips described in greater detail in this description detect glucose in the blood. A person of ordinary skill in the art could readily adapt the information in this description to detect other analyte / biological fluid combinations. A test strip of the present invention provides a relatively simple and rapid determination of the concentration of glucose in a sample of blood not measured. The strip comprises a lower layer with a hole through which a sample can be introduced to the sample side of a porous matrix, whose opposite side is the test side. The matrix is generally a membrane and the two terms are used interchangeably in the present description and in the appended claims. A test reagent is applied to the matrix and, to a lesser or greater degree, impregnated into the pores of the matrix. For simplicity some reference will sometimes be made to the reagent present in the matrix as a "coating"; in this specification and the appended claims, recognizing that the reagent coating penetrates the matrix. An intermediate layer extends between the lower layer and the matrix. In one embodiment, cutouts are aligned in the intermediate layer with the non-blistered areas of the membrane to guide the sample to a series of bibulous analysis areas that are disposed along the strip. (As it is used in eeta descriptive memory and in the claims that come at the end, "bib? Oso" is understood with the meaning of absorbent). A series of notches in the middle layer surround the space around and above the analysis areas to constrict the flow of the sample to those areas. In another embodiment, a substantially rectangular, elongated slot in the intermediate layer guides the sample to a succession of bibulous areas that are separated by a non-bibulous region. A sample volume, typically whole blood that includes both red blood cells and glucose, is then directed to the sample side of the membrane in each of a number of test areas. The porosity of the matrix allows the fluid to pass from the sample side to the test side, by capillary action. Thus, the test reagent can react with the glucose present in the blood to cause a color change on or near the test side. Since the strongly colored red blood cells may make it more difficult to detect the color change, preferably the anisotropic matrix, the pore sizes being graduated from large pores on the sample side to smaller pores on the test side, in order to catch the red blood cells outside the test side. A variety of materials can be used for the various components of the test strip and the time controller of this invention. Some of these materials are described in U.S. Patents 5,306,623 and 5,418,142, issued April 26, 1994 and May 23, 1995, respectively, to Kiser and co-inventors, which are incorporated herein by reference. The test reagent comprises a component for converting glucose to hydrogen peroxide, such as glucose oxidase; one or more components for detecting hydrogen peroxide produced from the glucose present in the sample; and an inhibitor. The components for detecting hydrogen peroxide can be a peroxidase, preferably horseradish peroxidase, together with an "indicator" that changes color in the course of the reaction. The indicator can be an oxidizable dye or a coloring pair. Peroxidase catalyses the oxidation of the indicator in the presence of hydrogen peroxide. The final reagent element is an inhibitor that retards the indicator's color change oxidation. The strip is segmented throughout its length such that adjacent membrane segments have different concentrations of inhibitor. Each segment has a bibulous analysis area that only changes color if and when enough glucose is present to first cause all the inhibitor to be consumed and then oxidize the indicator and thereby cause the characteristic color change. Thus, a change of color in a particular area evidences a minimum glucose concentration in the original blood sample. Along the strip, in a particular direction, each successive segment has a gradually greater concentration of inhibitor, which corresponds to a gradual increase in the threshold glucose concentration. The indicator concentration is the same for all segments. In principle, other variable inhibitor / indicator equilibria 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 an adjacent area is not. This result indicates that the concentration of glucose in the sample is at least equal to the minimum or threshold concentration required to change the color of an area., but not as large as the one required to change the color of the adjacent area. To monitor blood glucose, a coating of the optional time-controlling segment comprises the elements of the indicator strip, a porous matrix having a test reagent applied thereon, and additionally glucose. In the dry state, the chemistry of the reagent is not activated by glucose, but when a sample is applied to the strip, the time-controlling coating and the glucose in the coating are hydrated after a predetermined time, causing the indicator to change color . Preferably, the glucose is present in the time controller in an amount well in excess of that required to overcome the inhibitor. In that case, the time required is greater or lesser, depending on whether more or less inhibitor is present. The color changes in the strip and in the time controller can be observed directly with the naked eye or with an optical instrument that detects the changes of the mirror.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of the matrix in a reactive, direct reading test strip of the present invention. Figure 2 is a bottom plan view, cut away, of the sample side of a direct reading reactive strip of the present invention. Figure 3 is a fragmentary, enlarged perspective view of the interior of the test strip of Figure 2, partially cut away. Figure 4 is a section of the strip of Figure 2, taken along line 4-4. Figure 5 is a bottom plan view of the test strip of Figure 2. Figure 6 is a top plan view, showing the test side of the test strip of Figure 5. Figure 7 is the strip from figure 6 after a sample has been applied. Figure 8 is a cutaway perspective view of another embodiment of the test strip of Figure 2. Figure 9 is a bottom plan view of the test strip of Figure 8. Figure 10 is a top plan view of the test strip of Figure 8. Figure 11 is a section of the strip of Figure 10, taken along line 11-11.

