MXPA97001795A - Coloring copuard for anali spectrophotometric determination - Google Patents

Abstract

The present invention relates to a coupled dye compound for use in a test de that contains a reagent system for detecting the presence of an amount of an analyte in a sample. The reagent system comprises one or more enzymes which, in the presence of the analyte, produce an oxidizing agent in amounts indicative of the amount of analyte in the sample, the compound of choice being meta-methyl-2-benzothiazolinone-hydrozone] N-sulfonylbenzenesulfonate -monosod

Description

COLORONTE COPUlDOR Pft Q SPECTRROFOTOflETRICAL DETERMINATION PE ñNLITQS CflflPQ TÉCNICO The present invention relates to a device and a test method for the color determination of chemical compounds and chemistries (analytes) in aqueous fluids, such as whole blood and, more particularly, to a pair of colorants for use on said device. and everything.

Lñ TECHNICAL PE BACKGROUND The quantification of chemical and biochemical components in colored aqueous fluids, in particular colored biological fluids, such as whole blood and urine and in biological fluid derivatives, such as serum and plasma, are of increasing importance. There are important applications in medical diagnosis and treatment and in the quantification of exposure to therapeutic drugs, intoxicants, dangerous chemical substances and the like. In some cases, the quantities of ina + enals to be determined are + an lowercase (on a scale of 1 μg or less per deciliter) or so difficult to determine with precision that the apparatus used is complicated and only useful. for expert laboratory personnel. In that case, the results are usually not available for a few hours or days after the sample is taken. In other cases, there is often an emphasis on the ability of lay operators to routinely perform the test quickly and reproducibly outside a laboratory facility, with rapid and immediate display of information. A common medical test is the measurement of blood glucose levels by diabetics. Current teaching advises diabetic patients to measure their blood glucose level two to seven times a day, depending on the nature and severity of their individual cases. Based on the pattern observed in the measured glucose levels, the patient and the doctor together make adjustments in diet, exercise and ingestion of insulin for the best management of the disease. Clearly this information must be available immediately to the patient. Many blood glucose test methods and test items have been known in the art; They all suffer from a variety of limitations. A great improvement is described and claimed in U.S. Patent Nos. 4,935,345; 5,049,487; 5,059,394 and 5,179,005 of R. Phillips and co-inventors, and that the same successor as the present application was assigned. The method described and claimed in these patents involves reading a reflective reader from an inert porous matrix impregnated with a reagent that will interact with the analyte to produce a light absorbing reaction product, when the fluid being analyzed is applied to the analyte. another surface and rotate through the matrix through the surface that is being read. The reagent includes glucose oxidase, an enzyme that consumes glucose in the sample to produce hydrogen peroxide which, in the presence of another enzyme, horseradish peroxidase, oxidizes a dye pair comprising 3-rnet? L-2-benzot hydrochloride azolmone hydrazone (M1T?) and acid 3-d? met? larn? nobenzo? co (DMPIB) to produce a blue tint. The refle measurement is carried out at two separate wavelengths. The concentration of glucose in the blood is determined based on the intensity of the color of the dye, with the help of a LED spectrototrórnet ro. In the co-pending U.S. patent application, serial number 245,940, filed May 19, 1994 (LF 30), a dye pair comprising 3-met? 1-2-benzoth-azolene hydrazone in free form or in the form of acid (MBTH) and 1-naphthalenesulphonate 1-an? Lmo in the form of acid or in salt form (RN5) to be used in place of the coloring BTH-DMAB, such as described above. The MBTH-RNS dye pair is less soluble by oxidation and, therefore, provides a stable end point with minimal colorant dimming, in comparison with the oxidized MBTH-DriRB colorant. While these prior systems have actually been employed to produce test devices useful for determining the presence or amount of glucose, several drawbacks have been noted. Test devices in which several pairs are employed are designed for both domestic and institutional use and are thus sold by manufacturers and distributors in the hope that they remain in the user's inventory for a substantial period of time. time and, of course, must remain effective during that period of time. This need for a substantial shelf life has caused difficulties in formulating products that employ MBTH as one of the components of a dye pair. First, it has been found that MBTH stability decreases with increasing temperature and alkalinity. The free acid form of MBTH is very susceptible and tends to sublimate. In an attempt to account for this, a preterm form is ol acid hydrate of MBTH, for example, the hydrochloride of 3-rnet? ] -2-benzot? Azol? Nona hydrazone. Unfortunately this hydrate itself is unstable as the temperature increases and it dissociates easily to the free acid MBTH and HC1 when heated. In addition to having low stability at a high pH, the efficiency of MBTH to react oxidant with its counterpart of the pair, greatly decreases with increasing alkalinity, so that at a high pH there is essentially little or no color from the pair Colorant.

