DE2213721A1 - Glucose detection - Google Patents

Description

2 1st M & IB72

Dr. f rui; / foursome ο ο ι ο * 7 ο 1

PATENT ADVOCATE! LL \ 0 IL I

RAN 4090/33

F. Hoffmann-La Roche & Co. Aktiengesellschaft, Basel / Switzerland

Glucose detection

The present invention relates to a means and a method for the detection of glucose in liquids. The invention also relates to a method for producing such an agent. The agent according to the invention is characterized in that it contains glucose oxidase, a phenazine and / or phenoxasin compound and a tetrasolrum salt.

To determine the glucose the method of the erizymatic Oxidation with glucose oxidase and oxygen and subsequent detection of the resulting reaction products Peroxide or gluconic acid by means of a color reaction has become more and more popular in recent years. An essential one The disadvantage of this method is that the oxygen content can vary greatly in different samples It is never certain whether the reaction will proceed quantitatively. Attempts have been made to solve the problem by adding an endogenous one

209841/1056 ■

Oxygen donor (peroxide or the like) to bypass the reaction solution. This naturally makes the glucose determination limited to the relatively insensitive gluconic acid detection. (U.S. Patent No. 3,164,534.) In the agent of the present invention the phenazine or phenoxazine compound (Phen) functions in the enzymatic reaction as hydrogen acceptor. The thereby themselves The reduced form that forms then reacts in a rather rapid, non-enzymatic reaction with the tetrazolium derivative, reduces this to the corresponding Pormazan and is itself oxidized again to the starting compound in order to be used again for the the same reaction cycle to be available:

Glucose + phen> gluconic acid + phen

ox reu

Phen, + tetrazolium > Vhen + formazan

red ox

The resulting formazans, which are very colorful and stable, are used to detect the glucose present.

As phenazine compounds come for the inventive Means phenazinium salts, especially metho- or etho-compounds

considerations, e.g. the commercially available phenazinium methosulfate. Phenoxazinium salts, in particular, are used as phenoxazine compounds T-Diraethylamino-l ^ -benzophenoxazinium chloride (Meldola blue) or 7-dimethylamino-2 '-hydroxy-1-4 -benzophenoxazinium chloride into consideration.

As examples of tetrazolium salts, thiazolyl blue (2,5-diphenyl-3- (4,5-dimethy.l.-2-thiazolyl) -mono-tetrazolium bromide), nitrotetrazolium hose chloride (3,3'-dianisyl-4,4 · - bis [2 - (4-nitrophenyl) -5-phenyl] tetrazolium chloride), iodonitrotetrazolium chloride (2- (p-iodophenyl) -3- (p-nitrophenyl) -5-phenyltetrazolium chloride), neotetrazolium chloride (2,2 ', 5,5'- Tetraphenyl-3- (4,4 ^! -Biphenylene) -ditetrazolium chloride), tetranitrotetrazo.Lium blue {z, Z ^x , 5,5'-tetranitrophenyl-3- (3,3'-dimethoxy-4,4'-biphenylene) - ditetrazolium chloride), triphenyltetrazolium chloride, tetrazolium violet (2,5-diphenyl-3- (naphthyl) tetrazolium chloride) and tetrazolium blue (3,3'-dia-

2098A1 / 1056

nisyl-4, k '-bis- (3,5-diphenyl) -tetrazoliumchlorid) can be mentioned. Thiazolyl blue, nitrotetrazolium blue chloride and iodonitrotetrazolium chloride are preferred.

The agent according to the invention can be in solid form, e.g.

in the form of a powder or tablet. Preferably it is as

Solution before or is by impregnation on an absorbent

Carriers such as paper, cardboard, textile fabric, wood, etc. applied.

The solutions are mainly aqueous solutions, but it is two-fold to use an organic solvent, e.g. a lower alcohol such as ethanol, or a solubilizer such as polyethylene glycols or polyoxysorbitanic acid esters add in order to convert the formazans, which are insoluble in water and which are formed in the course of the detection reaction, into Hold solution. Instead of a solvent, it is also possible to use a suspension for this purpose effect of the dye, such as gelatin or alginates.

The concentration of the individual components of the agent according to the invention in solutions is not critical in the narrower sense; however, the concentration of glucose oxidase is preferably 0.5-5 mg / ml, of phenaain or phenoxazine compound 0.1-2.5 mmol / ml and of tetrazolite salt 1-10 mmol / ml.

For two purposes, the glucose production according to the invention is carried out in a pH range between approximately pH 4 and pH 8, preferably pH 5.5 ^and pH 7.5. The agent according to the invention therefore preferably contains a corresponding buffer system, for example a phosphate buffer, the molarity of which is 0.1-0.4 in two dimensions.

Body fluids, in particular, are used as the test substrate such as urine, blood, or serum or plasma and liquor.

2098A1 / 1056

-H-

The evaluation of the detection method according to the invention takes place, as already indicated, on the basis of the color and the Color intensity of that formed from the tetrazolium component Formazans.

As a variety of tetrazolium compounds are available there are various possibilities for following the reaction. So can be for the semi-quantitative ülucose determination uses the tetrazolium compound whose formazan absorbs light in the most sensitive spectral range for the eye, but for the quantitative Determination of the glucose by means of a photometer the compound whose formazan at a given by the device Maximum wavelength absorbed. Some examples are given in the table.

