RU1768642C - Indicator test-device for assay of enzymatic reaction substrates - Google Patents

Abstract

The invention relates to analytical biochemistry and can be used in clinical studies, in the food and microbiological industry, in fermentation. The purpose of the invention is to reduce the cost and simplify the manufacturing technology of the indicator test device. The indicator test device for determining the concentrations of enzymatic reaction substrates is a capsule made of a transparent heat-resistant material, the components of which contain a layer of true jelly for applying the sample, and the invention relates to the field of analytical biochemistry and can be used in clinical studies, and food and microbiological industry, fermentation. The purpose of the invention is to simplify manufacturing techniques and reduce costs. The indicator test device is in the form of a capsule, in which a layer for reagents, which can also be included in the jelly. Moreover, the layer for applying the sample and the jelly layer containing the reagents are spatially separated from each other and are in different parts of the capsule. After contact with the test sample for several minutes, the part of the capsule containing the layer for applying the sample, the capsule is sealed and immersed in warm water, the temperature of which does not exceed 60 ° C. After a few minutes under such conditions, the jelly in the capsule is melted, mixed, which initiates a reaction accompanied by a change in color. Visual assessment of the color intensity allows one to judge the concentration of the analyte in the test sample with an error of no more than 25%. If a photometer is used with an indicator test capsule, a quantitative analysis can be performed. Gelatin, agar-agar and some other compounds are used to form jellies. The design of indicator test capsules actually allows any spectrophotometric method to analyze substrates of enzymatic reactions. 3 ill. the sample is spatially separated from the reagents, some of the reagents, like the layer for applying the sample, are made on the basis of true jellies. An indicator test capsule device is shown in FIG. 1 and 2, where 1 are capsule parts, 2 are caps used to seal reagents inside capsule parts during transportation or storage. In FIG. 3 shows the capsule assembly, where 1 is the cuff E "." A H O 00 o N N5

Description

any design used to seal the reagents inside the capsule during the analysis,

The capsule may be made of any heat-resistant transparent material, for example glass or plastic. The size of the capsule, with the exception of its inner diameter, is not critical. The inner diameter determines the amount of substrate transferred from the sample to the capsule. Each part of the capsule is used to form a layer in it for depositing a sample of either the reaction layer, or for storing reagents not included in the reaction layer. The choice of storing the reagents in dry form or placing them in the reaction layer, as well as the number of reaction layers, depending on the number of reagents, are determined by the conditions necessary for the long-term preservation of the reagents and the reaction conditions. The formation of these layers is carried out on the basis of compounds that form true jellies at temperatures up to 30 ° C, capable of changing their state of aggregation depending on temperature (gel-solution). Such compounds include gelatin, agar-agar, polyvinyl alcohol, methyl cellulose and some others. Thus, for example, at temperatures up to 30 ° C, a 10% gelatin solution is jelly, and with increasing temperature it easily melts to form a solution in which the diffusion coefficient of low molecular weight compounds does not practically differ from the diffusion coefficient in water.

Determination of the concentrations of the substrates of enzymatic reactions using indicator test capsules is carried out as follows: a few drops of the test solution are placed on the layer for applying the sample for a certain time. During this time, diffusion of low molecular weight compounds and occurs in the jelly layer. in particular the test compound. The amount of diffused substrate will be directly proportional to its concentration in the sample / and the diameter of the portion of the capsule containing the sample coating layer /, which is removed by rinsing with a soft stream of water or shaking. The capsule is sealed, connected to its other parts containing reaction layers and reagents, and placed in warm water / with a temperature not lower than 37 ° C and not higher than 60 ° C /, As a result of which the jelly is melted, the layer for applying the sample and reaction the layers are mixed, the solution is dry reagents, which initiates the enzymatic

reaction. 5-15 minutes after the start of the reaction, the color of the indicator test capsule is compared with a standard scale.

Example 1. Indicator test capsules for determining glucose concentration. Preparation of the reaction layer: 1 g of gelatin, 20 mg of glucose oxidase, 10 mg of phenol, 20 mg of peroxidase, 20 mg of 4-aminoantipyrine and 10 mg of sodium azide are dissolved at 40-45 ° C in 10 ml of phosphate buffer solution / 0.05 M pH 7.2 /. 100 μl of the prepared solution is placed in one of the halves of the indicator test capsule / height 15 mm, inner diameter 4 mm /.

Preparation of the sample application layer: 1 g of gelatin, 10 mg of phenol and 10 mg of sodium azide are dissolved at 40-45 ° С in 10

ml of phosphate buffer solution / 0.5 M pH 7.2 /. 100 μl of the prepared solution is placed in the other half / height 10 mm, inner diameter 4 mm / indicator test capsules.

