Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS3016292 A
Publication typeGrant
Publication dateJan 9, 1962
Filing dateMay 15, 1959
Priority dateMay 15, 1959
Also published asDE1284124B
Publication numberUS 3016292 A, US 3016292A, US-A-3016292, US3016292 A, US3016292A
InventorsBauer Robert, Ray L Mast
Original AssigneeMiles Lab
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Diagnostic composition
US 3016292 A
Images(3)
Previous page
Next page
Description  (OCR text may contain errors)

nit-ed States atent Q 3,016,292 r v DIAGNOSTIC COMPOSITION Robert Bauer, Kalamazoo, Mich., and Ray L. Mast, Elkhart, Ind., assignors to Miles Laboratories, Inc., Elkhart, Ind., a corporation of Indiana No Drawing. Filed May 15, 1959, Ser. No. 813,336

6 Claims. (Cl. 23 -253) This invention relates toa new and improved diagnostic 'guided in this regard by a regular check on urine glucose.

But beyond its usefulnesspin regular urine testing on 'known diabetics by both patients and physicians, this glucose indicator may also be used efficiently inroutine urinalyses in hospitals and physicians offices, in diabetes detection screening programs, in the dilferentiation of glucosuria from other meliturias, and the like.

Because early diagnosis and continued control are so important in diabetes, a urine-sugar test, to be of greatest value, must be conveniently rapid, simple enough for any patient to learn with ease, accurate enough to serve the clinician, and sensitive enough to reflect variations in the patients condition. 7 i must be adequately stable.

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- "ice k i The degreeof blue color formation ismediately but accurately indicative of the" glucose content-ofthe fluid being tested. I

The principle underlying the "function of th'ejiodid'eand molybdate salts as wellas their coacti'on or interaction is not yet fully understood. However, it appears that, ana-' lytically, it is theirrole to serve as electron-transfer agents. We have found that'due totheir collective presence-with one intensifying the effect of the other-the reaction'proceeds at "a'faste'r rate and lesser concentrations of hydrogenperoxide and, accordingly, glucose may be determined. These salts may therefore be referred to as'accelerators and promoters which hasten and enhance the oxidation of the indicator substance by hydrogen peroxide.

The incorporation and use in this new glucose diag-.

nostic' of potassium iodide and sodium molybdate, 'both of which are commercially available, represents a substantial and significant advance in the artpertaining to glucose diagnostics." Glucose indicators in stick form present-ly' on the market depend for the detection of hydrogen peroxide on the action of enzymes,-s uch asperoxidases. The glucose indicator of the present invention, on the contrary, makes use of a non-enzymatic agent which permits a more accurately controllable and more definite composition. A very sensitive test for hydrogen peroxide and,faccordingly, glucose is'thus made possible.

Moreover, the reagent composition heating alkaline copper reduction test in tablctfdrmiUzS.

Ser. No. 514,395, filed June 9, 1955, by Alfred l l. Free and assigned to the assigneeof the present invention); These'and other procedures have greatly contributed to the art of diagnosing for urine glucose. v

We have now found a novel and highly useful glucosedetecting means which represents an important improvement and a freshapproaeh to the problem of determining glucose in various materials including body fluids, such as urine, by a technique that utilizes a diagnostic composition which rivals even the latest and most commonly used glucose tests.

Specifically, we have now found that a unique combination of a glucose oxidase'system for determining glucose with a novel indicator system using an iodide-molybdate accelerator for detecting hydrogen peroxide offers a superior means for testing biological fluids for their glucose content. p

A diagnostic composition according to the present'im vention comprises as essential constituents glucose oxidase, an iodide salt, such as potassium iodide, a molybdate salt, such as sodium molybdate, and a color-forming substance or indicator, such as 2,7-diaminofluorene.