DETAILED DESCRIPTION OF THE INVENTION The present invention is a reactive test strip, direct reading, for measuring the concentration of analyte in a biological fluid. The key element of said test strip is a porous matrix that incorporates a test reagent that undergoes a color change in response to the analyte present in a sample of biological fluid that is applied to the sample. The matrix may be of a uniform composition or may be a coated substrate and may be isotropic or anisotropic. It has a sample side, to which a sample and a test side are applied, in which the color change is observed. Preferably the matrix is an anisotropic membrane; most preferably an anisotropic membrane having a wide scale of pore sizes. For example, a gradient of pore sizes from about 0.1 millimeters to about 150 millimeters may extend across the membrane. On the large pore side, the pore size preferably is on the approximate scale of between 30 micron and 40 micron. On the side of the membrane where the pores are smaller, the hollow volume is relatively small and the material of the membrane is generally quite dense, within a layer which can typically constitute up to 20% of the thickness of the membrane. Within that layer, the pore size preferably is in the approximate range of 0.1 to 0.8 micrometers, with a nominal pore size of about 0.3 micrometers being preferable. When the biological fluid is applied to the sample side, the sample finds smaller and smaller pores as it penetrates the membrane. Eventually, solids, such as red blood cells, reach a position in the membrane, in which they can no longer penetrate. The rest of the sample, which still contains the dissolved glucose, penetrates through the test side. The amsotropic nature of the membrane and / or the use of a spacer component (discussed below) allows relatively rapid flow rates through the membrane, even while filtering the solids. When the sample passes through the matrix, the reaction with the reagent causes a light absorbing dye to form or decompose in the hollow volume near the test side, thereby substantially affecting the reflection of the matrix. Lae polysulfones and polyarnides (nylons) are examples of suitable matrix materials. Other polymers that have comparable properties can also be used. The polymers can be modified to introduce other functional groups that provide the charged structures, so that the surfaces of the matrix can be neutral, positive or negative. A preferred method for preparing the porous material forming the matrix is to mold the polymer without a supporting core. Said matrix, for example, is the anisotropic polysulfone membrane obtainable from Me tec, Inc., Tirnoniun, MD. A matrix of metals of about 200 micrometers in thickness is usually employed, with one of about 115 to 155 millimeters being preferred. What is most preferred is an approximate thickness of 130 to 140 millimeters, particularly when the matrix is nylon or amsotropic polysulfone. The membrane can be treated with the test reagent by dripping it into a mixture of the components, thereby saturating the membrane. Preferably, at least some of the components are applied sequentially to the membrane. The excess reagent can be removed by mechanical means. For example, by means of an air knife, a spatula or a glass rod. Then the membrane is dried. The reagent tends to concentrate near the small (test) pores side of the membrane. The test reagent comprises: (i) a component for converting glucose to hydrogen peroxide; (n) a component for detecting hydrogen peroxide; and (m) a component for inhibiting the component that detects hydrogen peroxide. The reagent may additionally optionally comprise a spacer component which causes the solids, such as red blood cells, to be trapped in the matrix, thereby effectively removing the solids from the biological fluid. Additional components may also be included, as described in the examples below. Preferred components for converting glucose to hydrogen peroxide include glucose oxidase, an enzyme that is usually obtained from Aspergillus niger or Pemcilliurn. Glucose oxidase reacts with glucose and oxygen to produce gluconoiactone and hydrogen peroxide. The optimal concentration of glucoea oxidase depends on the composition of the indicator system. For example, if the indicator system is MBTHSB-ANS (which will be described later), then the glucose oxidase is on the approximate scale of 500-10,000 U / ml, which is adequate, rn? And preferably 700 to 2,000 U. / ml and what is more preferred, around 1,000 U / ml. In general, higher concentrations of glucose oxidase cause the reaction to process more quickly and lower concentrations, faster. 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 uses hydrogen peroxide as an oxidant that is capable of removing hydrogen atoms from various substances. A suitable peroxidase may contain ferriprotoporphyrin, a red hernin obtained from plants. Peroxidases obtained from animals, for example, from the thyroid gland of animals, are also suitable. Horseradish peroxidase (HRPO) is especially preferred as a constituent of the component for detecting hydrogen peroxide. The hydrogen peroxide, preferably catalyzed by a peroxidase, reacts directly or indirectly to form or decompose an indicator dye that absorbs light on a predetermined wavelength scale. Preferably, the indicator dye absorbs strongly at a wavelength different from that to which the test reagent strongly absorbs. The indicator's rusty shape-may be the colored, faintly colored shape or a colorless end product that shows a color change on the test side of the matrix. That is, the test reagent can indicate the presence of an analyte in a tooth by a colored area that is bleached or, alternatively, by a colorless area that develops color. The indicators which are useful in the present invention include: (a) hydrazone hydrochloride of 3-rnethyl-2-benzothiazolinone (MBTH) combined with 3-dirnethylamininobenzoic acid (DMAB); (b) MBTH combined with 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS); (c) 4-arninoantipyrene (4-AAP) and 5-oxo-l- (p-sulfophenyl) -2-pyrazoline-3-carboxylic acid (OPSP); (d) 4-AAP and N- (rn-tolyl) -diethenol ina (NDA); (e) 2, 2'-azino-di (3-ethylbenzthiazoline) sulfonic acid (ABTS); (f) 4-AAP and -ethophtol; (g) pyrogallol red (PGR); (h) bromopyrogallol red (BPR); (i) acid green 25 (AG); or (j) rnonosodium salt of C3-methyl-2-benzothiazolinone hydrazone N-sulfonylbenzenes (C-methyl-2-benzothiazolinone) (MBTHSB), combined with 8-anilino-l-naphthalene sulfonic acid ammonium salt (ANS). MBTHSB-ANS is preferred. More information with respect to MBTHSB-ANS appears in U.S. Patent 5,563,031, issued October 8, 1996, which is incorporated herein by reference. The inhibitor component retards the reaction between hydrogen peroxide and the indicator, for example, by reducing hydrogen peroxide or reducing the oxidized indicator. In principle there are different modes of operation for an inhibitor. First, the inhibitor would compete with the indicator and thus slow down the rate at which the change of place in the indicator takes place. Secondly, the indicator could not be competitive, so that substantially all the inhibitor would be consumed before any substantial color change in the indicator was made. Other modes of inhibitor operation are also possible. Preferably, the inhibitors of the present invention are non-competitive. Among the scale of suitable inhibitors are the acid 2,3,4-tph? Drox? Benzo? Co; propyl gallate; 3,4-dihydroxymarnic acid; 3,4-d hydroxybenzaldehyde; Gallic acid; 5,6-d? Am? Nouraclo; ascorbic acid and isoascorbic acid. Ascorbic acid is preferred; however, the ascorbic acid is oxidized in solution and must be stabilized in order to allow the reagent to be applied. Preferred stabilizers are primary alcohols, such as ethyl, methyl or propyl alcohol. Ethyl alcohol is preferred, particularly in concentrated solutions, ie, solutions of 50% or more of ethanol. Although the amsotropic membrane which is the preferred matrix separates the red blood cells by filtration and keeps them away from the test side, optionally the test reagent may also contain a separating component. The separating com pound must be capable of producing and relatively clear colorless fluid from the fluid containing red blood cells, for example, whole blood, sequestering the red blood cells in the matrix. The spacer components for use in the present invention include, but are not limited to: polyethylene glycol, poly (methylmeryl ether / rnaleic ether), polypropylene glycol, polystyrene sulphonic acid, polyacrylic acid, polyvinyl alcohol and polyvinyl sulphonic acid at a pH of between about 4.0-8.0. Said separating components are present in the matrix in amounts that will vary depending on their charge and their molecular weight; the components are embedded in the matrix, the pH of the matrix and the pore size and the residual humidity of the matrix after drying. These parameters are easily determined by who is an expert in the field. For example, when polypropylene glycol is used as the separating component (for example, PPG-410 from BASF, lyandotte, MI) it is preferably