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Publication numberUS3654179 A
Publication typeGrant
Publication dateApr 4, 1972
Filing dateMar 1, 1971
Priority dateMar 1, 1971
Publication numberUS 3654179 A, US 3654179A, US-A-3654179, US3654179 A, US3654179A
InventorsRobert Bauer
Original AssigneeMiles Lab
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Indicator for detecting hydrogen peroxide and peroxidative compounds containing bindschedler's green
US 3654179 A
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Description  (OCR text may contain errors)

United States Patent O Int. Cl. C12k 1/04 US. Cl. 252-408 Claims ABSTRACT OF THE DISCLOSURE Bindschedlers Green has been found to be an excellent indicator for detecting hydrogen peroxide and peroxidative active compounds. For example, when said indicator is formulated with glucose oxidase and peroxidase, it provides a very sensitive test for glucose in urine and other body fluids.

BACKGROUND OF THE INVENTION The determination of glucose in urine is important since this test is employed to detect diabetes. Procedures for the detection of sugar in urine 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, while still another test depends solely on the action of enzymes. The diagnostic composition in most glucose tests comprises essentially glucose oxidase, peroxidase and an indicator which is oxidized by hydrogen peroxide and undergoes a color reaction during such oxidation. Typical indicators employed in the past include o-tolidine, benzidine, dianisidine and 2,7-diaminofluorene.

It is well known that glucose oxidase catalyzes the aerobic oxidation of glucose to gluconic acid and hydrogen peroxide, the latter of which oxidizes the indicator to produce a color change which is accurately indicative of the amount of H 0 present as well as of the glucose content of the fluid being tested. Since some of the indicators previously used are toxic, it has spurred a search for more suitable replacements which will still give satisfactory results in detecting H 0 generally and more specifically, in detecting glucose in urine or blood. In addition, such indicators can be used to detect peroxidase as well as peroxidative active substances such as hemoglobin in aqueous fluids.

SUMMARY OF THE INVENTION The present invention is based upon the discovery that Bindschedlers Green can be used as an indicator in formulations to detect hydrogen peroxide, hemoglobin or glucose in body fluids such as urine or blood. Said indicator has the formula and is known chemically as 4,4'-bis(dimethylamino) diphenylamine.

Although the test system may comprise the reagent composition in the form of a tablet, powder or solution. it is preferable to aflix said composition on bibulous base materials or carriers such as strips of filter paper by dissolving the components in a suitable solvent, impregnating the strips with the resulting solution and drying the im- 3,654,179 Patented Apr. 4, 1972 pregnated test strips. The types of compositions contemplated are set forth in the following examples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 A composition was prepared by mixing the following ingredients in the volumes indicated below:

Porous paper strips about one half inch wide and 3 inches long were dipped into the above solution so that by capillary action about one half inch of each strip at one end was completely impregnated. The strips were then dried at C. for 10 minutes. If desired, other porous materials such as Wood sticks or plastic strips can be employed as a carrier. When contacted with urine containing glucose such a test strip gave a positive reaction in one minute or less as evidenced by the change in color of the indicator from gray to green. The higher the concentration of the glucose, the deeper the color produced. When dipped into urine containing no glucose, the strips undergo no color change. A simple color chart can be prepared for use in estimating various concentrations of glucose in urine employing strips containing Bindschedlers Green and dipping them into solutions with known amounts of glucose. The color observed will vary from grayish-green at 0.002% of glucose to an intense green at 2% or more of glucose. Test strips prepared in this manner will change color when a drop of a blood containing urine and a drop of 3% hydrogen peroxide solution is applied thereto.

Example 2 A first solution was prepared containing 1.5 grams of carrageen, 15 grams of polyvinylpyrrolidone, 15 ml. of ethanol and 192 ml. of water.

A second solution was prepared containing 9.24 grams of citric acid, 40.79 grams of sodium citrate and 124.8 ml. of water.

