Composition for diagnosing glucose
US 3123443 A
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United States Patent 3,123,443 CGMPOSKTION FOR DHAGNGSING GLUCOSE Robert R. Srnehy, Cleveland, Ohio, assignor to Miles Lahoratories, EH2. Ellrhart, Ind, a corporation of Indiana No Drawing. Filed Apr. 18, 1960, Ser. No. 22,662 9 Claims. (til. 23-253) This invention relates to new and improved diagnostic compositions. Particularly the invention relates to diagnostic compositions which are useful for the quantitative determination of glucose in fluids, particularly body fluids such as urine, plasma, blood, and the like.
The detection of glucose in body fluids as well as the determination of its concentration therein is of great importance for diabetic patients who must control their diets so as to regulate their sugar intake and who must frequently be guided in this regard by a regular check on glucose in body fluids. But beyond its usefulness in regular testing no known diabetics by both patients and phys1- cians, this glucose indicator may also be used efficiently in routine glucose analyses of body fluids in hospitals and physicians oifices in diabetes detection screening programs, in the differentiation of glucose from other sugars, and the like.
Because early diagnosis and continued control are so important in diabetes, a sugar test, to be of greatest value, must be conveniently rapid, simple enough for the technician to learn with ease, accurate enough to serve the clinician, and sensitive enough to reflect variations in the patients condition. Moreover, the reagent composition must be adequately stable.
Procedures for the detection of sugar in various body fluids are well known in clinical chemistry. One such procedure utilizes Benedicts copper reduction test, another employs a self-heating alkaline copper reduction test in tablet form (US. Patent No. 2,387,244), still another involves a test which depends on the action of a double enzyme system (U.S. application No. 514,395, filed June 9, 1955, now abandoned, by Alf ed H. Free and assigned to the assignee of the present invention). These tests have greatly contributed to the art of diagnosing body fluids for glucose.
I have now found a novel and highly useful glucosedetecting means which represents an important improvement in the area of determining glucose in various materials including body fiuids, such as urine. This technique utilizes a diagnostic composition preferably impregnated on a paper strip, and more particularly a composition which in addition includes ingredients which result in the production of a diagnostic composition which is more stable than any heretofore prepared. Until the present improvement, paper strips which were to serve as carriers for the active ingredients had to be dipped into the solution within a relatively short time after the solution was made up in order to avoid impairment or even total loss in the glucose-detecting ability of the strips occasioned by the instability of the compositions as heretofore prepared. Such solutions as heretofore prepared, if left standing for even a short period lost much of their glucosedetecting ability.
Specifically, l have now found a unique combination of a glucose oxidase system with an indicator system which offers a superior means for testing biological fluids for their glucose content. This unique combination comprises an enzyme system having glucose oxidase activity, a substance efiective to carry out the necessary oxidation reaction, and an indicator material which is capable of being oxidized to a colored form in the presence of hydrogen peroxide and the substance for effecting the oxidation reaction. In a preferred embodiment bibulous strips are impregnated with this composition and then dried. This product represents an important improvement in more accurately determining the quantity of glucose in a specimen. With this unique combination, glucose present in a tested specimen is indicated by the formation, in a test strip dipped in the specimen, of a clearly perceptible single shade or color which may have various intensities correlated with the glucose concentrations present in the test samples.
When o-tolidine is the indicating material, glucose present in the urine specimen results in the production of a pure blue color. Then the nuances or variations in the intensities of the shades of blue formed are clearly perceptible and readily and visually differentiated by the average eye. These variations in intensity form a clear visible index of the glucose concentration present in the original fluid tested and thus allow a simple chart based on this intensity phenomenon to be conveniently prepared for use in determining the concentration of glucose present in the tested sample.
The principles underlying the basic reactions of enzyme tests for glucose are well known. Glucose oxidase catalyzes the aerobic oxidation of glucose to produce gluconic acid (gluconolactone) and hydrogen peroxide. In this reaction hydrogen removed from the glucose combines with atmospheric oxygen to form the hydrogen peroxide. This reaction may be represented by the following schematic equation:
glucose oxidase Glucose 02 gluconic acid H202 A substance effective to carry out the necessary oxidation reaction (such as horseradish peroxidase, iodide and molybdate salts, a molybdate salt alone, blood, etc.), in turn, is capable of inducing the oxidation of a class of indicators in the presence of the hydrogen peroxide formed in the conversion of glucose to gluconolactone. This latter reaction may be represented by the following schematic equation:
substance for eficcting the oxidation reaction (color change) Using, as an example, materials known to the art, glucose oxidase, peroxidase and o-tolidine, the reaction proceeds as follows:
The glucose oxidase reacts with the glucose present in the material being testedand there is formed, as indicated above, gluconic acid and hydrogen peroxide. The peroxidase in the presence of the hydrogen peroxide catalyzes the oxidation of the o-tolidine to its colored form, thus indicating the presence of glucose.
