US3092465A - Diagnostic test device for blood sugar - Google Patents
Diagnostic test device for blood sugar Download PDFInfo
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- US3092465A US3092465A US17501A US1750160A US3092465A US 3092465 A US3092465 A US 3092465A US 17501 A US17501 A US 17501A US 1750160 A US1750160 A US 1750160A US 3092465 A US3092465 A US 3092465A
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- glucose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
Description
United 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 blood, plasma, urine, 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 on known diabetics by both patients and physicians, this glucose indicator may also be used efiiciently in routine glucose analyses of body fluids in hospitals and physicians oflices, 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. 2,3 87,244), still another involves a test which depends on the action of a double enzyme system (US. application No. 514,395, filed June 9, 1955, by Alfred 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.
We have now found a novel and highly useful glucosedetectiug means which represents an important improvement in the area of determining glucose in various materials including body fluids, and especially blood. This technique utilizes a diagnostic composition preferably impregnated on a paper strip and which in addition is protected from the staining tendency of the hemoglobin of the body by means of a coating which functions as a semipermeable membrane surrounding the test composition and separating it from direct contact with the blood test specimen.
Specifically, we have now found a unique combination of a glucose oxidase system with an indicator system which oflfers a. superior means for testing biological fluids for their glucose content. This unique combination comprises an enzyme system having glucose oxidase activity, a material having peroxidative activity, an indicator material which is capable of being oxidized to a colored form in the presence of hydrogen peroxide and the material having peroxidative activity. In a preferred embodiment bibulous strips are impregnated with this comrates Patent 'ice position and then dried .after which in accordance with this invention they are provided with a protective coating in the form of a semi-permeable membrane which allows water and glucose to pass therethrough but restrains and screens out such large molecules as hemoglobin or proteins from direct contact with the test composition.
The screening or dialyzing effect of this semi-permeable membrane represents an important improvement in test strips for 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 coated test strip dipped in the specimen, of a clearly perceptible single shade or color which may have various intensities, the strip being uncontaminated by any red coloration due to hemoglobin or other coloring bodies found in whole blood.
When o-tolidine is the indicating material, glucose present in the blood specimen results in the production of a pure blue color uncontaminated with the stains accompanying hemoglobin or other color bodies. Then the nuances or variations in the intensities of the shades of blue formed are clearly perceptable and readily and visually diiferentiated 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 O: Gluconie acid H2O;
A substance having peroxidative activity (such as horseradish peroxidase, iodide and molybd-ate salts, blood, etc.), in turn, is capable of inducing the oxidation of a class of indicators by the hydrogen peroxide formed in the conversion of glucose of glyconolactone. This latter reaction may be represented by the following schematic equation:
Substance having peroxidative ctivity E10; oxldizable dye Oxidized dye 1120 (color change) Using, as an example, materials known to the art,
glucose oxidase, peroxidase and o-tolidine, the reaction The previously described compositions of the prior art have the disadvantage that there has been a tendency for the hemoglobin and other coloring bodies of the blood to stain the test strip so that it assumes a dark red color which is interblended with the color developed 5 on this stained strip by the glucose test composition to form a blended color which is difficult to read. As a result, color matching and determination after a smear test, especially for blood sugar, is beyond a normal or average persons ability to effect by mere visual p-ercephaving peroxidative activity, and an indicator material which is oxidized and changes color in the presence of hydrogen peroxide and the material having peroxidative activity. This mixture is used to impregnate bibulous filter paper strips which are dried and coated with a semipermeable coating material in film form which screens out and prevents hemoglobin or protein materials from contacting the test composition.
Although the principle or mode of action as well as the interaction of the combination of materials makingup 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 coated with a semi-permeable layer forming material such as cellophane, cellulose acetate, cellulose butyrate, cellulose nitrate and the like allow water and glucose to pass through the coating material and react with the test com position on the strip while screening or preventing the larger molecules such as hemoglobin or other coloring matter as well as other protein constituents of the blood from passing through to the test composition. Since the hemoglobin does not contact the test composition but remains separated therefrom by the coating film and remains on the outer surface thereof, it is readily wiped off with a tissue or washed from the coated strip with water. For example, if o-tolidine is present as the glucose indicator material, 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, and o-tolidine. A bibulous strip is impregnated with this composition and dried thereon. similar to that of the prior art is then coated with a semipermeable membrane in the form of a film of cellulose acetate by dipping the strip impregnated with the diagnostic composition into a solution of cellulose acetate in acetone followed by drying 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 Alcohol (95% ethanol) ml This test strip prepared in a manner Preparation of the impregnating Solution Five grams of sodium alginate was placed in a 1 liter beaker and to this was added 256 ml. of distilled water. 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 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 until used. 300 ml. of the citric acid-sodium citrate buffer solution and 2.5 g. of otolidiue dihydrogen chloride (the latter suspended in 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 bulfer-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 out 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.