present at about 2-30% w / v (w / v) and, better yet, from 8 to 10% by weight / volunen. Other separating components can also be used in an approximate concentration of 2 to 30% by weight / volume. It is possible to impregnate or embed the polymeric components in the matrix or they can be emptied into the membrane during manufacture. Some water soluble salts can also?: > effect the separation of the blood. Among the salts suitable for separating the blood components are the citrates, forrniates and sulphates, as well as certain acids, such as amino acids, citric acid, phytic acid and rnálico acid. (See, for example, U.S. Patent 3,552,928, issued January 5, 1971 to M.C. Fetter). An advantage of including the spacer component is that with solids such as red blood cells substantially removed from the biological fluid there are less background colors at the test site to obscure a change in coloration produced by the test reagent. Other components can be embedded in the matrix to increase the coloration and readability of the test strips and to preserve the uniformity and integrity of the matrix. For example, the test reagent may include salts and / or regulators to aid in separation of the dye in the matrix. Said regulators may contain, for example, citrate, present in solution of about 0.01 mol to about 1.0 mol and, preferably, about 0.1 mol. Other regulators can also be employed. 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 limited to: bovine serum albumin, polypeptides, and the low molecular weight protein obtainable as Crotein SPA (CRODA, Inc., New York, N.Y.). Such compounds are used at concentrations, for example, of about 1 rnG / ml to about 100 mg / ml. In the case of Crotein, about 30 mg / ml is preferred. Other stabilizers and preservatives in the coating for the matrix may also be included. For example, ethylenediarinotetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA) and related compounds can be used, for example, at concentrations of about 0.01 mg / rnl to 10 mg / ml. Among the aims of the conservatives is to help stabilize the inhibitor. Some of the indicators (for example, BPR) have an undesirable tendency to migrate in the matrix. When such an indicator is used, an ion-spacing agent is included to prevent such migration. For example, polyethylene glycol derivatives obtainable commercially as Polyquart (H) (Henkel, Inc., Ambler, PA) are particularly useful for their ability to facilitate the ionic pairing between the indicator and other substituents of the matrix. 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 increase the contrast in the non-colored environment. Organic solvents may also be used in the practice of this invention, and may be included in the formulation of the test reagent for the matrix; provided, of course, that they are compatible with the matrix and with the test reagent compositions. Potentially suitable organic solvents include: chloroform, acetone, alcohols, methylene chloride, diethyl and petroleum ethers, acetomyls and their mixtures. In the practice of the present invention, 70% ethanol in water is particularly preferred. The test reagent that is applied over, or impregnated into, the matrix is not uniform on the surface of the test strip. Rather, the reagent is preferably applied to the matrix in a series of parallel strips or "segments" that extend through the narrow dimension of the strip. The composition in the adjacent segments increases gradually in the concentration of the inhibitor. Each segment has a tubulous analysis area. It is in the areas of analysis that the test reagent reacts with any glucose present in the blood to cause a color change, provided that the glucose concentration is large enough to overcome the level of inhibitor present in that area of analysis. . In such a way, each successive analysis area requires, gradually, a higher concentration of glucose in the sample to cause a change of color in the area. Optionally, one of the analysis areas is adapted to serve as a time controller, to indicate that enough time has elapsed for the reactive reactions with glucose in each of the areas of analysis. The time-controlling segment of the matrix is applied or impregnated with a composition consisting of the test reagent and, in addition, glucose. Since the purpose of the test reagent is to change color in response to glucose, the combination of the two without causing any color change - requires some care. An amount of inhibitor beyond that needed for the time control function must be present to compensate for this effect. The speed at which the controlled time segment dries, after the solution containing glucose is applied, is controlled. In practice, the membrane is first coated with a solution containing regulators, stabilizers and enzymes, and that coating is dried to form a first layer. Then a solution containing the indicator, the inhibitor and the glucose is applied in a second coating step. Parameters such as continuous strip velocity, furnace temperature and air flow, as well as the amount of coating solutions deposited, will have to be fixed in advance and appropriate adjustments will be made for inhibitor concentrations. and / or glucose. Instead of applying the second coating directly, a less preferred alternative involves forming the second coating on a separate strip 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 forcing reaction to proceed. The time it takes for the time-controlling segment to change color is then determined by the temperature and by the characteristics of the test reagent, particularly the concentration of the inhibitor, the amount of glucose and the rates of hydration and diffusion of oxygen. The color change time of the time controller may be made to depend on the concentration of glucose present in the sample or, alternatively, to be independent of that concentration. By incorporating a large excess of glucose in the time controller, the time is substantially independent of the glucose concentration in the sample. By incorporating less glucose into the time controller, the time can be made to depend on the glucose present in the sample, i.e., 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 greater than about 1,500 rng / dl, which makes the time controller substantially independent of the glucose concentration in the sample, on the approximate scale of 40 to 400 ng / dl. The composition of the time-controlling segment includes excessive amounts of the component (such as gl? Oxidase) that converts glucose to hydrogen peroxide and glucose. The time-controlling composition must then include at least as much inhibitor or less than that which results in the segment having the highest concentration of inhibitor (which corresponds to the highest glucose reading). The time controller also serves an important quality control function by making it apparent when a test strip has been compromised by exposure to moisture. A test strip must be kept dry until the moment it is going to be used, because the components that convert glucose to hydrogen peroxide (usually enzymes) tend to degrade by exposure to moisture. So thatIf the strip is exposed prematurely to moisture, it will be compromised. However, the damage to the test strip is not obvious to the user who, therefore, could 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. Other information that relates to the time controller appears in the co-pending US patent application serial number 08 / 706,753, filed on September 3, 1996 and incorporated herein by reference. In addition to the matrix containing the reagent, the strip of the present invention includes a lower layer that supports the matrix. The lower layer is preferably a thermoplastic sheet, better still, a polyester, generally 0.05 to 0.2 rnm thick and has a hole through which the sample can be applied to the sample side of the matrix. From the sample hole the blood sample is distributed throughout the matrix. If the lower layer is generally opaque, then one or more transparent window sections can be located at an appropriate distance from the sample hole, and the appearance of the sample in the window or windows confirms that the sample has been properly applied. to the strip. Distributing the blood from the sample hole to the areas of analysis involves an intermediate layer that lies between the bottom layer and the membrane and, optionally, is adhered to both. The intermediate layer is preferably a thermoplastic sheet, better still, a polyester, generally about 0.05 to 0.2 mm thick. In one embodiment, the cuts in the intermediate layer guide the sample along the length of the strip along the non-biblical trajectories of the membrane and direct the membrane to each of the analysis areas. Notches in the intermediate layer are aligned with the analysis areas, so that each analysis area is substantially surrounded by the walls of the intermediate layer. In another embodiment, the intermediate layer has an elongate, substantially rectangular groove, which guides the sample through the membrane surface to the analysis areas. The width of the groove is generally in the approximate range of 0.5 and 3 m.