The previously described compositions of the prior art have necessitated careful preparation in order to obtain consistently reproducible and invariable color reactions in the reagent strips for predetermined glucose concentrations in test specimens. In particular, the paper strips which are to act as carriers for the active test ingredients had to be dipped into the solution within a relatively short time after the solution is made up, otherwise there is a loss of precision and sensitivity in the glucose-detecting ability of the strips. Furthermore, paper test strips prepared from such prior art compositions are likewise unstable, particularly when subjected to exposure to moisture, light and heat. The result is a non-uniform response in the diagnostic test strips which renders color matching and determination of glucose clearly beyond a normal or average persons ability to effect by mere visual perception, and produce readings which are clearly irreconcilable and inconsistent. The composition and combination of elements forming the present invention, on the contrary, make use of a more stable and more accurately controllable technique as set out in detail below.
The present diagnostic test embodies the use of an enzyme system having glucose oxidase activity, a substance eilective to carry out the necessary oxidation reaction, and an indicator material which is oxidized and changes color in the presence of hydrogen peroxide and the substance for effecting the oxidation reaction and a material or materials capable of stabilizing the mixture. This mixture is used to impregnate bibulous filter paper strips which are dried, after which they are ready for use in dip and read tests for urine sugar.
Although the principle or mode of action as well as the interaction of the combination of materials making up the novel diagnostic system is not yet fully understood, it appears that in the instant invention the test strips prepared in a manner similar to those of the prior art but containing an aliginate-alcohol addition results in a more highly stabilized product than heretofore obtained. For example, if o-tolidine is present as the glucose indicator material, in the product of this invention, the pure unblended blue color of the oxidized o-tolidine is easily read from the strip and any variation in its intensity of color development is visually distinguishable. These variations in intensity form a clear visible index of the glucose concentration present in the original fluid tested and thus allow a simple chart based on this intensity phenomenon to be conveniently prepared for use in determining the concentration of glucose present in the tested sample.
In one of the preferred examples the test composition comprises glucose oxidase, peroxidase, o-tolidine, sodium alginate and ethyl alcohol. A bibulous strip is impregnated with this composition and dried thereon. This test strip is prepared in a manner similar to that of the prior art as specifically described in Example 1 below.
The invention is illustrated in greater detail, but not limited, by the following examples:
EXAMPLE 1 Formulation of impregnating Solution Sodium alginate grn 5.0 Tween 81 (1% solution) ml 50.0 Gelatin gm 12.0 o-Tolidine.2HCl gm 2.5 Bufier (pH 4.8-5.0) ml 300.0
Citric acid 22.2 gm./ 300 ml.
Sodium citrate 97.8 gm./ 300 ml. Glucose oxidase gm 18.2 Peroxidase (horseradish) mg 380.0 Alcohol (95% ethanol) ml 125.0
Preparation of the Impregnation Solution Five grams of sodium alginate was placed in a 1 liter beaker and to this was added 230 ml. of distilled water together with 50 ml. of a 1% solution of a surface active agent such as Tween 81. This was allowed to stand with occasional stirring until free from lumps. An enzyme solution was then prepared by placing 19.2 grams of glucose oxidase and 400 mg. peroxidase into a 250 ml. beaker and a small volume of distilled water was added. This was mixed with a spatula until a smooth paste was formed whereupon more water was added until the volume totaled 100 ml. Then 95 ml. of the enzyme solution was added to the sodium alginate solution and the mixture stirred until free from lumps.
A gelatin solution was prepared by adding 300 ml. of boiling water to 14.4 grams of gelatin. This gelatin solution was kept warm (circa 40 C.) until used. 300 ml. of the citric acid-sodium citrate butter solution and 2.5 g. of o-tolidine dihydrochloride (the latter suspended in 100 ml. of 95% ethanol) were poured rapidly into 250 ml. of the warm gelatin solution and the mixture was stirred rapidly while the additions were being made. As soon as it was prepared, this buffer-tolidine-gelatin solution was poured slowly and with rapid stirring into the sodium alginate-enzyme solution prepared as described above to complete the preparation of the impregnating solution for use in the making of reagent impregnated test strips.