After drying, the impregnated strips are dipped into a 2% cellulose acetate solution in acetone to cover the impregnated portion of the strip with a thin layer of the cellulose acetate solution. This is followed by air drying the strips for about 30 minutes to form a semi-permeable coating of cellulose acetate over the impregnated end of the strips. The air dried strips are preferably stored in brown glass bottles until used. In use, an impregnated and coated strip is smeared with the liquid specimen (i.e. blood) to be tested. After maintaining the test strips of this invention in contact with a blood specimen for about one minute, any excess blood is. readily washed from the strip with water or may be wiped ofi the strip with tissue paper. No staining of the bibulous strip material by the hemoglobin or other coloring materials of the blood is evident. The distinct color intensities of progressively increasing amounts of oxidized indicator dye formed in the coated strips herein described because of the presence of correspondingly increasing amount of glucose in blood samples are clearly distinguishable by the average eye. ,This is possible only because of the absence of the confused color masking usually accompanying the staining of an uncoated strip by hemoglobin and other coloring bodies present in the blood sample being tested. When tested or smeared with blood containing no glucose, the coated 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 blood glucose with test strips prepared in accordance with this disclosure.
Instead of the cellulose acetate coating of the above detailed example, a number of other illustrative embodiments of this invention are possible using, however, such coating compositions as cellophane, cellulose butyrate, cellulose nitrate, cellulose acetate propionate, and the like to form the semi-permeable coating or film.
The examples may be still further varied by adding to the testing composition from 0 to 240.0 mg. of ascorbic acid which reacts preferentially with the peroxide to predetermine the level at which the color change occurs in the indicator in accordance with the disclosure in US. Patent No. 2,893,844.
Still other examples may be prepared by making up laminated test strips using precut film as the coating material. Thus, instead of a coating formed in situ by dipping a completed test strip into a cellulose acetate or similar coating solution, films such as cellophane, cellulose acetate sheets, etc., may be laminated on the surface of a test strip. In addition various dyes serving as light filters may be incorporated in these surface laminations to screen out undesirable light rays which have an accelerating effect in decomposing the diagnostic composition.
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, we 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, impregnating the bibulous strips with the resulting solution, and after drying the impregnated test strips a semipermeable film is applied as a coating thereon. Embodying the diagnostic composition in the form of test sticks insures ease and simplicity of test procedure.
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 having peroxidative activity may be substituted for those given in the examples.
Among the materials having peroxidative activity 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, lyophylized whole blood and like substances having peroxidative activity. Inorganic compounds having peroxidative activity 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. In addition, urohemin and a number of other porphyrin substances having peroxidative activity may be used. In addition to the urohemin mixture mentioned a number of other porphyrin substances having peroxidative activity 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 Z-aminobenzothiazole, pyridine, bipyridyl, bipyridylpyridine, nicotinic acid or the like. Other substances which are not enzymes but have peroxidative activity 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 having peroxidative activity are 2,7-diaminofluorene, o-dianisidine, leucoindophenols, etc., all of which are satisfactory indicators.
6 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:
( 1) An enzyme having glucose oxidase activity 4000-16,000 units. (2) A substance having peroxidative activity 0.25l.5 gm. (3) Indicating material 0.2-1.0 gm. (4) A buffer 0.1-0.3 molar (pH. 4.0 to 7.5).
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.