A preferred structure for the non-bibulous paths in the membrane is formed by crushing the pore structure of the membrane. This can be achieved by heating either directly or using a laser or ultrasound and, preferably, including pressure. However, the preferred method is crushing. Thus, the membrane is crushed to make it non-bibulous (but still hydrophilic) in all parts except in the areas of analysis. In one embodiment of the invention, the membrane is flattened between flat plates with a die that prevents the areas of analysis from being crushed. Pressures of at least 80,000 kPa are preferred. Optionally, the plates can be heated to at least about 100 ° C. The pressures and the preferred temperatures depend, of course, on the crushing mechanism and the residence time, as well as on the parameters of the membrane. The optimal values can be determined by routine experimentation. The manner in which the membrane is crushed in this way produces areas of analysis which extend into the lower layer and is used with the grooved intermediate layer, as discussed below. For precise measurements, the volume of blood provided for each area of analysis is preferably reducible. If the notches completely surround the analysis areas, then, assuming a hermetic seal in liquid between the intermediate layer and both the lower layer and the crushed membrane, each analysis area would be associated with a closed volume (cylindrical) whose walls are formed by the intermediate layer and whose ends are formed by the membrane and lower layers. However, a distribution channel runs along the strip and feeds the sample to each area of the analysis areas. Preferably the lower layer has vent holes in alignment with the analysis areas to facilitate the filling of the channel and the analysis areas, uniformly. The high precision requires that the distribution channel provide a fixed volume of sample to each analysis area, but then it does not provide more, at least not in the time frame of the measurement, around 2 minutes, from approximately 90 seconds after the blood is applied. Since the initial sample volume is variable, there is preferably an absorbent layer at each end of the membrane to remove the excess sample from the ends of the distribution channel. The absorbent layers at the ends of the channel also increase the capillary effect of the sample throughout the length of the strip. Non-woven fabrics, well known in the art, form the preferred absorbent layers. In another embodiment of the invention, the membrane and the cover sheet are pressed between rollers. The cover sheet has holes placed to accommodate the analysis areas, and those areas then extend into those holes, remaining without being crushed. For this embodiment, a die is not necessary, and the crushing is preferably obtained by rollers, with an applied force of at least about 4,450 Newtons. Note that the analysis areas in this modality extend in the opposite direction to those of the modality previously described. Since the sample is taken to the upper layer, open to the outside, no vent holes are used in the lower layer. This embodiment is used in the intermediate layer having a substantially rectangular slot to guide the sample to the analysis areas. Since that embodiment has a sample hole located near the end of the strip having the "high glucose content" analysis areas, only a single absorbent layer is used near the opposite end of the strip. The color change caused by the glucose in the test sample appears on the test side of the membrane. In the modality in which the analysis areas extend towards the lower layer, it is convenient to overlap the test side of the membrane with an upper layer that has holes that align with the areas of analysis. The holes make color changes visible and also allow oxygen to reach the reaction sites. When the analysis areas extend in the opposite direction, the holes in the upper layer define the areas of analysis during the crushing process, as described previously. In both cases, the top layer is preferably a thermoplastic sheet, better still, a polyester, generally around 0.05-0.2 mm thick. The top layer can be attached to the membrane, for example, with an adhesive. Any adhesive is preferably limited to non-biblical areas if it interferes with the glucose measurement reactions. However, if the adhesive does not interfere with the reactions, its placement is less critical. Since the areas of analysis, when they contain the preferred reagent, slowly undergo a color change when exposed to light or oxygen and since the optional time controller is sensitive to moisture, the strips are preferably packed in an envelope opaque, impervious to oxygen and moisture, such as a sealed metal foil wrapper. If the strips are individually packed, the strip can remain in the open wrapper during use. The invention will now be described further with reference to the figures. Figure 1 shows a matrix 10 of the present invention for measuring the amount of analyte in a biological fluid. Although shown in an arched position, the matrix 10 is flexible and is generally in a straight plane when used. The matrix includes a sample side 12, to which the biological fluid sample is applied, and a test side 14, at or near which a color change indicates the presence of the analyte. The color change is the result of the interaction of the analyte with the reagent impregnated in the pores 16. Preferably, to measure the concentration of glucose in the blood, the pore sizes are relatively large near the test side 12 and decrease in size as they approach the test side 14. The pore size gradient serves to trap the red blood cells near the sample side 12, so that their color does not interfere with the ability to see the color change, which indicates the presence of the analyte. Three parallel segments, a, b and c, are shown schematically. Each successive segment has gradually more inhibitor than the preceding one. In a preferred embodiment, after the reagent has been applied to the membrane in parallel segments, as shown, the membrane is crushed at all points, except in the areas of analysis, where the analyte reactions take place. reagent. One mode of a pattern of areas of bibulous analysis, a single area located in each of the parallel segments, and the non-bibulous squashed areas, is illustrated in the plan view of Figure 2 and in the enlarged fragmentary perspective view, of Figure 3. Figure 2 is a bottom plan view, in partial section, of the sample side 12 of the membrane 10 and the absorbent layers 20 and 22, which lie on top of the intermediate layer 24 and the lower layer 26. The membrane 10 and the absorbent layers 20 and 22 are preferably supported by an upper layer, not shown. Absorbent layers 20 and 22 are preferably located at the ends of the membrane (beyond dotted lines A and B) to absorb the blood sample which is an excess of the volume needed for the