In view of these relationships, in practice, the MBTH should be used in large excesses and at a low pH to minimize the effects of instability and inadequacy. Ideally, a pH of less than 2.0 would be preferred from the point of view of the sublimation ba a and the high efficiency of the compound. Unfortunately, for the systems that are being considered in the present, said ideal low pH can not be employed. As described above, the reactive systems used depend on enzymes that act on the substrate analyte and generate oxidation agents in amounts indicative of the amounts of the analyte present in the sample being tested. The low pH, which would be ideal with respect to the MBTH reagent, is totally inadequate for such enzymes as, for example, glucose oxidase and horseradish peroxidase, said low pH, many of the commercially available enzymes have little or no activity. Consequently, the technique has been forced to consider and select a moderate pH, for example 4 and a large excess of reagents to guarantee the efficiency of its test devices, for the required storage life.

BRFVF DESCRIPTION OF THE INVENTION In accordance with the teachings of this invention, a highly stable component of a color-ante pair is provided in a test device containing enzymes. The component, in contrast to those employed in the above test devices, is capable of oxidatively coupling efficiently with a wide variety of coupling counterparts at relatively high pH conditions compatible with the high efficiency of the enzymes. Specifically, the dye couple compound of this invention is to be used in a test device that contains a reactive system for detecting the presence or amount of an analyte in a sample, wherein the reactive system comprises one or more enzymes that, in the presence of the analyte, they produce an oxidizing agent in amounts indicative of the amount of the analyte present in the sample. In accordance with the teachings herein, the reactive system further comprises a dye pair capable of forming a chromium forum when oxidized by the oxidizing agent produced; and the dye pair comprises the compound: wherein R is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heterocyclic, quaternary amine or organic acid portions; and Y is selected from the group consisting of NOs, S03-, H, halide, alkyl or 5? Za, wherein Z is alkyl or aryl. Preferably, Y is H. In a preferred embodiment, R is: wherein any of R, Rs "and Ra is independently selected from the group consisting of H, alkyl, aplo, silyl, halide, hydroxide, ercaptide, alkoxide, thioalkoxide, amine, sulfonate or carboxylate; and X is selected from the group consisting of amine, sulfonate or carboxylate. In a specific embodiment, the test device is provided to determine the presence or amount of analytes such as glucose, cholesterol, alcohol, unco acid, formaldehyde or glycerol 3-phosphate, all blood analytes, commonly measured. In such cases, the enzyme system comprises enzymes selected from the group consisting of glucose oxidase, cholesterol oxidase, alcohol oxidase, upcase, aldehyde oxidase and glycerophosphate oxidase; together with peroxidase or an inorganic complex that has peroxidase-like activity, for example, atma, hernin and tetrakis-csulf- "ofeml3-orf? r? na-rnanganeso." A selection peroxidase is horseradish peroxidase.

BREVF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of an embodiment of a test device containing a reaction pad to which the liquid sample being analyzed is applied; and Figure 2 is a perspective view of a second embodiment of the test device of Figure 1.