The impregnating solutions prepared in accordance with any of the above examples as well as test strips impregnated 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 blood, consisting of a bibulous material such as a strip which has been impregnated with a composition comprising an enzyme system having glucose oxidase activity, a substance having peroxidative activity (such as horseradish peroxidase, an iodide-molybdate salt, a urohemin and like substances having peroxidative activity), a color forming indicator (such as 2,7-diaminofluorene, o-tolidine, etc.), which is oxidizable in the presence of hydrogen peroxide and said substance having peroxidative activity and in combination a semi-permeable membrane coating superposed over the test composition and separating it from direct contact with a tested specimen such as blood. In making a test this coated strip remains uncontaminated with the red coloration due to blood pigments and as a result the indicator materials when dipped and read produce color changes of varying intensities of a single shade or one color. Any attached blood particles are readily wiped ofl or washed ofl the coating and the underlying strip shows variations in intensity forming a clear visible index of the glucose concentration present in the original fluid tested. This allows 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.
What is claimed is:
1. A test article for detecting glucose in a fluid which comprises a bibulous material containing impregnated therein a mixture which comprises an enzyme system having glucose oxidase activity, a substance having peroxidative activity, an indicator material which is oxidized in the presence of peroxide and said substance having peroxidative activity and changes color thereupon and a semi-permeable coating of transparent film forming a continuous smooth protective layer superposed over the impregnated bibulous material.
2. A test article for detecting glucose in accordance with claim 1, wherein the semi-permeable coating is cellulose acetate.
3. A test article for detecting glucose in accordance with claim 1, wherein the semi-permeable coating is cellulose acetate propionate.
4. A test article for detecting glucose in accordance with claim 1, wherein the semi-permeable coating is cellulose butyrate.
5. A test article for detecting glucose in accordance with claim 1, wherein the semi-permeable coating is cellulose nitrate.
6. A test article for detecting glucose in accordance with claim 1, wherein the semi-permeab1e coating is regenerated cellulose.
7. A test article in accordance with claim 1, wherein the bibulous material is a paper strip. a
8. 'A test article in accordance with claim 1, wherein the bibulous material is an inert solid support material.
9. A test article for detecting glucose in accordance with claim 1, wherein the semi-permeable coating is a plastic film laminated to the impregnated bibulous material and selected from the group consisting of cellulose acetate, cellulose acetate propionate, cellulose butyrate, cellulose nitrate and regenerated cellulose.
10. A test article in accordance with claim 1 wherein said enzyme is glucose oxidase, wherein said substance "8 having peroxidative activity is peroxidase, and wherein said indicator material is o-tolidine.
References Cited in the file of this patent UNITED STATES PATENTS 2,140,341 Wallach et al Dec. 13, 1938 2,567,445 Parker Sept. 11, 1951 2,912,309 Free Nov. 10, 1959 FOREIGN PATENTS 571,181 Great Britain Aug. 10, 1945
Claims (1)
1. A TEST ARTICLE FOR DETECTING GLUCOSE IN A FLUID WHICH COMPRISES A BIBULOUS MATERIAL CONTAINING IMPREGANTED THEREIN A MIXTURE WHICH COMPRISES AN ENZYME SYSTEM HAVING GLUCOSE OXIDASE ACTIVITY, A SUBSTANCE HAVING PEROXIDATIVE ACTIVITY, AN INDICATOR MATERIAL WHICH IS OXIDIZED IN THE PRESENCE OF PEROXIDE AND SAID SUBSTANCE HAVING PEROXIDATIVE ACTIVITY AND CHANGES COLOR THEREUPON AND A SEMI-PERMEABLE COATING OF TRANSPARENT FILM FORMING A CONTINUOUS SMOOTH PROTECTIVE LAYER SUPERPOSED OVER THE IMPREGNATED BIBULOUS MATERIAL.
Priority Applications (2)
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US17501A US3092465A (en) | 1960-03-25 | 1960-03-25 | Diagnostic test device for blood sugar |
GB10456/61A GB911181A (en) | 1960-03-25 | 1961-03-22 | Diagnostic composition for glucose |
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US17501A US3092465A (en) | 1960-03-25 | 1960-03-25 | Diagnostic test device for blood sugar |
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US17501A Expired - Lifetime US3092465A (en) | 1960-03-25 | 1960-03-25 | Diagnostic test device for blood sugar |
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Cited By (70)
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EP3694322A1 (en) | 2017-10-09 | 2020-08-19 | Terumo BCT Biotechnologies, LLC | Lyophilization container and method of using same |
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