The glucose oxidase (glucose aerodehydrogenase), as is well known, catalyzes the aerobic oxidation of glucose to produce gluconic acid (gluconolactone) and hydrogen peroxide. The iodide and molybdate salts, in turn, are capable of inducing the oxidation of indicators by hydrogen peroxide formed in the oxidation of glucose. The latter reaction may be represented by the following schematic equation: a

Oxidized diar'ninofiuorene (blue color) In place of the potassium iodide and sodium molybdate it isalso possible, and therefore contemplated, to employ other equivalent iodide and molybdatelsalts. For example, other iodides, such as sodium and ammonium iodides and other molybdates, such as potassium 'and ammonium molybdates may also be used. In lieu of the i2,7-diaminofluorene, o-tolidine, o-dianisidine, leucoindophenols, etc. would be satisfactory indicators.

From the detailed examples below it is evident that such additives are incorporated in the reagent composition as suitable protective, thickening, wetting, suspending agents and the like as well as inert dyes to impart thereto a uniformrcolor background.

' Furthermore, it'i's important to include an appropriate 7 buffer system to maintain a'desired pH range in the presence of urine. Although the pH range may vary between 4 to 7 .5, an especially preferred pH is 6.7.

Although the test device itself may comprise the reagent composition in the form of a'tablet, powder or other embodiment, we prefer to afiix 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 drying the impregnated test strips. Embodying the diagnostic composition in 'the form of test sticks insures ease and simplicity. of test procedure.

The invention will now be illustrated in greater detail, but not limited, by the following examples:

EXAMPLE I Formulation of the impregnating solution Polyvinyl'alcohol (PVA) gm 1 95% ethanol ml 15 Glycine gm 3 KI gm 1 Na MoO .2H O gm 0.5 Gelatin gm 0.4 FD & C Red #3 cm 0.015 Glucose oxidase gm 0.5 ,2,7-diaminofluorene.2HCl gm 0.3 I Phosphate buffer-pH 6.5 ml 20 Water ml 50 Total ml 3 Preparation of impregnating solution (1) 1 gm. of PVA is dissolved in ml. of water by heating.- To thisis added ml. of 95% ethanol and 10 ml. ofa gelatin-dye solution prepared by dissolving 4 gm. of gelatin and 0.15 gm. of FD & C Red #3 in 100 ml. of boiling water.

(2) 3 gm. of glycine and 1 gm. of KI are dissolved in 10 ml. of boiling water and added to solution No. v1 and cooled.

(3) 0.5 gm. of glucose oxidase (16,000 units per gram) are dissolved in.10 ml. of water and added to solution No. 2.

(4) 0.3 gm. of 2,7-diaminofiuorene dihydrochloride are dissolved in 5 ml. of Water and added to solution No. 3.

(5) m1. of phosphate butter of pH 6.5 (prepared by dissolving 43.55 gm. of dibasic potassium phosphate (K HPO and 39.0 gm. of monobasic sodium phosphate (NaH PO;.H O) in water and bringing. to a volume of 500 ml.) are added to solution No. 4 with constant stirring.

(6) Finally, 0.5 gm.-of Na MoO .2H O are dissolved in 5 ml. of 'water and added slowly to solution No. 5 with constant stirring. The impregnating, solution or mix is now ready for use.

Preparation of reag'ent'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 attrac-' tion about one-half inch of the strip at one end is completely impregnated. These strips are then dried in a. drying tunnel or in aforced draft oven. The test portion of these strips is of a red color. It will be under stood that other porous or absorbing materials, 'such as small sticks of wood, etc. and other methods of applying the impregnating solution and of drying the impregnated sticks may likewise be employed.

EXAMPLE II Formulation of the impregnating solution I No. 5.

4 solving 4 gm. of gelatin and 0.15 gm. of PD & C Red #3 in 100 ml. of boiling Water) are added to solution (7) 0.5 gm. of Na MoO .2H O are dissolved in 5 ml.-

of water and added to solution No. 6. The impregnating solution or mix is now ready for use.

Test strips are then prepared in accordance with the procedure recited in Example I.

Algin gm... 0.3 Polyvinyl alcohol (PVA) gm 1. Glycine gm 3- KI gm 1 Buffer ml 1 0 Glucose oxid am 0.5 2,7-diaminofiuorene.2HCl gm 0.3- 95% ethanol ml 15 Gelatin gm 0.4 FD & C Red #3 gm 0.015 Na MoO .2H O gm 0.5 Water ml 65 Total volume ml 100 Preparation of the impregnatingsolutio'n 1) 0.3 gm. of algin are dissolved in 20 ml. of water.