DISCUSSION PETRLLRPñ PE Lñ INVENCIÓN As described above, the invention involves an improved dye compound for use in a test device for determining the presence or amount of an analyte in a liquid sample. With reference to Figure 1, in a preferred embodiment of this invention, the test device comprises a porous matrix 10 having a chemical reagent system incorporated therein and which is adhered to a support 12. It is provided with an opening 16 through the support, by which a liquid sample can be applied to a sample receiving surface 17 of the matrix 10. The chemical system is provided to react with any analyte present in the liquid sample and as a result of which a surface of test 19 of the matrix with a manifest luminous reflection, indicating the amount of analyte present in the liquid sample. The test surface can be read at a glance, but is preferably read by using a photometpco spectrum matrix device. The elements of said devices are shown schematically in FIG. 1 and comprise a light source 18, such as a light emitting diode for preferably directing a light of uniform wavelength on the test surface 19. The light detector 20 for detecting light reflected from surface 19 and producing a signal 22 indicating the amount of light detected, which signal can be processed, for example, by a microprocessor incorporated in the reading device? ar-to calculate the amount of analyte present in the sample. Systems such as that described above are known in the art and are well described in U.S. Patent Nos. 4,935,345; 5,049,487; 5,059,394 and 5,179,005. Said systems contemplate that these test devices will be inserted into the reader section and then a sample, for example blood, will be applied to the sample receiving surface 17. Figure 2 represents an alternative to this, where the blood is first applied to the sample receiving surface 7 and only then is the test surface 19 presented to the display for reading. In all other senses, the elements numbered in Figure 2 are identical to those in Figure 1. The reflective properties of the test surface vary with the amount of analyte present in the sample by the operation of a series of reactions. between the analyte present in the liquid sample and the chemical reagents present in the porous matrix. In particular, the matrix includes one or more enzymes which, together with the analyte substitute, result in the production of hydrogen peroxide or other strong oxidizing agents. A colorant is included in the matrix, ie, two compounds that are capable of being oxidized to form a chromophore that absorbs light at a specific wavelength, in proportion to the amount of chromophore present. The oxidizing agent formed by the enzyme-catalyzed reaction then reacts with the dye sample to produce the chromophore. The selection of the enzymes, the resulting oxidizing agent and the selection of the coloring pair vary widely in the art and, to a large extent, are a function of the analyte that is being determined. For example, in the case of the determination of cholesterol as in a blood sample, an enzyme oxidase, such as cholesterol oxidase can be employed. Similarly, alcohol oxidase can be used in the determination of methanol or ethanol; formaldehyde determinations may employ aldehyde-oxi dasa; or glycerol 3-phosphate determinations may employ glycerophosphate oxidase. The hydrogen peroxide product of these enzyme-catalyzed reactions can be further modified by a subsequent reaction catalyzed by an enzyme to produce a reactive oxidizing agent to react with the dye pair to form the chronophore. physiological, for example, the reaction of hydrogen peroxide to form an active oxidant reagent can be catalyzed by the horseradish peroxidase enzyme. Accordingly, while it will be understood that the teachings of this invention are broadly applicable, for the purpose of the discussion that follows, the analyte will be exemplified by glucose in a liquid sample of whole blood. The preferred chemical system will then be exemplified by the enzyme glucose oxidase which acts on the glucose substrate to form hydrogen peroxide. The hydrogen peroxide, in turn, is converted to the active oxidant reagent by reaction with another enzyme, horseradish peroxidase. Heretofore, the dye pair widely used in a glucose test of the type described above, was the combination of 3 ™ rnethyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH hydrochloride hydrate) (formula I), together with dirnetyl-n-benzene (formula II). These compounds undergo the following oxidation reaction to form a blue chromophore (formula III): CO] = hydrogen peroxide / horseradish peroxidase As described above, this system suffers from several disadvantages. MBTH, even in the form of hydrochloride hydrate, is relatively unstable under the action of heat and alkalinity. Additionally, the above reaction is very inefficient under strongly acidic conditions, for example, pH 2 or less. Unfortunately, at those conditions the enzymes employed in the test devices, for example, glucose oxidase and horseradish peroxidase, have little or no activity. Accordingly, commercial practice has dictated that, in order to obtain a relatively stable system, an optimum pH be used, for example, about 4, and large amounts of enzymes and the coloring pair are used to constitute the decreased activity of the enzymes. and the reduced efficiency of the oxidation of the coupling reaction. In accordance with this invention, it has now been discovered that modified forms of MBTH can be provided that solve the stability problem hitherto encountered and are also efficiently reactive in an environment that further leads to the activity of the enzymes employed in the test employed here as, for example, at pH values ranging from 4 to 7, approximately. Some preferred derivatives, moreover, have been found to be highly reactive for the suitable coupling partners, the aromatic amines. The derivatives of the invention have the general structure indicated in formula IV that follows: wherein R is selected from the group consisting of the alkyl, substituted alkyl, aplo, substituted aryl, heterocyclic, quaternary amine or organic acid moieties; and Y is selected from the group consisting of NOs ", S03-, H, halide, alkyl or S? Za, wherein Z is alkyl or ryl. Preferably Y is H. In a preferred embodiment R is: wherein any of R, 2 and Ra is independently selected from the group consisting of H, alkyl, aryl, silyl, halide, hydroxide, cyclopentadide, alkoxide, thioalkoxide, amine, sulfonate or carboxylate; and X is selected from the group consisting of amine, sulfonate or carboxylate. The MBTH derivatives of this invention can be subjected to an oxidation reaction with a broad scale of dye pair partners, such as aromatic amines, phenols and substituted phenols. In addition, said reactions can proceed efficiently at room temperature, at pH values that can vary from 4 to 11. In the preferred form of the derivatives of this invention, the oxidation reaction is optimal at approximate pH values. from 4 to 7 and, therefore, is particularly useful in conjunction with the amine dye couple partners of interest in diagnostic chemistry, such as 3-d-r-ilanoinobenzoic acid and 1-naphthalensulphonates of 8-an ? l? no. In contrast to MBTH, either in the form of free acid or in the form of the acid hydrate, these derivatives are remarkably stable even when heated at 10 ° C for up to 1 (5 hours. oxidation, enzymes that catalyze peroxide, such as horseradish peroxide, are especially effective to carry out the coupling reaction by oxidation.

