DD235115A1 - METHOD FOR QUANTITATIVE TRACE DETECTION OF HYDROGEN PEROXIDE - Google Patents

Abstract

The invention relates to a method for quantitative trace detection of hydrogen peroxide by oxidative formation of photometrically evaluable dyes from dye precursors (leuco dyes). The aim of the invention is the simple quantitative determination of H2O2 traces without great expenditure on equipment. The object of the invention is to reinforce the oxidative formation of the dye in such a way that a photometrically evaluable dyeing results. The object is achieved by using a Leukotriphenylfarbstoffes which is irradiated after formation of the dye by the presence of H2O2 traces in the presence of polyhalocarbon with light of the absorption wavelength of the corresponding dye. In this case, an amplification effect of the dyeing occurs, which can be evaluated photometrically. The invention is used in medical diagnostics, for example in the determination of glucose and in biotechnological process control.

Description

Field of application of the invention

The invention relates to a method for quantitative trace detection of hydrogen peroxide. It is used in medical diagnostics, for example in the determination of glucose and in biotechnological process control.

Characteristic of the known technical solutions

Hydrogen peroxide has as a carrier of the analytical information importance in the detection of substrates or in the determination of the activity of specific oxidases, for example in the glucose determination by glucose oxidase in physiological fluids such as blood and urine and in biotechnological process control. Hydrogen peroxide also acts as an information carrier in the use of specific oxidases, for example glucose oxidase as a marker enzyme for immunological tests ("enzyme immunoassays") The application of dye precursors ("chromogens"), for example of leuco dyes, by hydrogen peroxide, usually under catalysis of peroxidase, more or less quantitatively converted to dyes has long been known. The total amount of dye required for the photometric evaluation must be formed by oxidation by means of hydrogen peroxide, which limits the sensitivity of the systems, either because of the low concentration of the primary analyte or the limited functional stability or the low concentration of the marker enzymes, for example glucose oxidase or peroxidase, or the low specific absorption of the dye formed.

Object of the invention

The aim of the invention is H ₂ O ₂ traces, as may occur in the execution of the above tests with lowest Analvtkonzentrationen, to determine quantitatively without much equipment.

Explanation of the essence of the invention

The invention is based on the object in the quantitative H ₂ O ₂ detection, the concentration of the dyes normally formed by the H ₂ O ₂ oxidatively from dye precursors ("chromogens") in the presence of traces of H ₂ O ₂ proportional to H ₂ O to reinforce ₂ amount so that a photometrically evaluated amount of dye is produced. the object is achieved by a method for the quantitative detection of H ₂ O _2, which is characterized in that there are used as chromogens Leukotriphenylmethanfarbstoffe that in aqueous solutions in the presence of peroxidase (s) are stoichiometrically converted to the corresponding dyes These dyes are extracted with water immiscible solvents, preferably ketones such as cyclohexanone, and aliquots of the extract are removed in the absence of light in a 0.01 to 0.5 molar solution of Leukotriphenylmethanfarbstoffs, in the molar ratio of 1:10 to 10: 1, vorzu 1: 1 to the leuco triphenylmethane dye containing a bromine-containing polyhalogenated hydrocarbon, preferably tetrabromomethane, in a ketone, preferably acetone. By irradiation with light of the absorption wave length of the triphenylmethane dye, leuco dye is converted into the dye in proportion to the initial amount of the Ti'-diphenylmethane dye, whereby an enhancement effect occurs which is greater the longer the irradiation time and intensity, and therefore the irradiation duration and intensity are as follows be chosen that after 10 to about 30 minutes, depending on the concentration of the initial analyte extinctions between 0.1 and 1.5 are obtained. By control measurements, a check of the required extinction is readily possible. The calculation of the H ₂ O ₂ content or the content of H ₂ O ₂ -generating analyte in the sample is carried out by comparing the time course of an identically treated sample known content of H ₂ O ₂ or primary analyte, advantageously two measurements at a time interval be made of about 10 minutes. This procedure also has the advantage that the result is not affected by small amounts of possibly autoxidatively developed dye. In principle, however, it is also possible to carry out a single measurement and to calculate the analyte content on the basis of a calibration curve to be set up beforehand.

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As water-miscible solvents ketones, alcohols or ethers are used and as polyhalogenated hydrocarbons bromine-containing poly-halogenated hydrocarbons, such as carbon tetrabromide, Hexabrommethan, Dichlordibrommethan, (tribromomethyl) benzene.

Suitable triphenyl dyes are compounds of the general formula I, for example rosaniline, malachite green and crystal violet.