(2) 1 gm. of PVA is dissolved in 10 ml. of water by heating and added to solution No- 1.

(3) 3 gm. of glycine and 1 gm. of KI are dissolved in 10 ml. of boiling water. 10 ml. of butter (prepared by dissolving 436 gm. of dibasic potassium phosphate (K HPO in 1000 ml. of water and adding thereto 290 ml. of 2.5 M phosphoric acid) are added-to the glycine- KI solution which is then added to solution No. 2.,

(4) 0.5 gm. of glucose oxidase (16,000 u./grn.) are dissolved in 5 ml. of Water and added to solution No. 3.

(5) 0.3 gm. of 2,7-diaminofluorene dihydrochloride are dissolved in 5 ml. of water and 15 ml. of 95% ethanol. This is added to solution No. 4 with constant stirring.

(6) -10 ml. of gelatin-dye solution (prepared by dis- (2) 1 gm. of KI, 3 gm. of glycine and 0.5 gm. of Na MoO .2H O are dissolved in 20 ml. of hot water. 10 ml. of 4% gelatin-dye solution is added to this solution. The gelatin-dye solution is prepared as described in Example I. v

(3) Solution No. 2 is now added to solution No. 1 to give solution No. 3. i

(4) 0.5 gm. of glucose oxidase (16,000 units per gram) is dissolved in 10 ml. of water and added to solution No. 3. Y

(5) Finally, there is added to theimpregnating mixture 4 gm. of Cellex-p, treated with 2,7-diaminofluorene in accordance with the following procedure:

Two grams of 2,7-diaminofluorene dihydrochloride are dissolved in ml. of water. Twenty grams of cation exchange cellulose (Cellex-p, Bio-Rad Laboratories), are mixed with this solution and left standing for 30 minutes. The mix is periodically agitated during this time. The cellulose is filtered through a Biichner tunnel .and washed four times by resuspending .it in 100 ml. portions of water. The cellulose is then dried before use.

The use in this embodiment of cation exchange cellulose has a two-fold purpose: it maintains the indicator in suspension so as to prevent it from precipitating, and it serves as a buffer for the reagent composition. These functions in the embodiments of Examples I and II are performed, respectively, by polyvinyl alcohol and the buffer system.

Procedure of testing In use, an impregnated strip, made as described in the above examples, 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 5 to 30 seconds evidenced by various shades of blue color as. follows: I

With urine containing 1% or 2% glucose an intensedeep blue color develops, while with urine containing: as little as 0.1% or even less of glucose, the red color of the impregnated strip changes to a distinctly light bluecolor. With urine s containing from 0.1 to 1% of glucose the blue colors which develop are of intermediate intensities: the urine having the higher glycose content produces the deeper color. When dipped in urine con taining 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.

In addition to the compositions described in the above examples illustrating optimum contents variations in amounts of components are permissible as shown by the following chart:

The impregnated 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. However, it is recommended that the strips be stored in brown bottles containing desiccant packs.

In summary, this invention pertains to a diagnostic test for the detection of glucose in body fluids, especially in urine, consisting of a bibulous strip or stick that has been impregnated with a composition comprising glucose ox1- dase, potassium iodide, sodium molybdate and 2,7-diaminofluorene which is oxidizable by hydrogen peroxide formed in the glucose oxidase-catalyzed decomposition of glucose in the presence of said iodide and molybdate salts.

What is claimed is:

1. A diagnostic composition for detecting glucose which comprises glucoseoxidase, an iodide salt selected from the group consisting of potassium iodide, sodium iodide, and ammonium iodide, a molybdate salt for intensifying the action of the iodide and selected from the group consisting of sodium molybdate, potassium molybdate, and ammonium molybdate and a color-forming substance oxidizable by hydrogen peroxide in the presence of said iodide and molybdate salts.

2. A diagnostic composition for detecting glucose which comprises glucose oxidase, an iodide salt selected from the group consisting of potassium iodide, sodium iodide,

lected from the group consisting of 2,7-diaminofluorene, o-tolidine, and dianisidine.

