MXPA98004424A - Diagnostic compositions and devices utilizing same - Google Patents

Abstract

A dry chemistry dye indicator composition provides improved shelf life, stable color indication end point and capability for a system at near normal pH. The novel dry chemistry dye indication system comprises 3-Methyl-6-(sodium sulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S). Preferred dye systems are based on the dye couple (MBTH-S) and 8-anilino-1-naphthalenesulfonate (ANS), and the dye couple MBTH-S and N-(3-sulfopropyl)analine. These dye indicator systems are used in conventional blood chemistry test strips and are particularly preferred for indication of glucose in blood.

Description

COMPOSITIONS AND DEVICES FOR DIAGNOSIS DESCRIPTION OF THE INVENTION The present invention relates to dye compositions used for the colorimetric determination of a chemical or biochemical compound (analyte) in an aqueous body fluid, such as blood. In particular, the present invention relates to the field of dry reagent compositions for use in test strips adapted to receive a liquid sample of body fluid wherein the dye indicator composition wetted by the fluid reacts with the analyte and provides a visual indication or color of the presence or concentration of the analyte. Compositions of indicators for use in devices for color indication of various analytes in various body fluids are well known in the art and are exemplified in numerous commercial products. Typically, a dye composition or an assembled dye composition is formulated into a solution which is applied to a test strip matrix then dried to form a dry chemical system on the test strip. The dry chemical system commonly involves an oxidizable dye or a dye assembly in combination with an oxidase or peroxidase specific for the analyte to be tested. The analyte reacts with the corresponding oxidase or peroxidase producing hydrogen peroxide which in turn oxidizes the dye or dye assembly to produce the desired color change for the indication of the presence or concentration of the analyte. Examples of such dyeing and dye assembly systems are described by Phillips et al. In U.S. Patent No. 4,734,360; and No. 5,304,468; by Yu in U.S. Patent No. 5,453,360; Hoens in U.S. Patent No. 5,334,508 and by Hochstrasser in U.S. Patent No. 3,964,871 and No. 4,059,407. The descriptions of the above patents are incorporated herein by reference. A number of indicator systems are incorporated in the above references in various commercial products. While the products generally provide acceptable indication and testing under the same conditions, there are certain problems with the previous dyeing systems. For example, in some dye systems the existing chemical dye system on the test strip will allow the dye compounds to be sublimated from the test strip in such a way that when the consumer uses the test strip it can not provide an indication adequate This limits the half-life of the test strips. In some dye systems the color change provided by the dye system continues to change over time rather than reaching a stable end point. In those dye systems, the color indication must be read exactly at specific time intervals in order to obtain an accurate indication of the presence or concentration of the analyte. Some dye systems require a low pH to provide the necessary stability of the dye system. If the pH is below the desired pH level for stability of the enzymes used in the dye system, the amount of enzymes used in the formulation must be increased. In such a case this may cause the enzymes to produce unwanted indications due to the instability of the enzymes at the low pH required for the dye system. In view of the foregoing it is an object of this invention to provide an indicator dye system with improved stability to avoid sublimation of the dye system from the dry chemical test strips. It is an object of this invention to provide a dyeing system which will rapidly produce a stable end point whereby a final color indication is produced in a short period of time thereby eliminating the time-dependent or user-determined measurement. It is a further object of this invention to provide a dye system which can be formulated and used at a more normal pH to provide a more stable system for the enzymes present during the manufacture or use of the dye system.