measurement. That volume should be sufficient to provide sample to each of the analysis areas and, if present, also to the time control area. In general, a strip that has fewer areas of analysis does not require as much sample, but provides a smaller scale of glucose values and lower accuracy. The figure 2 shows 9 bibulous areas representing 8 areas of analysis (numbered 1 to 8) and a time controller (T) that provides adequate scale and adequate accuracy although it does not require an unacceptably large sample volume. The intermediate layer 24 has a notch 28 which aligns with the sample hole 30 in the lower layer 26. The sample is introduced through the sample hole 30 and is directed by capillary action along the central channel 32 of the intermediate layer 24 to each of the analysis areas and the time control area, any excess sample being absorbed in the absorbent layers 20 and 22. The appearance of the sample through the optional clear windows 34 and 35, confirms that sufficient sample has been provided for the measurement. Preferably the intermediate layer 24 forms a seal with the sample side 12 of the membrane, so that the sample, for example, can not flow directly between the adjacent analysis areas. Figure 3 is a fragmentary, enlarged perspective view, illustrating parts of three analysis areas 6, 7 and 8 seen through the lower layer 26 and separated by intermediate layer projection 24. The optional adhesive layers 24a join the intermediate layer 24 with the lower layer 26 and the membrane 10. The vent holes 40 in the layer 26 facilitate the flow of the sample within the strip. The holes, such as 38, in the upper layer 36, are aligned with the bibulous areas, making any color change visible in the bibulous 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 section taken along the line 4-4 of FIG. 2, which shows the upper layer 36 in addition to the layers shown in FIG. 2. The vent holes in the lower layer 26, such as or 40, are aligned with the analysis and control areas of FIG. time and facilitate the filling with a sample of the volume surrounding each of these areas. The volumes to be filled are limited by the membrane 10, the intermediate layer 24 and the lower layer 26. Note that the columnar analysis area 3 extends towards the lower layer 26 and the minimum separation between the analysis area and the The lower layer is typically only around 12 millimeters. The separation is shown larger than scale, for clarity. Figure 5 is a bottom plan view of a strip of the present invention, showing the sample hole 30 and graphs instructing the user to introduce the sample through the hole. When you see the 3f > Through the clear windows 34 and 35, it is confirmed that the proper sample has been applied to the strip. FIGURE b is a plan view of the top layer 36 of a strip that has been calibrated to associate the test areas with the glucoea concentration. Figure 7 shows the strip of Figure 6 after a blood sample has been applied to the opening 30 (Figure 2), the sample has been spread along the central channel 32 and the glucose of the sample has reacted with the reactive in the areas of analysis. Since the lower analysis area has the least amount of inhibitor, that area will have changed color first. Subsequently the second and third areas will change color, successively. The upper circles did not change color because there was too little glucose in the sample. Since enough time had elapsed for the time control area 42 to change color, the strip can be read. Thus, the result illustrated in Figure 7 indicates that the glucose concentration in the sample is at least 120 rng / dl but is less than 150 rng / dl. The reading may be returned at any time after the time control area 42 changes color *. Note that in Figure 7 the color change-caused by the reaction with glucose is from white to colored. However, the system could alternatively function with an indicator dye that is destroyed by glucose-induced oxidation, with a corresponding color change from colored to white. Figure 8 is a cutaway perspective view of another embodiment of the strip of this invention. The lower layer 126 has the sample hole 130 for introducing the blood sample. Contrary to the embodiment of Figure 2, where the sample hole 30 is located near the middle (between the ends) of the strip, the sample hole 130 is preferably located near the end of the strip having areas of analysis to indicate an elevated glucose concentration, as well as an optional time controller. The placement of the sample hole at that end provides two advantages. In the first place, the time needed to measure glucose is reduced in a short time so that the blood reaches the "high glucose content" analysis areas (which take longer to respond). Second, the variability of the time controller is reduced because the sample is applied essentially directly to the time controller, eliminating the variability in time for the blood to reach the time controller. The intermediate layer 124 has an elongated slot 132 that runs along the strip from a cutout that generally corresponds to, and is aligned with, the sample hole 130. The slot channels the blood sample throughout the length of the sample. the strip, on the membrane 110, towards the absorbent layer 120. When the sample passes over the membrane 110, part of it is deposited in the time controller T 'and in each of the eight areas of analysis (numbered 101 -108). The time controller and the analysis areas are viewed through the corresponding holes in the upper layer 136, that are aligned with them. The appearance of blood through the clear window 135 confirms that sufficient sample has been provided for the measurement. Figure 9 is a bottom plan view of the strip of Figure 8, where the graphs (such as the one illustrated in Figure 5) instruct the user to introduce the sample through hole 130 in the lower layer (and the coalmeated hole 128 in the middle layer) have been omitted. Figure 10 is a plan view of the upper layer 136 showing the time control charts as well as the calibration of the analysis areas. Figure 11 is a section taken on line 11-11 of Figure 10, showing the upper layer 136, the membrane 110, the intermediate layer 124 and the lower layer 126. The arrow illustrates the direction of introduction of the sample into the hole 130, in the lower layer 126 and the co-aligned hole 128 in the intermediate layer 124. Note that the time zone T 'of the colurnar time controller extends upwards, towards and preferably within, the corresponding hole 138, which is aligned with the time controller T 'and is one of the nine holes of the upper layer 136 which are aligned with the corresponding time controller and the corresponding analysis areas. For a better understanding of the present invention the following examples will further illustrate various embodiments of the invention. The examples are not intended in any way to be limiting.