The leuco form is converted by oxidation into the dye

Leuco Dye (DH) Dye (D ⁺ X ") X "- anions, for example, phosphate" halide

The oxidizing agent is the H ₂ O ₂ H ₂ O ₂ -generating system H ₂ O _{2 which is to} be detected

2DH ₊ H ₂ O ₂ ² < ^{D + X} ") ^{+ 2 H} ₂ °

The amplification reaction is initiated by the irradiation with light of the wavelength 600 nm ,,. + Ν-. Irradiation ( ^D * J Ö'ÖÖ nro

⁺ X ") + DH + CBr 2 (D ⁺ X)

(D ⁺ X ") + DH + CBr ₄ 2 (D ⁺ X") * CHBr ₃

The invention allows in a simple manner the quantitative determination of H ₂ O ₂ in such low concentrations, the determination of which was hitherto only possible by means of special ultramicroanalytical methods, such as immunotechniques or the like, in specially equipped laboratories.

embodiments example 1 Determination of the activity of glucose oxidase as H ₂ O ₂ -forming enzyme in the pico-unit range

Solutions:

I. phosphate buffer 0.1 molar, pH = 7.0, saturated with air

II. Leukomalachite Green Solution 0.005 Molar (165 mg / 100 mils)

III. Glucose solution 0.5 molar (9 g / 100 ml I)

IV. Horseradish peroxidase (EC 1.11.1.7.); (prepare fresh) about 10 units in 0.1 ml of I

V. Prepare amplifier solution (with exclusion of light and store it!)

1.2 g leucomalachite green + 1.2 g tetrabromomethane in 18 ml acetone p. A., freshly distilled, dissolved.

VI. Serial dilution: 0.01 to 0.001 units of glucose oxidase (EC 1.1.3.4.) Each dissolved in 1 ml of I. Temperature: 25 ° C

2.47 ml of the solution II are mixed with 0.5 ml of the solution III and 0.01 ml of the solution IV added. By adding 0.02 ml of the samples of the dilution series VI, the reaction is started and after 5 minutes by addition of 0.5 ml of cyclohexanone p. A., freshly distilled, stopped. Briefly shake, separate the phases in the laboratory centrifuge and, under exclusion of light, add 0.25 ml of the upper phase to 2.5 ml of the amplifier solution V, with a 100 W collimator lamp which is opaque with a filter which is opaque below 600 μm, for 30 minutes with extraneous light irradiate. Thereafter, in the spectrophotometer at a wavelength of 625 nm, layer thickness 1 cm, the absorbance against a blank, for the production of which only buffer was used in place of glucose oxidase solution, measured. Results s. Fig. 1.

The determination of unknown glucose oxidase activity succeeds with an accuracy of ± 15%, based on the actual activity obtained.

Example 2 Determination of glucose oxidase as H ₂ O ₂ -forming enzyme in the nano-unit range

As in Example 1, except that instead of the amplifier solution V, the amplifier solution Va of the following composition is used:

Va amplifier solution 0.3g leucomalachite green

0.3 g of tetrabromomethane

in 18ml acetone p. A., freshly distilled, dissolved.

The dilution series VI is used in the concentration range of 0.1 to 10 units of glucose oxidase in each of 1 and I. Results: Fig. 1 The determination of unknown glucose oxidase activities is thus achieved with an accuracy of ± 10%.

Example 3 Determination of glucose in the nano-mol range by means of glucose oxidase / peroxidase

Solutions I, II, IV and V as in Example 1

Instead of solution III, a solution of about 5 units of glucose oxidase in 0.1 ml of I (solution purple) VIa. Dilution series of 5 x 10 ^-9 to 10 ^-7 moles of glucose dissolved in 1 ml each I Temperature: 25 ° C

2.47 ml of the solution II are admixed with 0.5 ml of the respective sample of the calibration solution VIa and 0.01 ml of the solution IV are added.

By adding 0.02 ml of III a, the reaction is started. Continue as in Example 1.

Results: Fig. 2

The determination of unknown glucose concentrations (amounts) thus succeeds in the specified concentration range with an accuracy of ± 10%.

Claims (4)

-1- 737 64 Invention claim: 1. A method for the quantitative trace detection of hydrogen peroxide by oxidative formation of photometrically evaluable dyes from dye precursors (leuco dyes), characterized in that as dye losses a Leukotriphenylmethanfarbstoff the general formula I is used, that this dye precursor in aqueous solution in the presence of peroxidase by the H ₂ O ₂ crystals is stoichiometrically converted to the dye, the dye formed is extracted by a H ₂ O immiscible solvent and an aliquot of the extract in the dark in a 0.01 to 0.5 molar solution of Leukotriphenylmethanfarbstoffes in a mixable with H ₂ O. organic solvent is added, this solution in a molar ratio of 1:10 to 10: 1 to the Leukotriphenylmethanfarbstoff a polyhalogenated hydrocarbon, and then irradiating the sample with light of the absorption wavelength of Triphenylmethanfarbs Toffes done 2. The method according to item 1, characterized in that the irradiation of the sample is carried out for about 10-30 min until an extinction of about 0.1-1.5 is formed. 3. The method according to item 1 + 2, characterized in that the water-miscible organic solvent is a ketone, alcohol or ether. 4. The method of item 1-3, characterized in that the polyhalogenated hydrocarbon is a bromine-containing polyhalogenated hydrocarbon such as carbon tetrabromide, hexabromomethane, (tribromomethyl (-benzene, dichlorodibromomethane. For this 3 pages drawings