The above objects as well as others are achieved by the compositions and systems of this invention as described herein. In one aspect this invention provides a dye composition comprising 3-methyl-6 (M-sulfonate) -benzothiazoline- (2) -hydrazone (MBTH-S) where M is a positive ion that provides a stable sulfonated salt and an enzyme oxidase or a peroxidase enzyme. This dye composition is formulated in a dry chemical indicator system including other dye compounds to form a conventional dye and binder assembly system, chelating agents, buffers and the like, as is known in the art. The MBTH-S in the composition is used in the form of a stable sulfonated salt, of which the sodium salt is preferred, but the sulfonated potassium, ammonium or other ionic salt form can be used. In a preferred aspect of this invention MBTH-S is used with another dye compound to form a dye assembly for an improved indication and a desired pH range in the range of about 6. In another aspect this invention provides a device for testing a fluid for the presence or concentration of an analyte comprising a support for the dye composition wherein the dye composition comprises MBTH-S and an enzyme oxidase or enzyme peroxidase as summarized above. The various mechanical configurations of such devices in the art are known and various configurations can be used incorporating the dye system with MBTH-S of this invention adapted as desired to carry out a particular test. In another aspect this invention provides a method for testing a fluid for the presence or concentration of an analyte comprising contacting a fluid sample with a dry chemical system comprising MBTH-S and an enzyme oxidase or a peroxidase enzyme as summarized above. . BRIEF DESCRIPTION OF THE INVENTION Figure 1 shows a comparison of the reflectance at certain wavelengths of the dye systems based on (MBTH-S) -ANS and based on MBTH-DMAB. Figure 2 is an illustration of the relative general activity of enzymes over a range of pH. Dry chemical indicator systems for use in test strips such as for testing blood glucose are well known, as illustrated by the prior art patents referenced above in the background section of this specification. Therefore, this description is directed to a person skilled in the art who has knowledge of how to formulate a dye composition or dye assembly in a dry chemical reagent system on a test strip. The test strip is typically in the form of an absorbent matrix to contain the dry chemical reagent indication system and to receive the fluid sample to react with the dry chemical reagent indicator system. This invention provides an improved dye indicator system based on the use of 3-methyl-6- (M-sulfonate) -benzothiazoline- (2) -hydrazone (MBTH-S), wherein M is a positive ion that provides a salt stable sulfonated, preferably a sodium, potassium, ammonium or other equivalent ion. Dry chemical reagent systems formulated based on the MBTH-S provide a dry chemical system on the test strips which are resistant to dye sublimation in the dry chemical system and thus provide long life and improved reliability of the test strips that contain the MBTH-S dye system. In addition, the dry chemical reactive dye systems formulated based on the MBTH-S of this invention can be formulated and buffered to operate in a pH range of about 6, which provides additional stability of the oxidases enzymes and peroxidase enzymes present in the dye indicator system. In addition, the dry chemical reactive dye systems formulated based on the MBTH-S of this invention also provide a stable colored reaction endpoint which is reached in a short period of time after applying the fluid sample. This allows the user to read and interpret the color indication if accurate timing dependence or take readings at specific time intervals, which usually requires the use of an electronic meter for accurate measurement and synchronization. This stability of the end point of the dye system of this invention allows the use also of the test strip as at least in semi-permanent recording of the test results. The dye systems of this invention are useful in devices and systems of the prior art including those involving the reaction of whole blood or other unfiltered fluid with the dry chemical reactive dye system. In such devices and systems the presence of whole blood color obscures the visual inspection of the color change of the indicator, but these systems can be read and measured by reflectance at certain specific wavelengths by an appropriate electronic measurement system. Dye systems according to this invention are particularly useful in devices and systems that separate blood solids such as red blood cells from blood fluids and allow them to make contact and react clear blood fluids with the dry chemical reactive dye system , thus providing a visually readable color change, not obscured. In particular, the dye system of the present invention is useful in the devices and systems described in the Copending Request Serial No. 08 / 628,489 filed on the same date of this application, the description of which is incorporated herein by reference. In general, the person skilled in the art will recognize that the dye system with MBTH-S of this invention can be formulated and implemented in many of the systems of dyes previously based on MBTH by making the appropriate adjustment in the buffer and other components to accommodate the different pH range and other properties of the MBTH-S compared with the conventional MBTH. The dry chemical reactive dye system of this invention is formulated to allow the analyte to react with a specific oxidase enzyme to produce hydrogen peroxide which reacts with the indicator system with MBTH-S according to this invention to produce a color change which is read visually or measured electronically with a meter. The enzyme oxidase may be selected from glucose oxidase, cholesterol oxidase, uricase, alcohol oxidase, aldehyde oxidase, glycerophosphate oxidase or other similar oxidases enzymes known in the art to react with the particular analyte. The system may also include the presence of a peroxidase enzyme such as horseradish peroxidase or other known peroxidase to produce or increase the desired color change in the indicator. The indicator reactive dye system is formulated in a solution and is typically impregnated in a porous matrix or membrane such as a polyethersulfone membrane available from