Preparation of Reagent Strips Bibulous strips, such as filter paper cut into narrow strips, are dipped into the impregnating solution so that through the process of submersion and capillary attraction about /2 inch of the strip at one end is completely impregnated. These strips are then dried in a drying tunnel or in a forced draft oven. It will be understood that other porous or absorbent materials such as small sticks of wood, etc. and other methods of applying the impregnating solutions and of drying the impregnated sticks may likewise be used. The dried strips are preferably stored in brown glass bottles until used.
Procedure for Testing In use, an impregnated and coated strip is dipped into the liquid specimen (i.e. urine) to be tested by maintaining the test strips of this invention in contact with a urine specimen for from 10 to 60 seconds. Thus, the distinct color intensities of progressively increasing amounts of oxidized indicator dye formed because of the presence of correspondingly increasing amounts of glucose in urine samples are clearly distinguishable by the average eye. When dipped in urine containing no glucose, the strips undergo no color change. A simple color intensity chart based on distinct color intensities developed by various glucose concentrations may be conveniently prepared for use in testing for urine glucose with test strips prepared in accordance with this disclosure.
EXAMPLE 2 Formulation of lmpregnating Solution Sodium alginate gm 5.0 Tween 81 (1% solution) ml 50.0 Gelatin grri 12.0 o-Tolidine gm. 2.5 Buffer (pH 4.85.0) ml 300.0
Citric Acid (11.1 gm./150.0 ml.) Sodium Citrate (48.9 gm./150 ml.) Glucose oxidase gm 18.2 Peroxidase (horseradish) mg 380.0 Alcohol ethanol) ml 125.0 Gallic acid mg 6080 Preparation of the Impregnating Solution Five grams of sodium alginate was placed in a 1 liter beaker and to this was added 230 ml. of distilled water together with 50 ml. of a 1% solution of a surface active agent such as Tween 81. This was allowed to stand with occasional stirring until free from lumps. An enzyme solution was then prepared by placing 19.2 grams of glucose oxidase and 400 mg. peroiddase into a 250 ml. beaker and a small volume of distilled water was added. This was mixed with a spatula until a smooth paste was formed whereupon more water was added until the vol ume totaled ml. Then 95 ml. of the enzyme solution was added to the sodium alginate solution and the mixture stirred until free from lumps.
A gelatin solution was prepared by adding 300 ml. of boiling water to 14.4 grams of gelatin. This gelatin solution was kept warm (circa 40 C.) until used. 300.0 ml. of the citric acid-sodium citrate bufier solution and 2.5 g. of o-tolidine dihydrochloride (the latter suspended in 100 'ml. of 95 ethanol) were poured rapidly into 250 ml. of the warm gelatin solution and the mixture was stirred rapidly while the additions were being made. As soon as it was prepared, 25 ml. of 95 ethanol to which 60-80 mg. of gallic acid had been added was mixed with this buffer-tolidine-gelatin solution and the resultant mixture was poured slowly and with rapid stirring into the sodium alginate-enzyme solution prepared as described above to complete the preparation of the impregnating solution for use in the making of reagent imprenated test strips.
Preparation of Reagent Strips Bibulous strips, such as filter paper cut into narrow strips, are dipped into the impregnating solution so that through the process of submersion and capillary attraction about /1 inch of the strip at one end is completely impregnated. These strips are then dried in a drying tunnel or in a forced draft oven. It will be understood that other porous or absorbent materials such as small sticks of Wood, etc. and other methods of applying the impregnating solutions and of drying the impregnated sticks may likewise be used. The dried strips are preferably stored in brown glass bottles until used.
Procedure for Testing In use, an impregnated strip, made as described in the above example, is dipped into the liquid specimen to be tested. When contacted with urine containing glucose, a test strip will give a positive reaction in about to 60 seconds evidenced by various shades of blue color.
With urines containing 0.1% to 1.0% of glucose in steps of 0.1, 0.25, 0.5 and 1.0 of glucose present, color changes of distinguishable intermediate intensities are developed by the indicators of this invention corresponding to positive indicator numbers of l, 2, 3 and 4 respectively. Urines having the higher glucose content produce the deeper color and accordingly At 0.1% glucose, a light purple color 1 At 0.25% glucose, a grey purple color 2 At 0.50% glucose, a blue purple color 3 At 1.0% glucose, a dark blue color 4 are the corresponding indicator colors and numbers developed in the test strips. When dipped into urine containing no glucose, the strips undergo no color change. A simple color chart based on this phenomenon may be conveniently prepared for use in testing for urine glucose.