EXAMPLE 1 BPR INDICATOR The following solution was prepared: Distilled water 83.5 g EDTA Na2 at 1% (w / w) 23. T g Aconitic acid 6.0 g NaOH (solid) 2.2 g Crotem SPA 4.2 g Irnidazole 0.6 g Mamtol 3.0 g Surfactol 01 at 5% ( p / p) 3.0 g Adjusts pH to 4.80 ethyl alcohol 40.0 g PPG-410 5.6 g Enzyme solution 28.0 g Enzyme solution 0.2 mole of Aconite acid 27.0 g Glucose oxidase 165,000 U HRPO 340,000 U Me tec BTSH 55 membrane was coated by immersion in this solution and the excess was expelled by expression with glass rods. The coated membrane was dried in a flotation dryer at 82 ° C, under moderate air flows, so that the core was substantially dry in a period of 20 seconds. The continuous tape was wound up in preparation for the second coating, which is described below. The following solutions were prepared: Ascorbate storage solution (inhibitor) Diluent Distilled water 190 g 370 g EDTA Na2 1% 55 g 107 g BPR 0.36 g 0.71 g PolyOuart® H 6 g 11.8 g PPG-410 14.2 g 27.8 g Ascorbic acid 1.37 g Ethyl alcohol 243 g 477 g Diluent solvent solution (according to 120 g the above formula) Ascorbic acid 0.885 g Glucose solution * 17.25 g * The glucose solution is a solution of 16.0 g / dl of glucose in water, which is allowed to stand for 24 hours, and stored refrigerated. The following dilutions of the stock material were formed: 0.0405: 1, 0.108: 1, 0.236: 1, 0.369: 1, 0.569: 1, 1.260: 1. This gradual increase in the concentration of inhibitor corresponds to the gradually higher concentration of glucose reported by the areas of analysis. These solutions, together with the time controller solution, were applied side by side on the large-pored side of the membrane loaded with enzyme, in order to deposit approximately 1.2 x 10- * ml per square millimeter of membrane. The membrane was wetted approximately 15 seconds before subjecting to the same drying conditions as described above for the passage of enzyme coating. The results showed that the time controller reacted around 70 seconds with around 95% of the resistances within 64 to 79 seconds.