Gelman Science, Ann Arbor, Michigan, or a glass fiber matrix available from AhlstromFiltration, Inc. , Chattanooga, Tenn. , and dried to provide a dry chemical system useful in conventional test strips. The MBTH-S provided by this invention can be formulated in the dry chemical reactive dye system alone or in combination with other dye components to form dye assembly systems which preferably include 3, 3-dimethylaminobenzoic acid (DMAB), acid 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCBS), 8-anilino-naphthalenesulfonate (ANS), ON- (3-sulfopropyl) aniline. Other dye compounds can be used which provide a sufficiently high extinction point (approximately 7.0 or higher is preferred) and which form an appropriate dye assembly with MBTH-S in the dye system of this invention. The formulation thereof will be apparent to a person skilled in the art following the teachings herein. Reference is made to the reagents catalog of Doj indo Laboratories, Tokyo, Japan, and to a document entitled "Reagents Used for Detection Substances in Biological Matrix by Enzymatic Methods" published by Doj indo Laboratorires in 1995 Pacific Rim Conference, for dyes of appropriate properties for use in this invention. Using MBTH-S and ANS can be used a dye assembly which exists at a pH of 6 which allows the dry chemical dye assembly system to be used at this higher pH. It has been found that the formulation of MBTH-S and N- (3-sulfopropyl) aniline is another preferred embodiment for the indicator dye system in the devices and methods of this invention. This creates a stable end-point chemical which is soluble in water and not sublime during the time when it is applied and dried in the membrane matrix. The MBTH-S coupled with ANS provides a flat spectral absorption in the region of about 580 to 650 nm. The MBTH-S coupled with ANS provides good spectral absorption, is soluble in water and does not sublimate under dry chemical storage conditions. It will be apparent to a person skilled in the art that the selection of the additional dye components to be combined with the MBTH-S or providing the dye assembly systems with MBTH-S will depend on the analyte to be detected, the conditions under which the which the test strip will be stored and used and other conventional considerations . However, it has been found that the dye assembly formed from the combination of MBTH-S and ANS provides a preferred dye assembly system particularly for formulation with glucose oxidase for glucose detection and measurement in blood fluids. The preparation of 3-methyl-6- (sulfonate of M) -benzothiazoline- (2) -hydrazone (MBTH-S) for a person skilled in the art will be apparent as the sulfonation of MBTH. The preferred MBTH-S is where M is Na. In this example, the sulfonated sodium salt is preferred and is prepared as follows. 85 grams of 3-methyl-benzothiazolinone- (2) -hydrazone (MBTH) are dissolved in 750 grams of oil placed in an ice bath in such a way that the temperature is not allowed to exceed 30 ° C. After standing at room temperature for 12 hours, complete dissolution is obtained. The solution is emptied into 8 liters of water at 0 ° C containing excess ice to maintain the temperature at 0 ° C. After standing for approximately 8 hours, the precipitate of free sulphonic acid is filtered off, wash and dry. Approximately 85 grams of unpurified product are obtained, which is purified by repeated extractions with boiling methanol. The resulting solid is washed with water and dissolved in an equimolar amount of 3N NaOH with heating. This solution is filtered on activated carbon and treated with a double volume of dioxane to then allow recrystallization overnight at about 5 ° C. The product is then subjected to repeated recrystallizations from 10% Na acetate with recurrent activated carbon filtration until the material is almost white. The final water material is recrystallized and washed with 70% dioxane, then with pure dioxane, then with ether. The resulting crystals of 3-methyl-6- (sodium sulfonate) -benzothiazoline- (2) -hydrazone are air-dried. A dry chemical reagent indicator is formulated as follows: Reagent lb 20 ml water 420 mg citric acid (a buffering agent). The pH of the citric acid solution is adjusted with NaOH to a value of 4.25. 16.7 mg of EDTA 90 mg of Gantrez S95 available from GAF 250 mg of Crotein SPA 20,500 units of glucose oxidase 16,200 units of peroxidase Reagent 2b 10 ml of a mixture of 3 parts by volume of water and 7 parts by volume of isopropyl alcohol 13 mg of MBTH-S 40 mg of ANS A piece of polyethersulfone membrane from Gelman Science is coated uniformly with reagent lb, the excess is removed and the membrane is dried. The membrane is then coated with reagent 2b in the same form and dried. The membrane is then assembled in a test device as shown in Figure 2 of the Copending Request Serial No. 08 / 628,489 referred to above. Whole blood is applied to the test area and the glucose level is read by visual inspection of the color indication on the test side of the device. Color changes from light to purple to blue and final color end-point shapes from clear in about 45 seconds are reached and the color of the end point is calibrated for known concentrations of glucose. Figure 21 shows the comparative spectral reflection in the range of 520-720 nm for the dye system of the present invention based on the MBTH-S-ANS dye assembly compared to the MBTH-DMAB dye assembly, as described in U.S. Patent De Kiser No. 5,306,623. Figure 2 is a general illustration of the peroxidase activity of enzymes in a pH range, as it can be observed that it is preferred to have systems which can operate close to the pH between 6 and 7. The dyeing system according to the present invention it is stable at a buffered pH of about 6 thus allowing the dye system of the invention to operate in a more favorable pH range for the stability and activity of the oxidases enzymes and peroxidase enzymes present in the dye indicator system. It will be apparent to a person skilled in the art that the MBTH-S dye indicator can be formulated in various systems with various oxidase and peroxidase materials to provide desired indication of several analytes. It will also be apparent that the systems can be formulated at buffered pH levels which provide a favorable environment for stability of the enzymatic components in the dry chemical reagent system and in the fluid to be analyzed.