E3E11PLQ 1 SYNTHESIS BEL PERIVRPO PE? BTH SYNTHESIS OF N-SULFONILBENCENSU FONRTO MONOSODIC C21 OF METAC3-METHYL-2-ENZOTITIZED INONfl HI RPZON 1 REACTION SCHEME Material 3-Rhenethyl-2-benzothiazolinone hydrochloride (MBTH-HC1), Nal, hydroxide and tetrabutylammonium, methylene chloride and n-methyl-2-? -rolidolone were purchased from Rldrich Company of Milwau ee, Wisconsin, and are used without purification. Tpeti lamina is obtained from Baker Chemicals and distributed by Baxter-Company of Phillipsburg, New Jersey. Benzene 1,3-disulfonylchloride was purchased from Fluka Chemical, from Ronkonkona, New York or Lancaster Chemical from Umdharn, New Hanpshire.

Synthesis of (1) A sample of 4 g of MBTH-HC1 was loaded into an Erlen eyer 150-ml flask., equipped with magnetic stirring bar, and added 50 rnl of l-rnet? i ~? -p? rrol? dona and 5 ml of triethylanine. The flask was covered with a rubber septum and placed on a hot plate with magnetic stirring. The mixture was heated at 60-70 ° C while stirring vigorously for 0.5 hours, which produced a yellow suspension. The flask was placed in an ice bath to cool. A 5 g sample of 1,3-d-sulphonyl benzene fomlchloride was added to a 250 ml Erlenmeyer flask equipped with a magnetic stir bar. The flask was lowered onto an ice bath and 20 ml of n-met l-2-pyrrolidone was added. The mixture was stirred until all the solids had dissolved (approximately 15 minutes). The free base suspension of MBTH, which was previously obtained, was decanted over the solution. The resulting light yellow mixture was reacted at an ice bath temperature for 1.5 hours. After that time the reaction was quenched with 10 ml of 2 N HCl and stirred for another 30 minutes at room temperature. 50 ng of Nortí (activated carbon pellets) of 12 meshes were introduced into the solution, which produced a light yellow solution after 10 minutes of stirring. It was filtered through a fine grade fry with the help of a vacuum cleaner. A uniform yellow to light brown solution was obtained. 300 ml of 2 N HCl was added to the yellow solution which was stirred, resulting in the precipitation of a white matte powder. The solid was collected by vacuum filtration and the solid was washed three times with 25 ml of deionized water. Upon drying at 110 ° C under vacuum for 2 hours, 5.6 g of the white matte product was obtained. The product was analyzed with NMR with H and HPLC, yielding a purity of 97%. The compound (1) is not very soluble in most common organic solvents or in water. However, it is soluble in the basic solution and in polar solvents, such as DMSO, NMP and DMF.

Synthesis of (2) 2.0 g of crude oil sample (1) was suspended in 50 ml of methylene chloride. 4 ml of 1 M tetrabutyranonium hydroxide was slowly added over the course of 2 minutes to the suspension under stirring; which produced a light yellow solution. The solution was washed with 10 ml of deionized water and dried over anhydrous sodium sulfate. The sulfate was removed by gravity filtration and the resulting mixture was evaporated to dryness in a rotary evaporator. The thick yellow oil was collected. The oil was collected with 13 125 ml of acetone and 10 ml of 20% Nal in acetone was added over the course of 5 minutes. A white precipitate was evident. The mixture was allowed to react for another 20 minutes and the precipitate was collected by vacuum filtration with a fine grade fritter. The resulting white-matt solid was washed three times with 20 ml of acetone. On drying at 110 ° C for 45 minutes 1.3 g (65%) of the desired product was obtained. The compound (2) is very soluble in water and in a water-alcohol mixture. The solid is stable in air and light, but its solution slowly decomposes to a turbid, light yellow mixture when exposed to light for extended periods of time.