Tabel

Tetrazolium component

Thiazolyl blue

Te tr anitro tetrazo].! umblau

NitrotetrazoliUiTiblauchlorid

Iodonitrotetrazine chloride

Neotetrazolium chloride

Tr 5 phenyltetrazolium chloride

ΧΚΪ LJ- aiUlllllllVlUlC LL-Totrazoln ur.iblau

Wavelength of the Color of Abs or pti ons maxim urns
of formazan in Formazans Polyethylene glycol 570 nm violet ^ ₁ ) ^ j Kf I j in blue red b8o nm blue 456 nm blue ^> 6u nm Red ca «^ _r .,
} <J ^ J J Jill I ¹ Ot 'j iO ilia ν i υ 1 a LL 570 nm blue

209841/1056

A major advantage of the new glucose detection is that the reaction is both aerobic and in particular also expires under anaerobic conditions. Another, Particularly in the case of clinical use, the significant advantage is that in the presence of ascorbic acid no a negative result is simulated.

The following examples illustrate the invention:

example 1

For the quantitative determination of glucose one prepares the following reagent solution:

Buffer (0.3 M potassium phosphate pH 7.0) 1.0 ml

Phenazinium methosulfate (2 mg / ml) 0.05 ml

Thiazolyl blue (10 mg / ml) 0.4 ml

Glucose oxidase (20 mg / ml) 0.05 ™ 1

1 ml of the sample solution is added to this solution. The mixture is then left to stand at room temperature for about 2 minutes the reaction mixture with the same volume of ethanol and measures the extinction difference against one in a photometer Blind var search in the wavelength range from about 500 - 600 nm The glucose content of the sample can then be taken from a calibration curve.

Example 2

The working procedure in Example 1 is modified by adding 1 g of gelatin to 100 ml of said buffer is resolved. The resulting formazan is thereby in suspension göhsxten and. can dxrekt goiuessen wsrusn.

Example ρ

The work;.; Rule in example 2 becomes rnod.i 2098 4 1/1056 "

confirmed that gelatine was replaced by Tween 8l.

Example 4
A solution is being prepared

Glucose oxidase 500 mg

Phenazinium methosulfate 10 mg

Thiazolyl blue 500 mg

0.5 M phosphate buffer pH 6.6 50 ml

Water 40 ml

Absorbent paper is impregnated with this solution and dried in the dark. By dipping the so impregnated Paper in the sample to be examined, its glucose content can be determined semi-quantitatively.

Example 5
A solution is being prepared

GluoosGOxidase (20 mg / ml) 0.05 ml

Meldola blue (2 mg / ml) 0.05 ml

Iodonitrotetrazolium chloride (2.5 mg / ml) 0.40 ml buffer (0.2 M potassium phosphate pH 7.0,

Vfo of polyethylene glycol 1500) 1.50 ml

1 ml of the sample solution is added to this solution and the glucose content from the absorbance differs, for example at 492 nm, measured photometrically.

2 09841/1056

Claims (13)

Claims means for the detection of glucose in liquids, characterized in that it contains glucose oxidase, a phenazine and / or contains a phenoxazine compound and a tetrazolium salt. 2. Agent according to claim 1, characterized in that it contains a phenazine salt as the phenazine compound. 3. Means according to claim 2, characterized in that it contains a phenazinium motho or • etho compound as a phenazinium salt. 4. A composition according to claim 1, characterized in that it contains _f as a phenoxazine compound, a Phenoxaziniumsalz. 5. Agent according to claim 4, characterized in that it contains a TD.imethyl-amino-l ^ -benzophenoxazinium salt or a 7-Mmethylaniino ~ 2 '-hydroxy-l ^ -benzophenoxazinium salt, in particular a chloride daΛ ^r on, as the phenoxazinium salt. 6. Agent gomäcs one of claims 1-5 »characterized in that it is thiazolyl blue, nitrotetrazoliuablau, iodonitrotetra; io3.iu: nchlorid, fsotetrasolium, triphenyltetra ^ oliumchlorid as tetrazolite salt. Tetrazoliunivi oil ett _; Contains zolive blue or tetranitrotetrazolium blue. 209 8 Λ 1/1056 7. Agent according to claim 6, characterized in that it is thiazolyl blue, tetranitrotetrazolium blue or iodonitrotetrazolium chloride .contains. 8. Agent according to claims 1-7, characterized in that it contains a buffer system for maintaining a pH of K to 8. 9. Means according to claims 1 - 8 ^ characterized in that that it is applied to an absorbent material. 10. Means according to claims 1-8, characterized in that that it contains water and a water-miscible organic solvent. 11. Agent according to claim 10, characterized in that it is a lower alcohol, a polyglycol, polyoxysorbitan ester, Contains gelatin or alginates. 0 9 8 4 1/1056 12. Method for the determination of glucose in liquids, characterized in that the material to be examined is brought together with an agent according to claims 1-11. 13. Process for the preparation of an agent according to claim 1, characterized in that one glucose oxidase, a phenazine or phenoxazine compound and a Tetrazolium salt mixed together. 209841/1056