US3654179A - Indicator for detecting hydrogen peroxide and peroxidative compounds containing bindschedler's green - Google Patents

Indicator for detecting hydrogen peroxide and peroxidative compounds containing bindschedler's green Download PDF

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Publication number
US3654179A
US3654179A US3654179DA US3654179A US 3654179 A US3654179 A US 3654179A US 3654179D A US3654179D A US 3654179DA US 3654179 A US3654179 A US 3654179A
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Prior art keywords
indicator
green
glucose
hydrogen peroxide
peroxidative
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Robert Bauer
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Bayer Corp
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Bayer Corp
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2326/00Chromogens for determinations of oxidoreductase enzymes
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/805Test papers
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/14Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333Saccharide [e.g., DNA, etc.]
    • Y10T436/144444Glucose
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/20Oxygen containing
    • Y10T436/206664Ozone or peroxide

Abstract

BINDSCHEDLER''S 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.

Description

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

US3654179A 1971-03-01 1971-03-01 Indicator for detecting hydrogen peroxide and peroxidative compounds containing bindschedler's green Expired - Lifetime US3654179A (en)

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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3917452A (en) * 1973-12-20 1975-11-04 Boehringer Mannheim Gmbh Diagnostic agent for the detection of peroxidatively active substances
US3986833A (en) * 1975-09-08 1976-10-19 Miles Laboratories, Inc. Test composition, device, and method for the detection of peroxidatively active substances
US4010104A (en) * 1974-05-08 1977-03-01 Bayer Aktiengesellschaft Solutions of new complex antimony compounds
US4017261A (en) * 1974-10-16 1977-04-12 Lachema, Narodni Podnik Biological diagnostic test strip and method of producing same
US4066408A (en) * 1974-10-16 1978-01-03 Ab Kabi Chromogen-reactive-indicator preparations containing a 3,3'-di(carbonyloxy- or sulfonyloxy-group-containing) benzidine derivative chromogen
US4175923A (en) * 1978-06-26 1979-11-27 Friend William G Method and apparatus for occult blood testing in the home
EP0038205A1 (en) * 1980-04-14 1981-10-21 Kyowa Hakko Kogyo Co., Ltd Method for determination of peroxide and test reagent therefor
US4302537A (en) * 1979-02-21 1981-11-24 Boehringer Mannheim Gmbh Reagent and method for the determination of peroxidase
EP0045220A1 (en) * 1980-07-30 1982-02-03 Kyowa Hakko Kogyo Co., Ltd Method and test composition for determination of hydrogen peroxide
US4363874A (en) * 1981-08-07 1982-12-14 Miles Laboratories, Inc. Multilayer analytical element having an impermeable radiation nondiffusing reflecting layer
US4390343A (en) * 1981-07-06 1983-06-28 Miles Laboratories, Inc. Multilayer analytical element having an impermeable radiation diffusing and blocking layer
EP0124287A2 (en) * 1983-03-31 1984-11-07 Kyowa Medex Co. Ltd. Method and test composition for determination of hydrogen peroxide
US4956300A (en) * 1982-01-05 1990-09-11 Helena Laboratories Corporation Aid for determining the presence of occult blood, method of making the aid, and method of using the aid
US5081040A (en) * 1987-06-29 1992-01-14 Helena Laboratories Corporation Composition and kit for testing for occult blood in human and animal excretions, fluids, or tissue matrixes
US5196167A (en) * 1989-04-04 1993-03-23 Helena Laboratories Corporation Fecal occult blood test product with positive and negative controls
US5217874A (en) * 1989-04-04 1993-06-08 Helena Laboratories Corporation Fecal occult blood test product with positive and negative controls
US5273888A (en) * 1984-01-16 1993-12-28 Helena Laboratories Corporation Chemical test kit and method for determining the presence of blood in a specimen and for verifying the effectiveness of the chemicals
US5445755A (en) * 1994-05-31 1995-08-29 The Procter & Gamble Company Detergent compositions containing a peroxidase/accelerator system without linear alkylbenzenesulfonate
US5451337A (en) * 1994-05-31 1995-09-19 The Procter & Gamble Co. Dye transfer inhibition system containing a peroxidase/accelerator system
US5702913A (en) * 1983-12-21 1997-12-30 Helena Laboratories Corporation Chromgen-reagent test system
US5942754A (en) * 1994-11-25 1999-08-24 Kyoto Dai Ichi Kagaku Co., Ltd. Method of and apparatus for determining hydrogen peroxide
US6063631A (en) * 1997-05-21 2000-05-16 3M Innovative Properties Company Sterilization indicator
US6238623B1 (en) 1997-05-21 2001-05-29 3M Innovative Properties Company Labels and tracking systems for sterilization procedures
US6287518B1 (en) 1997-06-25 2001-09-11 3M Innovative Properties Company Sterilization monitors
US9034593B2 (en) 2010-11-22 2015-05-19 Kimberly-Clark Worldwide, Inc. Vaginal indicator to detect biomarkers of good health