Claims (8)

1. CLAIMS 1. A device for testing the presence or concentration of an analyte in a fluid sample characterized in that it comprises: A support member, A composition placed in or impregnated in the support, the composition comprising a composition for a dry chemical reagent indicator comprising 3-methyl-6- (M-sulfonate) -benzothiazoline- (2) -hydrazone, wherein M is a positively charged ion that provides a stable aqueous salt thereof, a second dye component selected from the group consisting of 3, 3-dimethylaminobenzoic acid, 3,5-dichloro-2-hydroxybenzenesulfonic acid, 8-amino-1-naphthalenesulfonate and N- (3-sulfopropyl) aniline and an enzyme oxidase or a peroxidase enzyme; Wherein the support member is adapted to receive a fluid sample in such a way that the fluid sample makes contact with the composition and is adapted in such a way that the support provides the inspection or reading of the color change produced by the composition after making contact with the fluid sample.
2. 2. The device according to claim 1 characterized in that M is a sodium, potassium or ammonium ion.
3. 3. The device according to claim 1, characterized in that the second dye component is 8-anilino-1-naphthalene sulfonate.
4. 4. The device according to claim 1, characterized in that the second dye component is N- (3-sulfopropyl) aniline.
5. 5. A method for testing a fluid for the presence or concentration of an analyte characterized in that it comprises: Applying a fluid sample to a support member having thereon or impregnated therein a composition comprising a composition for an indicator of dry chemical reagent comprises 3-methyl-6- (M-sulfonate) -benzthiazoline- (2) -hydrazone, wherein M is a positively charged ion that provides a stable aqueous salt thereof, a second dye component selected from the a group consisting of 3, 3-dimethylaminobenzoic acid, 3,5-dichloro-2-hydroxybenzenesulfonic acid, 8-anilino-1-naphthalenesulfonate and N- (3-sulfopropyl) aniline and an oxidase enzyme or a peroxidase enzyme; Wherein the support member is adapted to receive a fluid sample in such a way that the fluid sample makes contact with the composition and is adapted in such a way that the support provides the inspection or reading of the color change produced by the composition after making contact with the fluid sample; and Read or measure the color indication provided by the composition after making contact with the fluid sample 6.
6. The method according to claim 5 characterized in that M is a sodium, potassium or ammonium ion.
7. The method according to claim 5 characterized in that the second dye component is 8-anilino-1-naphthalenesulfonate.
8. 8. The method according to claim 5 characterized in that the second dye component is N- (3-sulfopropyl) aniline.