Classifications

• • 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
• • Y—GENERAL 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
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/805—Test papers

Definitions

• This invention relates to a new and improved diagnostic composition and is particularly concerned with a diagnostic test which is useful for the qualitative detection and quantitative determination of glucose in biological fluids, such as urine, and wherein the reagent composition is incorporated upon a bibulous carrier.
• glucose indicator may also be used efiiciently in routine urinalyses in hospitals and physicians oflices, in diabetes detection screening programs, in the difierentiation of glucosuria from other meliturias, and the like.
• 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.
• the reagent composition must be adequately stable.
• metalloporphyrins which are not operable per so, are, however, when activated by certain nitrogenous substances, surprisingly and unexpectedly capable of acting as catalysts to induce the oxidation of indicators by hydrogen peroxide formed when glucose is aerobically oxidized in the presence of glucose oxidase.
• the principles underlying these reactions are well known and need not be expounded.
• a diagnostic composition according to the present invention comprises as essential constituents glucose oxidase, a metalloporphyrin, such as hemin, complex-forming compounds containing amino-donor groups, such as Z-aminobenzothiazole, pyridine, bipyridyl, bipyridylpyridine, nicotinic acid or the like, and color-forming substances or indicators, such as 2,7-diaminofluorene, o-tolidine, leucoindophenols, etc.
• a metalloporphyrin such as hemin
• complex-forming compounds containing amino-donor groups such as Z-aminobenzothiazole
• pyridine bipyridyl, bipyridylpyridine, nicotinic acid or the like
• color-forming substances or indicators such as 2,7-diaminofluorene, o-tolidine, leucoindophenols, etc.
• the metalloporphyrin used in the preferred embodiments of this invention is hemin, which is commercially available and whose full chemical name is 1,3,5,8-tetramethyl-2,4-diviny1porphine-6,7-dipropionic acid ferrichloride, (C H N O FeCl).
• the following structural formula characterizes its complex chemical nature:
• H3C CH CH n 1 CH3 l /N Fe 4% nooccn cn 2 Haw H nooccn ca CH3 Hernin and the other 'metalloporphyrins that can be 0 employed in accordance with the inventive concept react wherein the 4-membered ring represents an octahedral porphyrin ring system, M represents Fe, Co, Ni, Mn, Mg,
• a metalloporphyrin it is also contemplated to employ other equivalent metallic complexes or chelates, such as phthalocyanines, the structure of which is closely analogous to that of the porphine nucleus present in hemin.
• phthalocyanines the structure of which is closely analogous to that of the porphine nucleus present in hemin.
• Further catalytic materials useful in the practice of this invention are ferrocene compounds, the ferrous salt of dimethylglyoxime and the like.
• the diagnostic composition of our invention is prepared, due to incompatibility of ingredients, by way of two separate solutions, one comprising the glucose oxidase and the indicator substance dissolved in 1:1 alcohol-water and the other containing hemin and the nitrogenous substance in an alcoholic medium.
• this glucose test may contain a suitable inert dye to impart to the composition a uniform background color as well as an appropriate buffer system to maintain a desired pH range (pH 3 to 7 with a pH of 5.4 being preferred).
• a pH 5.4 buffer solution (prepared by dissolving 352.8 g. of sodium citrate monohydrate and 25.6 g. of anhydrous citric acid in water and diluting to 1000 ml.) was added with stirring.
• a solution of 2 g. of gelatin and 150 mg. of ED. & C. Red No. 3 (disodium salt of 9-o-carboxyphenyl 6 hydroxy 2,4,5,7 tetraiodo-3-isoxanthone) in 100 ml. of hot water was prepared and 20 ml. of this solution was added to the algin-bufier solution with stirring. Then a solution of 200 mg. of 2,7diamino-fiuorene dihydrochloride in 20 m1.
• Solution B.Hemin, 50 mg, and 500 mg. of 2-aminobenzothiazole were added to 50 ml. of 95 ethanol and 50 ml. of 10% aqueous polyvinyl alcohol and the mixture was heated to boiling and shaken for two minutes. The excess hemin was removed by filtration, and the filtrate was allowed to cool to room temperature.
• Bilbulous strips such as filter paper cut into narrow strips, small sticks of wood, or other porous or absorbing material with a water-impervious barrier of ethyl cellulose, one-half inch from the tip, were dipped into 'solution A so that through the process of submersion Procedure of Testing In use, an impregnated strip made as described above is dipped into the liquid specimen to be tested. When contacted with urine containing glucose, test strips gave positive reactions in about one minute evidenced by various shades of blue color as follows:
• EXAMPLE II Twelve ml. of a buffer solution (which was prepared by dissolving 7.4 g. of anhydrous citric acid and 32.6 g. of trisodium citrate dihydrate in 100 ml. of water) was added rapidly with good stirring to 10 ml. of a warm gelatin solution (which was prepared by dissolving 4.8 g. of gelatin in 100 ml. of boiling water). One hundred mg. of o-tolidine dihydrochloride was mixed with 10 ml. of 95% ethanol (2B), and the mixture was added rapidly to the gelatin-buffer solution with good stirring. Then 10 ml. of a glucose oxid-ase solution was added, which was prepared by dissolving 0.5 g. of glucose oxidase (having an activity of 16,000 units per gram) in 50 ml. of water, and the mixture was stirred until well mixed. Paper strips were dipped into this solution and dried at 100 C. for 10 minutes.
• EXAMPLE III A mixture of 50 mg. of hemin, 500 mg. of nicotinic acid, 50 ml. of ethanol and 50 ml. of 10% aqueous polyvinyl alcohol was warmed and shaken for 5 minutes. The excess hemin was removed by filtration. Strips described in Example IIA were impregnated with this mixture and dried at for 10 minutes. These strips developed a blue-green color after 5 minutes with urine containing 1% glucose and only a very faint green color with urine containing glucose.
• EXAMPLE IV When 50 mg. of Z-aminobenzothiazole was substituted for the nicotinic acid in Example III, the strips were a little more active, and a blue-green color developed with 1% glucose in urine in 3 minutes.
• EXAMPLE V (A) Twenty ml. of hot buffer solution (which was prepared by dissolving 352.8 g. of sodium citrate monohydrate and 25.6 g. of anhydrous citric acid in water and diluting to 1000 ml.) was added with stirring to a solution of 200 mg. of algin in 20 ml. of water. Then 20 ml. of a hot gelatin-dye solution (Which was prepared by dissolving 2 g. of gelatin and mg. of ED. & C. Red No. 3 dye in 100 ml. of water) was added with stirring and followed by a solution of 2,7-diaminofiuorene dihydrochloride (200 mg.) in 20 ml.
• hot buffer solution which was prepared by dissolving 352.8 g. of sodium citrate monohydrate and 25.6 g. of anhydrous citric acid in water and diluting to 1000 ml.
• this invention pertains to a diagnostic composition for the detection of glucose in body fluids
• a bibulous strip or stick that has been impregnated with a composition comprising glucose oxidase, a metalloporphyrin, such as hemin, a complex-forming compound containing aminodonor groups, such as 2-aminobenzothiazole, nicotinic acid or pyridine to activate the metalloporphyrin and a color-forming substance, such as 2,7-diaminolluorene or o-tolidine, which is oxidizable by hydrogen peroxide in the presence of the activated metalloporphyrin.
• the composition containing glucose oxidase, hemin, Z-aminobenzothiazole and 2,7-diaminofiuorene constitutes the preferred embodiment of this invention.
• a diagnostic composition for detecting glucose which comprises glucose oxidase, a metalloporphyrin, a complexforming compound containing amino-donor groups selected from the class consisting of 2-aminobenzothiazole, nicotinic acid and pyridine to activate said metalloporphyrin and a color-forming substance taken from the group consisting of 2,7-diaminofluorene and o-tolidine, oxidizable by hydrogen peroxide in the presence of the activated metalloporphyrin.
• a diagnostic composition for detecting glucose which comprises glucose oxidase, hemin, a complex-forming compound selected from the class consisting of Z-aminobenzothiazole, nicotinic acid and pyridine to activate said hemin and a color-forming substance taken from the group consisting of 2,7-diaminofiuorene and o-tolidine, oxidizable by hydrogen peroxide in the presence of the activated hemin.
• a diagnostic composition for detecting glucose which comprises glucose oxidase, hemin, Z-aminobenzothiazole to activate said hemin by forming a complex therewith and 2,7-diaminofluorene oxidizable by hydrogen peroxide in the presence of the activated hemin.
• a glucose-detecting means including a bibulous carrier impregnated with a diagnostic composition comprising glucose oxidase, hemin, Z-aminobenzothiazole to activate said hemin, and 2,7-dia-minofluorene oxidizable by hydrogen peroxide in the presence of the activated hemin.
• a glucose-detecting means including a bibulous carrier impregnated with a diagnostic composition comprising glucose oxidase, hemin, an amino-donor selected from the group consisting of Z-aminobenzothiazole, nicotinic acid and pyridine to activate said hemin and 2,7-diaminofluorene oxidizable by hydrogen peroxide in the presence of the activated hemin.
• rier impregnated with a diagnostic composition comprising glucose oxidase, hemin, pyridine to activate said hemin and 2,7-diaminofiuorene oxidizable by hydrogen peroxide in the presence of the activated hemin.

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• General Engineering & Computer Science (AREA)
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Cited By (12)

• 0
  • NL NL102893D patent/NL102893C/xx active
• 1959
  • 1959-03-27 US US802281A patent/US3042496A/en not_active Expired - Lifetime
• 1960
  • 1960-03-19 DE DEM44720A patent/DE1185841B/de active Pending
  • 1960-03-25 GB GB10679/60A patent/GB886778A/en not_active Expired

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