A third solution was prepared containing 4.5 grams of a maleic anhydride-methylvinylether copolymer, 1.5 grams of sodium lauroyl sarcosinate and ml. of water.

Still a further solution was prepared containing 0.5 gram of peroxidase and 76 ml. of an aqueous solution of glucose oxidase containing 1,000 International units per ml. of water.

A composition suitable for detecting H 0 and glucose was then prepared containing 9 ml. of a 1% ethanol solution of Bindschedlers Green, 9 ml. of ethanol, 5.5 ml. of water, 34.5 ml. of the first solution above, 20.8 ml. of the second solution above, 17.5 ml. of the third solution above and 7.6 ml. of the fourth solution previously prepared. Bibulous paper strips were dipped in said solution and thereafter dried for 10 minutes at 100 C. These strips readily turned from gray to green when contacted with urine containing glucose and the intensity of the green color increased with the glucose concentration. It was found that 0.002% of glucose could be detected in urine within a minute whereas a similar strip impregnated with o-tolidine as the indicator barely detected 0.005% of glucose in urine in 3 minutes.

In addition to the compositions set forth in the foregoing examples, it was found that the amount of indicator employed could be varied from about .005 to 0.15% by weight in such compositions whereas the glucose oxidase concentration could vary from 40 to 300 International units per ml. and the peroxidase concentration from 0.01% to 0.05% by weight at a pH of from 5 to 8 adjusted with tris-malonate buffer of about 0.05 to 0.2 molar. Other peroxidative active compounds such as water soluble molybdates and iodides may be substituted for the peroxidase if desired.

The impregnating solutions prepared as shown herein as well as test strips impregnated therewith were found to be very stable. However, it is preferred that the strips be stored in brown bottles containing a desiccant.

What is claimed is:

1. In a composition for detecting hydrogen peroxide or peroxidative active compounds utilizing the catalytic oxidation of an indicator dyestuff by hydrogen peroxide in the presence of the peroxidative active compound, the improvement which comprises the use of Bindschedlers Green as the indicator dyestuff.

2. A composition as claimed in claim 1 in which Bindschedlers Green is present in about .005% to 0.15% by weight of said composition.

3. A composition as claimed in claim 1 in which the peroxidative activecompound is selected from the group consisting of peroxidase, hemoglobin and molybdate.

4. A composition as claimed in claim 3 in which the peroxidase is present in about 0.01% to 0.05% by weight of said composition.

5. A composition for detecting glucose in aqueous fluids which comprises glucose oxidase, a peroxidative active material and Bindschedlers Green.

References Cited JOHN T. GOOLKASIAN, Primary Examiner M. E. MCCAMISH, Assistant Examiner US. Cl. X.R.

