Patent GB-784548-A (GB)
Title: Improvements in or relating to glucose indicator
Inventor: KESTON ALBERT S
IPC: C12Q1/54
Filed: 1955-07-07
Abstract: An indicator composition for detecting or estimating glucose in industrial or body fluids, e.g. urine or blood, comprises an enzyme capable of catalysing a reaction of glucose, and a substance producing a colour change in the presence of a product of the reaction.  A suitable enzyme is glucose oxidase of the flavoprotein type, obtained from moulds; glucose reacts with the oxygen of the air in the presence of this enzyme to produce gluconic acid and reduced flavin; the latter is oxidized to flavin, hydrogen peroxide being formed.  The indicator may include a substance which changes colour on oxidation or reduction by hydrogen peroxide; such substances are potassium iodide, which may be used alone or together with starch, ortho-phenylene diamine, alcoholic gum guaiac, or o-tolidine; lead iodide or sulphide; or a mixture of a ferrous salt and tannic, gallic, or pyrogallic acid.  Alternatively substances which change colour on reduction by reduced flavin may be used; examples are methylene blue, thionine, sodium 2:6-dichloro-benzenone indophenol, sodium 2:6-dibromo-benzene indophenol, and brilliant cresyl blue; in this case the enzyme catalase should preferably be present to destroy the hydrogen peroxide formed.  Another suitable enzyme for the reaction of glucose is glucose dehydrogenase, a pyridino-protein type enzyme which converts glucose to gluconic acid, the pH of the solution being determined colorimetrically; diphosphopyridinonucleatide should be added to this indicator composition.  A further enzyme is hexokinase which is used in conjunction with adenosine triphosphate to produce an increase in acidity in the presence of glucose.  A preferred indicator composition comprises two enzymes, one of which catalyses the formation of hydrogen peroxide from glucose, and the other, e.g. peroxidase, catalyses the oxidation of another substance by hydrogen peroxide; in place of peroxidase, substances possessing similar activity may be used, examples being hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemochromogen, alkaline hematin, hemin derivatives, iron sulphocyanate, iron tannate, ferrous ferrocyanide, and potassium chromic sulphate on silica gel.  Suitable colour-forming substrates are aniline, ortho-toluidine, para-toluidine, ortho-phenylene diamine, N,N1-dimethylpara - phenylenediamine, N,N1 - diethyl - phenylenediamine, benzidine, dianisidine, phenol, thymol, ortho-, meta- and para-cresols, alpha- and beta-naphthols, guaiacol, catechol, orcinol, pyrogallol, p,p - dihydroxydiphenyl, phloroglucinol, benzoic, salicylic, pyrocatechnic, and gallic acids, leucomalachite green, leucophenolphthalein, 2,6-dichlorophenolindophenol, adrenaline, the flavones, tyrosine, dihydroxyphenylalanine, tryptophane, gum guaiac, guaiaconic acid, nadi reagent, water-soluble iodides, and bilirubin.  The indicator composition may include a phosphate buffer.  The indicator may be in the form of a treated paper, bottled reagent, frangible capsule, a pill or tablet, or a solid polyethylene glycol gel containing the reagent; a heat-generating substance, e.g. lithium chloride, may be included in the pill or tablet.  The paper may be treated in strips, each strip containing a reagent of different sensitivity or giving different colours.  The colour produced by the test may be measured photo-electrically, colorimetrically, or spectrophotometrically, and may be compared with standards produced by known concentrations of glucose.  In examples: (1) an aqueous solution containing ethyl alcohol, o-tolidine, glucose oxidase, horseradish peroxidase, and tartrazine is applied to filter paper and dried; (2)-(8) liquid reagents are prepared from glucose oxidase solution and peroxidase extract, the other ingredients being gum guaiac, with or without phosphate buffer, ortho-phenylene-diamine with or without alpha-naphthol, and potassium or sodium iodide, with or without starch; (9) glucose oxidase solution is mixed with an aqueous solution of bromcresol purple indicator; (10) paper impregnated with a pH dye indicator is sprayed with glucose oxidase solution. Other indicator compositions are referred to, additional ingredients being diaphorase, xanthine oxidase, Zwischenferment, triphosphopyridinonucleotide, old yellow enzyme, and new yellow enzyme.ALSO:A peroxidase extract, for use in a glucose indicator composition (see Group III), is prepared by extracting grated horseradish with water, adding ammonium sulphate to 90 per cent saturation, filtering off the precipitated protein, and dissolving in water.  The resulting solution is dialyzed to remove ammonium sulphate and heated to 70 DEG  C. for a few minutes to destroy enzymes other than peroxidase.