EXAMPLE 2 PREPRENCIATION PE A PISPQSITIVQ PE PROOF A strip of polymer membrane (reaction matrix) is immersed in an aqueous immersion of table 1 until it becomes saturated. It is removed from the immersion and the excess reagent is squeezed with a glass rod. The strip is then hung inside a circulating air oven at 56 ° C for 5 to 10 minutes to dry, and after that time the strip is removed and immersed in the organic immersion described in table .1 until It is saturated. Again dry as in the previous step. The resulting strip is shaped as desired, to perform the test, OUADRO i FORMULATION OF REAGENTS Aqueous immersion Organic immersion (adjust pH to 4.25 with NaOH) Rgua 20 mi Rgua 3 mi Citric acid 420 my Ethyl diamine monoalonate alcohol 16.7 mg N-sulfonylbenzene 10-tetraacetic acid (EDTR) monosodium monohydrogen (2) meta-L "3-rnethyl-2-benzothiazolinone hydrazone] Gantrez S 5 90 rng flNS 10 - (obtainable from GRF, 100 mg New York, New York) Crotein SPA 250 ng (obtainable from Croda Co. New York, New York) Glucose-oxid sa 20,500 units Horseradish peroxidase 16,200 spicy units EXAMPLE 3 ETERMINATION PE 6LUCQSR A blood sample containing glucose is applied to the surface of the strip impregnated with the reagents.

The sample inside the matrix is immediately absorbed and a blue color is apparent. The intensity of color increases with time and is proportional to the concentration of the analyte. Based on the color intensity, the glucose concentration is determined by comparing with a standard calibration curve. Similarly, an aqueous solution of hydrogen peroxide (organic peroxides, ferric and quinone) also produces the desired blue color in the strip impregnated with reagent. The concentration of the analyte can be determined by the same means as above. The invention having been fully described, it will be evident to the person skilled in the art that any modification and change can be made therein without departing from the spirit and scope of the invention.

Claims (9)

NQVEPRP PE LR INVENTION CLAIMS

1. - In a test device containing a reactive system for determining the presence or quantity of an analyte in a sample, wherein the reactive system comprises enzymes to produce an oxidizing agent in amounts that indicate the amounts of the analyte present in the sample; the improvement characterized in that: the r-active system comprises a dye pair that forms a chromophore when oxidized by said oxidizing agent; the dye pair comprising a compound having the formula: wherein R is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heterocyclic, quaternary amine or organic acid portions; and Y is selected from the group consisting of NOa, SOa-, H, halogenide, alkyl or S? Za, wherein Z is alkyl or an ilo.

2. The test device according to claim 1, further characterized in that R is: wherein any of R, R "and Ra are independently selected from the group consisting of H, alkyl, aryl, silyl, halide, hydroxide, mercaptide, alkoxide, thioalkoxide, amine, sulfonate or carboxylate; and X is selected from the group consisting of amine, sulfonate or carboxylate.

3. The test device according to claim 2, further characterized in that Y is H.

4. The test device according to claim 1, further characterized in that the enzymes are selected from the group consisting of glucose oxidase. , cholesterol oxidase, alcohol -oxidase, uricase, aldehyde oxidase and glycerophosphate -oxidase.

5. The test device according to claim 4, further characterized in that the enzymes additionally comprise peroxidase or an inorganic complex having properties similar to peroxidase.

6. The test device according to claim 4, further characterized in that the enzymes comprise gl? Seoxidase and horseradish peroxidase.

7. The test device according to claim 1, further characterized in that the dye pair further comprises 3-dirnethylaminobenzoic acid.

8. The test device according to claim 1, further characterized in that the dye pair additionally comprises 8-anilino l-naphthalene sulfonate.

9. The test device according to claim 1, further characterized in that the compound is monosodium N-sulfonylbenzenesulfonate of metaC3-methyl-2-benzothiazolinone hydrazone.