Determination of glucose concentrations using indicator test capsules is carried out as follows: half of the capsule containing the image coating layer is placed in the test solution for 2 minutes. At the indicated time interval

this part of the capsule is removed, washed with water, combined with the other half,

containing the reaction layer, and placed

in the thermostat / 50 ° C / for 10 min, after which

compare the color of the capsule with a standard scale made using calibration solutions. The range of visually / stained / glucose concentrations analyzed using the indicator capsules thus prepared is 5-26 mM.

The material from which the test capsules are made is glass. Capsule sealing is carried out using small

tubes / not more than 4 mm long /, obtained by cutting a silicone hose, which are tightly put on both halves of the capsule when connecting them with the ends.

Example 2. Indicator test capsules for determining the concentration of ethyl alcohol.

Preparation of the reaction layer: 1 g of gelatin, 20 mg of alcohol oxidase, 10 mg

phenol, 20 mg of peroxidase, 20 mg of 4-amino-antipyrine, 10 mg of sodium azide and 10 mg of semicarbazide are dissolved at 40-45 ° C in 10 ml of phosphate buffer solution / 0.2 M pH 7.8 / containing 1 mm EDTA. 100 μl

the prepared solution is placed in one of the halves of the indicator test capsule / height 15 mm, inner diameter 4 mm /.

The design of the test capsule, the composition of the layer for applying the sample and the sequence of operations during the determination are similar to Example 1.

Example 3. Indicator test capsules for determining urea concentration.

Preparation of the reaction layer: 0.1 g of agar agar, 170 mg of urease, 20 mg of bromothymol blue, 15 mg of sodium azide are dissolved at 50 ° C in 10 ml of ammonium citrate buffer solution / 0.01 M pH 6.5 / containing 1 mM EDTA, 100 μl of the prepared solution is placed in one of the halves / height 15 mm, inner diameter 4 mm / indicator test capsule,

Preparation of the sample coating layer: 0.1 g of agar-agar and 15 mg of sodium azide are dissolved at 50 ° C in distilled water / using a weak alkaline solution, the pH of the water is adjusted to 6.2 / with a volume of 10 ml of 100 μl of the prepared solution placed in another part of the capsule / height 10 mm, inner diameter 4 mm /,

The design of the test capsule and the sequence of operations during the determination are similar to Example 1. The transition from light green to blue staining correlates with a change in the concentration of urea in the sample in the range of 2-13 mm.

PRI me R 4, Indicator test capsules for determining the concentration of ethanol.

Preparation of the sample application layer: 0.1 g of agar-agar and 10 mg of sodium azide are dissolved at 50 ° C ml of glycine-NaOH buffer solution / 0.5 M pH 9.4 /. 200 μl of the prepared solution is placed in one of the halves of the indicator test capsule / height 25 mm, inner diameter 4 mm /.

Preparation of the 1st reaction layer: 0.1 g of agar agar, 10 mg of nicotinamide adenine dinucleotide and 10 mg of sodium azide were dissolved at 50 ° C in 10 ml of glycine-HC buffer solution /0.02 M pH 3.5 /. 10 microliters of the prepared solution are placed in the other half of the indicator TCCI c-shsula

/ height 10 mm, inner diameter 4 mm /.

Preparation of the 2nd reaction layer:

20 mg of diaphorase / NADH-dehydrogenase /,

10 mg tetrazole, 10 mg semicarbazide, 20

mg of alcohol dehydrogenase and 10 mgae-acid sodium are dissolved at 45 ° C in 10 ml of a 1% agar-agar solution prepared on the basis of phosphate buffer (0.02 M pH 7.0). 100 mil solution is placed in the middle of the capsule / height 10 mm, inner

a diameter of 4 mm /, which is a glass tube of the indicated dimensions, closed on both sides by small plugs. Similar plugs also close the other two parts of the capsule when it

storage, transportation, which prevents mechanical damage to the layers, their overgrowth and pollution.

Claims (1)

The claims An indicator test device for determining the concentration of substrates of enzymatic reactions, consisting of a substrate, one or more layers of reagents and a layer for applying the analyzed sample, characterized in that, in order to simplify the manufacturing technology and reduce costs., The substrate is detachable capsule, filled in at least two parts and from a transparent heat-resistant material, the layer for applying the sample is made on the basis of true gelatin and placed in one of the parts of the capsule, and the layers of reagents, one or more of which are made also based on true jellies, placed in other parts of the capsule. l / Cf77W6 / t / c / vfftfef / b FIG. 4 T FIG. 2 -g I I I ..