From the detailed examples it is evident that additives are incorporated in the reagent composition such as suitable protective, thickening, wetting, suspending agents and the like as Well as inert dyes to impart thereto a uniform color background.
Furthermore, it is important to include an appropriate buffer system to maintain a desired pH range in a tested specimen. Although the pH range may vary between 4 to 7.5, an especially preferred pH range is 4.8 to 5.0.
Although the test device itself may comprise the reagent composition in the form of a tablet, powder or other embodiment, I prefer to impregnate the reagent composition on bibulous base materials or carriers, such as strips or sticks of filter paper, by dissolving the components in a suitable solvent and impregnating the bibulous strips with the resulting solution. Embodying the diagnostic composition in the form of test sticks insures ease and simplicity of test procedure.
In contrast to prior art strips containing no alginatealcohol ingredients, the strips prepared according to the description herein given in considerable detail showed higher resistance to the destructive effects of high temperature and humidity in accordance to the following table:
In the above table a reading of 60 is perfect, 50 to 60 is good and 30 to 50 fair.
Although the specific examples herein given illustrate certain preferred embodiments of the invention, a number of variations in the reagent materials are possible. For example any enzyme having glucose oxidase activity can be used. Likewise a number of other substances or materials effective to carry out the necessary oxidation reaction may be substituted for those given in the examples.
Among the substances or materials effective to carry out the necessary oxidation reaction may be included many organic and inorganic preparations. Thus various plant peroxidases, such as horseradish peroxidase or potato peroxidase may be used. There may also be used small measured portions, capable of producing little coloration or a definitely predetermined color background effect, of such substances as normal whole blood, red blood cells alone, lyophilized whole blood and like substances effective to carry out the necessary oxidation reaction. Inorganic compounds effective to carry out the necessary oxidation reaction such as mixtures of potassium iodide and sodium molybdate, as well as other iodides, such as sodium and ammonium iodides and other molybdates such as potassium and ammonium molybdates may be used. Sodium and other molybdates may also be used alone. In addition, urohemin and a number of other porphyrin substances for effecting oxidation reactions may be used. Thus in the metalloporphyrins, although hemin is preferred, various complex-forming compounds which activate certain other metalloporphyrins not operable per se, can be used therewith, such as 2- aminobenzothiazole, pyridine, bipyridyl, bipyridylpyridine, nicotinic acid or the like. Other substances which are not enzymes but which can carry out the necessary oxidation reaction include such compounds as iron sulfocyanate, iron tannate, ferrous ferrocyanide, potassium chromic sulfate, and others.
Among the indicator substances, other than o-tolidine, which are capable of being oxidized in the presence of hydrogen peroxide and a material for effecting the oxidation reaction are 2,7-diaminofiuorene, o-dianisidine, leucoindophenols, etc., all of which are satisfactory indicators.
In addition to the compositions described in the above examples illustrating optimum contents, variations in amounts of the four major components embodied in this invention are permissible as shown by the following chart:
An enzyme having glucose oxi- In addition various additives may also be incorporated in the reagent composition as suitable protective, thickening, and wetting agents as well as inert dyes to impart a uniform color background to the composition.
Besides sodium alginate other materials which can be used are carrageenin, gum guar, etc., among others.
While the product known as Tween 81 (polyoxyethylene substituted sorbitan mono-oleate) is a preferred surface active agent, other surface active agents such as Aerosol OT (sodium lauryl sulfate) among others are found suitable.
Gelatin is a preferred proteinaceous material although other soluble proteins including a protein degradation product, glutamic acid, glycine and other products such as polypeptides and the like are operable in this concept of the invention.
Among the various alcohols, other than ethanol which may be used are methanol, propanol, isopropanol as well as acetone among other solvents. Ethanol, however, is the preferred solvent.
The impregnated solutions prepared in accordance with any of the above examples as well as test strips impreg- 7 nated with any of these solutions were found to be very stable.
