US 3654179 A
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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