EJEI1PLO 2 INDICATOR MBTHSB-ANS The following solution was prepared: HPLC water 1500 ml Citric acid 16.92 g Sodium citrate 20.88 g Mannitol 15 g Disodium EDTA 1.26 g Gantrez S95 6.75 g Crotem 5PA 36 g Gl? Se-oxidase 1.69 MU HRPO 1.5 MU Carbopol 910 * 75 rnl Disodium Citrate ** 225 rnl * 11% solution in acetonitrile ** 0.1 moles, pH 5.0. A Meintec BTS 35 membrane was coated on a tundish, so that the large pore surface was in contact with the coating solution; the excess solution was removed with glass rods, as above. The membrane was dried and rolled as in Example 1. The following solutions were prepared: Solution A (indicator) Solution B (wetting agent) Ethanol at 2819 rnl Maphos * 60A 41 g 70% (v / v) 70% Ethanol (v / v) 205 rnl MBTHSB 2.98 g (NH *) ANS 25.83 g Solution B 205 ml 2% DTPA 51.25 rnl Solution C ( Warehouse of Solution D (ascorbate controller) of time) Water 115 rnl Water 53 mi Ascorbic acid 4.58 g Ascorbic acid 8.75 g Ethanol 267 ml Ethanol 123 rnl Bring the volume with 70% EtOH to 175 ml Glucose solution 40.5 rnl For each solution of inhibitor the volume of solution A was set at 263 rnl. For the various areas of analysis, the proportion of 70% EtOH: solution C varied from 58.9 to 0.200, so that the volume of EtOH at 70% + solution C added to solution A was 87.5 nm for all the inhibitor solutions. This effectively altered only the concentration of inhibitor in each solution. The solutions containing the gradually increasing inhibitor concentration and the time controller solution (solution D) were applied side by side on the large pore side of the membrane. The deposition was adjusted to obtain about 8 x 10-s rnl of inhibitor per square millimeter of membrian. 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 reacted in about 60 seconds with little effect of the blood protein, from 30 to 55% or glucose from 78 to 420 rng / dl. Those skilled in the art will understand that the foregoing description and examples are illustrative of the practice of the present invention but are by no means limiting. Variations can be made in the details presented here, without departing from the scope or spirit of the present invention.