In summary, this invention pertains to a diagnostic composition for the detection of glucose in fluids, and especially in body fluids such as urine, consisting of a bibulous material such as a strip which has been impregnated with a composition comprising an enzyme system having gluclose oxidase activity, a substance for effecting the oxidation reaction (such as horseradish peroxidase, a mixture of an iodide and a molybdate salt, a molybdate salt, urohemin and like substances efi'ective to carry out the necessary oxidation reaction), a color forming indicator (such as 2,7-diaminofluorene, o-tolidine, etc.), which is oxidizable in the presence of hydrogen peroxide and said substance for eifecting the oxidation reaction and in combination an alginate-alcohol mixture for stabilizing the composition. A strip prepared in accordance with this disclosure, when used in a test for glucose in urine, shows variations in intensity of color development forming a clear visible index of the glucose concentration present in the original fluid tested and thus a simple chart based on this intensity phenomenon may be conveniently prepared for use in determining the concentration of glucose present in the tested sample.
What is claimed is:
1. A diagnostic composition for detecting glucose which which comprises glucose oxidase, an oxidation catalyst selected from the group consisting of horseradish peroxidase, metalloporphyrin, urohemin, an iodide-molybdate mixed salt, a molybdate salt,blood, red blood cells, lyophilized whole blood, lyophilized blood cells, mixtures of lyophilized blood and lyophilized plasma, a color-forming substance oxidizable in the presence of peroxide and said substance for efiecting the oxidation reaction selected from the group consisting of 2,7-diarninofiuorene, o-toli dine, o-dianisidine and leucoindopheno-l, and an algin material selected from the group consisting of sodium a1- ginate, carrageenin and gum guar.
2. A diagnostic composition for detecting glucose which comprises glucose oxidase, urohemin, 2,7-diaminofiuorene and sodium alginate.
3. A diagnostic composition for detecting glucose which comprises glucose oxidase, potassium iodide, sodium molybdate, 2,7-diaminofluorene and sodium alginate.
4. A diagnostic composition for detecting glucose which 8 comprises glucose oxidase, horseradish peroxidase, o-tolidine, and sodium alginate.
5. A diagnostic composition for detecting glucose which comprises glucose oxidase, horseradish peroxidase, o-tolidine and carrageenin.
6. A diagnostic composition for detecting glucose which comprises glucose oxidase, horseradish peroxidase, o-tolidine and gum guar.
7. A composition comprising, in aqueous solution, an algin material, a Wetting agent, a soluble protein, an indicator which undergoes a color-reaction in the presence of hydrogen peroxide and peroxidase, glucose oxidase, peroxidase, a lower alkanol, and a bufier for maintaining the solution at a pH of about 4.8 to 5.0.
8. A composition comprising sodium alginate 5 .0 grams; gelatin 12.0 grams; o-tolidineZHCl, 2.5 grams; 300 ml. of water containing 22.2 grams citric acid and 97.8 grams sodium citrate; glucose oxidase 18.2 grams; horseradish peroxidase 380.0 milligrams; ethanol, 125.0 ml., and a small amount of a surface-active agent.
9. A test indicator for detecting glucose which comprises a bibulous based material containing the dried residue resulting from contacting said carrier with a composion comprising glucose oxidase, peroxidase, and an indicator which is oxidized in the presence of hydrogen peroxide and peroxidase and undergoes a color reaction during such oxidation, sodium alginate, a wetting agent, gelatin, a bufier for maintaining the pH of said composition between about 4.8 and 5.0 and ethanol.
References Cited in the file of this patent UNITED STATES PATENTS 458,244 Tyree Aug. 25, 1891 1,103,283 Gloess et al July 14, 1914 2,337,562 Lund Dec, 28, 1943 2,768,143 Henry Oct. 23, 1956 2,864,725 Sorg et al Dec. 16, 1958 2,893,844 Cook July 7, 1959 3,001,915 Fonner Sept. 26, 1961 FOREIGN PATENTS 203,451 Australia Sept. 27, 1956 OTHER REFERENCES Mantell: The Water-Soluble Guns, 1947 Reinhold Pub. Co., N.Y., pp. 75, 116, 117, 127.
UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No 3 l23 443 March 3 1964 Robert Ra Smeby It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.
Column l line 2O for "no" read on ---g column 3,, line 44 for "o-T0lidine.,2HC1" read o-To1idine 2HC1 3 line 52 for "Impregnationfi in italics, read Impregnating in italics; column 7 line 26 strike out "whichH Signed and sealed this 21st day of July 1964.,
ESTON G. JOHNSON EDWARD J BRENNER Lttesting Officer Commissioner of Patents