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3917452A (en) * 1973-12-20 1975-11-04 Boehringer Mannheim Gmbh Diagnostic agent for the detection of peroxidatively active substances
US4010104A (en) * 1974-05-08 1977-03-01 Bayer Aktiengesellschaft Solutions of new complex antimony compounds
US4017261A (en) * 1974-10-16 1977-04-12 Lachema, Narodni Podnik Biological diagnostic test strip and method of producing same
US4066408A (en) * 1974-10-16 1978-01-03 Ab Kabi Chromogen-reactive-indicator preparations containing a 3,3'-di(carbonyloxy- or sulfonyloxy-group-containing) benzidine derivative chromogen
US3986833A (en) * 1975-09-08 1976-10-19 Miles Laboratories, Inc. Test composition, device, and method for the detection of peroxidatively active substances
US4175923A (en) * 1978-06-26 1979-11-27 Friend William G Method and apparatus for occult blood testing in the home
US4302537A (en) * 1979-02-21 1981-11-24 Boehringer Mannheim Gmbh Reagent and method for the determination of peroxidase
EP0038205A1 (en) * 1980-04-14 1981-10-21 Kyowa Hakko Kogyo Co., Ltd Method for determination of peroxide and test reagent therefor
US4851353A (en) * 1980-04-14 1989-07-25 Kyowa Hakko Kogyo Co., Ltd. Method and test composition for determination of lipid peroxide
EP0045220A1 (en) * 1980-07-30 1982-02-03 Kyowa Hakko Kogyo Co., Ltd Method and test composition for determination of hydrogen peroxide
US4384042A (en) * 1980-07-30 1983-05-17 Kyowa Hakko Kogyo Co., Ltd. Method and test composition for determination of hydrogen peroxide
US4390343A (en) * 1981-07-06 1983-06-28 Miles Laboratories, Inc. Multilayer analytical element having an impermeable radiation diffusing and blocking layer
US4363874A (en) * 1981-08-07 1982-12-14 Miles Laboratories, Inc. Multilayer analytical element having an impermeable radiation nondiffusing reflecting layer
US4956300A (en) * 1982-01-05 1990-09-11 Helena Laboratories Corporation Aid for determining the presence of occult blood, method of making the aid, and method of using the aid
EP0124287A3 (en) * 1983-03-31 1988-01-13 Kyowa Medex Co. Ltd. Method and test composition for determination of hydrogen peroxide
US4916058A (en) * 1983-03-31 1990-04-10 Kyowa Medex Co., Ltd. Method and test composition for determination of hydrogen peroxide
EP0124287A2 (en) * 1983-03-31 1984-11-07 Kyowa Medex Co. Ltd. Method and test composition for determination of hydrogen peroxide
US5702913A (en) * 1983-12-21 1997-12-30 Helena Laboratories Corporation Chromgen-reagent test system
US5273888A (en) * 1984-01-16 1993-12-28 Helena Laboratories Corporation Chemical test kit and method for determining the presence of blood in a specimen and for verifying the effectiveness of the chemicals
US5081040A (en) * 1987-06-29 1992-01-14 Helena Laboratories Corporation Composition and kit for testing for occult blood in human and animal excretions, fluids, or tissue matrixes
US5217874A (en) * 1989-04-04 1993-06-08 Helena Laboratories Corporation Fecal occult blood test product with positive and negative controls
US5196167A (en) * 1989-04-04 1993-03-23 Helena Laboratories Corporation Fecal occult blood test product with positive and negative controls
US5445755A (en) * 1994-05-31 1995-08-29 The Procter & Gamble Company Detergent compositions containing a peroxidase/accelerator system without linear alkylbenzenesulfonate
US5451337A (en) * 1994-05-31 1995-09-19 The Procter & Gamble Co. Dye transfer inhibition system containing a peroxidase/accelerator system
US5942754A (en) * 1994-11-25 1999-08-24 Kyoto Dai Ichi Kagaku Co., Ltd. Method of and apparatus for determining hydrogen peroxide
US6238623B1 (en) 1997-05-21 2001-05-29 3M Innovative Properties Company Labels and tracking systems for sterilization procedures
US6063631A (en) * 1997-05-21 2000-05-16 3M Innovative Properties Company Sterilization indicator
US6287518B1 (en) 1997-06-25 2001-09-11 3M Innovative Properties Company Sterilization monitors
US6346417B1 (en) 1997-06-25 2002-02-12 3M Innovative Properties Company Sterilization monitors
US6440744B1 (en) 1997-06-25 2002-08-27 3M Innovative Properties Company Sterilization monitoring method
US6706537B2 (en) 1997-06-25 2004-03-16 3M Innovative Properties Company Sterilization monitors and method of use
US9034593B2 (en) 2010-11-22 2015-05-19 Kimberly-Clark Worldwide, Inc. Vaginal indicator to detect biomarkers of good health

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GB1380499A (en) 1975-01-15 application

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