Claims (31)

NOVELTY OF THE INVENTION CLAIMS

1. - A reactive test strip, multiple layers, elongated to measure the concentration of analyte in a sample of biological fluid that is applied to the strip, characterized in that it comprises: (a) a lower layer with a through hole to accept the sample; (b) a membrane layer having a sample side facing the lower layer and a test side opposite to it, and having a plurality of discrete, bibulous analysis areas disposed along its length; separated by a non-bibulous region; containing the reactive membrane that can react with the analyte to produce a color change; the reagent comprising: (i) a first component that interacts with the analyte to form hydrogen peroxide; di) a second component that interacts with hydrogen peroxide to undergo a color change; and (iii) a third component that inhibits the color change in the second component; (c) an intermediate layer between the lower and membrane layers; and (d) dosing means for distributing the sample along the strip; the dosing means comprising a fluid transport channel formed in the intermediate layer to guide the sample over the membrane surface towards the bibulous analysis areas; increasing the inhibitor concentration in a predetermined manner with the distance from a first end of the strip, so that a correspondingly increasing concentration of analyte should be contained in a sample if a color change is to be made; with which one or more analysis areas may change color when a sample 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, further characterized in that the analyte is glucose.

3. The strip according to claim 1, further characterized in that the biological fluid is blood.

4. The strip according to claim 1, further characterized in that the lower layer comprises a thermoplastic sheet.

5. The strip according to claim 4, further characterized in that the lower layer comprises polyester.

6. The strip according to claim 1, further characterized in that the lower layer further comprises a plurality of through holes in alignment with the areas of analysis.

7. The strip according to claim 1, further characterized in that the lower layer has a transparent section located at a predetermined distance from the sample acceptor hole, to ensure the proper size of the sample.

8. The layer according to claim 1, further characterized in that the membrane layer comprises a porous amsotropic membrane which has pores that are larger near the sample side and smaller near the test side.

9. The strip according to claim 8, further characterized in that the biological fluid is whole blood containing red blood cells.

10. The strip according to claim 9, further characterized in that the pore sizes are selected so that the red blood cells of the whole blood sample are trapped in the membrane.

11. The strip according to claim 8, further characterized in that the membrane comprises polyeulfone.

12. The strip according to claim 1, further characterized in that the fluid transfer channel is substantially rectangular.

13. The strip according to claim 1, further characterized in that the first component comprises glucose oxidase.

14. The strip according to claim 1, further characterized in that the second component comprises a peroxidase and a dye or indicator dye, which changes color when oxidized.

15. The strip according to claim 14, further characterized in that the peroxidase is horseradish peroxidase.

16. The strip according to claim 14, further characterized in that the dye or coloring dye indicators are N-sulfonylbenzenesulfonate of C3-met? L-2-benzothiazolone-5-hydrazone] rnonosodium, combined ammonium salt of the acid 8-an? L? No- l-naphthalenes? lfon? co (MBTHSB-ANS).

17. The strip according to claim 1, further characterized in that the third component comprises ascorbic acid.

18. The strip according to claim 1, further characterized in that the reagent further comprises a separating component selected from the group consisting of polyethylene glycol, polyalkyl ether (ethylene vinyl ether), polypropylene glycol, poly styrene sulphonic acid, polyacrylic acid, polyvinyl alcohol and polyvinyl sulphonic acid.

19. The strip according to claim 1, further characterized in that the intermediate layer comprises a thermoplastic sheet.

20. The strip according to claim 1, further characterized in that the intermediate layer comprises polyester.

21. The strip according to claim 1, further characterized in that the bibulous areas and the non-bibulous region comprise non-crushed and crushed regions of the membrane layer, respectively.

22. The strip according to claim 21, further characterized in that the non-collapsed tubular areas are substantially columnar, each with a base in the membrane and, opposite the base, an end joining with the lower layer.

23. The strip according to claim 21, further characterized in that the non-collapsed bibulous areas are substantially colunnar, each with a base in the membrane and, opposite the base, an end that is joined to the upper layer.

24. The strip according to claim 1, further characterized in that it comprises an upper layer that is contiguous with the upper surface of the membrane layer and has through holes that align with the areas of analysis.

25. The strip according to claim 24, further characterized in that the membrane layer is adhered to the upper layer.

26. The strip according to claim 25, further characterized in that the membrane layer is adhered to the upper layer with an adhesive that is restricted to the non-bibulous region of the membrane layer.

27. The strip according to claim 1, further characterized in that it comprises an absorbent layer that contacts the end of the membrane that is closer to the first end of the strip.

28. The strip according to claim 1, further characterized in that it additionally comprises absorbent layers, each of which makes contact with the end of the membrane layer.

29. The strip according to claim 30, further characterized in that the through hole for accepting the sample is near the end of the strip that is remote from the first end.

30. The strip according to claim 1, further characterized in that it comprises a time controlling element comprising an area of analysis that includes, in addition to the reagent, an amount of glucose that causes the area to change color in a predetermined time after the sample is applied to the strip. 31.- A method for measuring an analyte concentration in a biological fluid sample, characterized in that it comprises the steps of: (a) applying the sample to a reactive test strip that comprises: (i) a background layer with a through hole p > to accept the sample; (ii) a membrane layer having a sample side facing the lower layer and comprising a plurality of bibulous analysis areas, each of which changes color - when contacted with the fluid it contains when minus a predetermined amount of analyte, greater than the amount of analyte that causes a color change in the analysis areas that are closest to the first end of the strip; and (iii) dosing means for distributing the sample from the through hole along a predetermined path to each of the analysis areas; and (b) determining the analyte concentration by observing the analysis area which changes color and which is more distant from the first end of the strip.