3. A diagnostic composition for detecting glucose which comprises glucose oxidase, potassium iodide, sodium molybdate and 2,7-diaminofluorene.

4. A diagnostic composition for detecting glucose which comprises:

Parts Glucose oxidase 5 Potassium iodide 10 Sodium molybdate 5 v 2,7-diaminofluorene dihydrochloride 3 Potassium phosphate (dibasic) 33.8 Phosphoric acid 5.5 Algin 0-3 Glycine 30 Gelatin 4 Polyvinyl alcohol 10 'FD & C Red #3 0.15

5. A test indicator for detecting glucose which comprises a bi-bulous material which contains therein the dried residue resulting from deposition on said material, followed by drying, of a liquid comprising glucose oxidase, potassium iodide, sodium molybdate and 2,7-diamino-' fiuorene.

'6. A test indicator for detecting glucose which comprises a bibulous strip of paper which contains therein the diagnostic composition of claim 4.

References Cited in the file of this patent UNITED STATES PATENTS 2,848,308 'Free Aug. 19, 1958 2,881,213 Idol et a1. Apr. 7, 1959 FOREIGN PATENTS 203,451 Australia Sept. 27, 1956 and ammonium iodide, a molybdate salt for intensifying the action of the iodide and selected from the group con-' sisting of sodium molybdate, potassium molybdate, and

ammonium molybdate and a color-forming substance se-

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US2848308 *Dec 5, 1955Aug 19, 1958Miles LabComposition of matter
US2881213 *Dec 17, 1957Apr 7, 1959Standard Oil CoProcess for the conversion of acrolein and methacrolein to the corresponding unsaturated acids
AU203451B * Title not available
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3066081 *May 5, 1961Nov 27, 1962Edward S RoremMeans for detecting galactose
US3183173 *Jun 10, 1963May 11, 1965Miles LabTest composition for detecting hydrogen peroxide
US3266868 *Jan 24, 1962Aug 16, 1966Miles LabDiagnostic composition and test indicator
US3329486 *Sep 10, 1965Jul 4, 1967Miles LabComposition and device for testing for chloride, bromide, iodide, cyanide, and thiocyanate
US3350278 *Dec 2, 1963Oct 31, 1967Miles LabEnzymatic glucose test composition and device
US3371019 *Jan 8, 1965Feb 27, 1968Searle & CoDiagnostic indicator
US3453180 *Aug 2, 1965Jul 1, 1969Miles LabTest article
US3528780 *Apr 5, 1968Sep 15, 1970Us Air ForceVisual ammonia detector
US4143080 *Nov 11, 1977Mar 6, 1979The Goodyear Tire & Rubber CompanyMethod and reagent for the assay of hydroperoxide
US4331759 *Jul 3, 1980May 25, 1982E.N.I. Ente Nazionale IdrocarburiComposition suitable for testing biological tissues and/or liquids, and the method of use
US4376820 *Feb 23, 1982Mar 15, 1983E.N.I. Ente Nazionale IdrocarburiComposition suitable for testing biological tissues and/or liquids, and the method of use
US4654309 *Apr 9, 1985Mar 31, 1987Minnesota Mining And Manufacturing Co.Test method and article for estimating the concentration of free acid in liquid
US5382523 *Apr 13, 1993Jan 17, 1995Boehringer Mannheim GmbhUse of sparingly soluble salt of a heteropoly acid for the determination of an analyte, a corresponding method of determination as well as a suitable agent thereof
US5521060 *Oct 12, 1994May 28, 1996Boehringer Mannheim GmbhUse of a sparingly soluable salt of a heteropoly acid for the determination of an analyte, a corresponding method of determination as a suitable agent therefor
EP0266216A2 *Oct 30, 1987May 4, 1988Fuji Photo Film Co., Ltd.Control or calibration solutions for fluid analysis
WO1982002251A1 *Nov 9, 1981Jul 8, 1982Minnesota Mining & MfgMethod and article for determining free acid concentration in liquid
Classifications
U.S. Classification422/420, 436/95, 435/14, 435/805, 435/25
International ClassificationC12Q1/54
Cooperative ClassificationC12Q1/54, Y10S435/805
European ClassificationC12Q1/54