8-1 C, l D; 23230 B, 253 TP; l03.5 C

Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3917452 *Nov 25, 1974Nov 4, 1975Boehringer Mannheim GmbhDiagnostic agent for the detection of peroxidatively active substances
US3986833 *Sep 8, 1975Oct 19, 1976Miles Laboratories, Inc.Test composition, device, and method for the detection of peroxidatively active substances
US4010104 *May 8, 1975Mar 1, 1977Bayer AktiengesellschaftSolutions of new complex antimony compounds
US4017261 *Oct 16, 1975Apr 12, 1977Lachema, Narodni PodnikBiological diagnostic test strip and method of producing same
US4066408 *Feb 9, 1977Jan 3, 1978Ab KabiChromogen-reactive-indicator preparations containing a 3,3'-di(carbonyloxy- or sulfonyloxy-group-containing) benzidine derivative chromogen
US4175923 *Jun 26, 1978Nov 27, 1979Friend William GApplying developing solution to an absorbent material impregnated with a guaiac reagent
US4302537 *Feb 11, 1980Nov 24, 1981Boehringer Mannheim GmbhBy measurement of emission of light using 9-dimethylaminobenzo(f)phthalazine as light emitting compound
US4363874 *Aug 7, 1981Dec 14, 1982Miles Laboratories, Inc.Radioimmunoassay diagnosis
US4384042 *Jul 29, 1981May 17, 1983Kyowa Hakko Kogyo Co., Ltd.Method and test composition for determination of hydrogen peroxide
US4390343 *Apr 19, 1982Jun 28, 1983Miles Laboratories, Inc.Multilayer analytical element having an impermeable radiation diffusing and blocking layer
US4851353 *Feb 4, 1987Jul 25, 1989Kyowa Hakko Kogyo Co., Ltd.Method and test composition for determination of lipid peroxide
US4916058 *Jun 23, 1987Apr 10, 1990Kyowa Medex Co., Ltd.Chromogens
US4956300 *Oct 16, 1984Sep 11, 1990Helena Laboratories CorporationAdsorbent carrier having guaiac that will react with hemoglobin fresent to form a blue dye, oxidizers and binders
US5081040 *Jun 6, 1989Jan 14, 1992Helena Laboratories CorporationComposition and kit for testing for occult blood in human and animal excretions, fluids, or tissue matrixes
US5196167 *May 9, 1991Mar 23, 1993Helena Laboratories CorporationFecal occult blood test product with positive and negative controls
US5217874 *May 9, 1991Jun 8, 1993Helena Laboratories CorporationFecal occult blood test product with positive and negative controls
US5273888 *Apr 29, 1988Dec 28, 1993Helena Laboratories CorporationChemical test kit and method for determining the presence of blood in a specimen and for verifying the effectiveness of the chemicals
US5445755 *May 31, 1994Aug 29, 1995The Procter & Gamble CompanyContaining hydrogen peroxide source, phenothiazine or phenoxazine peroxidase accelerator; inhibit dye transfer
US5451337 *May 31, 1994Sep 19, 1995The Procter & Gamble Co.Dye transfer inhibition system containing a peroxidase/accelerator system
US5702913 *Jun 12, 1989Dec 30, 1997Helena Laboratories CorporationUsed for fetal specimens for occult blood
US5942754 *Nov 27, 1995Aug 24, 1999Kyoto Dai Ichi Kagaku Co., Ltd.In an aqueous sample solution
US6063631 *May 21, 1997May 16, 20003M Innovative Properties CompanyMonitoring a sterilization process using hydrogen peroxide vapor by exposing item and indicator containing acid fuchsin to vapor which causes indicator to become colorless; for medical equipment
US6238623Feb 5, 1998May 29, 20013M Innovative Properties CompanyLabels and tracking systems for sterilization procedures
US6287518Feb 5, 1998Sep 11, 20013M Innovative Properties CompanySterilization monitors
US6346417Jan 5, 2000Feb 12, 20023M Innovative Properties CompanySterilization monitors
US6440744Jan 5, 2000Aug 27, 20023M Innovative Properties CompanySterilization monitoring method
US6706537Aug 2, 2001Mar 16, 20043M Innovative Properties CompanyOf peracid sterilization vapor, using as indicator a colorant susceptible to halogenation and a halogen source to a peracid vapor, said colorant changes color when halogenated; e.g. phenol red and potassium bromide indicator; peracetic acid
EP0038205A1 *Apr 14, 1981Oct 21, 1981Kyowa Hakko Kogyo Co., LtdMethod for determination of peroxide and test reagent therefor
EP0045220A1 *Jul 30, 1981Feb 3, 1982Kyowa Hakko Kogyo Co., LtdMethod and test composition for determination of hydrogen peroxide
EP0124287A2 *Mar 30, 1984Nov 7, 1984Kyowa Medex Co. Ltd.Method and test composition for determination of hydrogen peroxide
Classifications
U.S. Classification435/28, 435/805, 8/401, 436/135, 436/95, 436/66, 8/400, 422/510
International ClassificationC12Q1/28, C12Q1/54
Cooperative ClassificationC12Q2326/00, C12Q1/28, Y10S435/805, C12Q1/54
European ClassificationC12Q1/28, C12Q1/54