KR20120103441A - Strip paper for analysis of creatinine - Google Patents

Abstract

PURPOSE: A piece of test paper for creatinine analysis in body fluid is provided to enhance sensitivity of creatinine concentration in urine. CONSTITUTION: A piece of test paper for creatinine analysis in body fluid comprises a spreading layer and 3,5 dinitrobenzene sulfonic acid and strong alkaline material which contacts with entire or at least a part of the spreading layer. The test paper further comprises a support for supporting the spreading layer. The body fluid is urine. The strong alkaline material is lithium hydroxiode. [Reference numerals] (AA) Colorimetric CMOS sensor measurement value; (BB) Creatinine concentration (mg/dL)

Description

Strip paper for analysis of creatinine

The present invention relates to a test paper and an analysis method for creatinine analysis in a body fluid, and more particularly, to provide a test paper with high stability that can be measured with high sensitivity even with creatinine in a body fluid with a strong buffering effect such as urine.

Creatinine is the final metabolite of the creatine pathway and has a structure in which creatine is dehydrated and cyclized. In the body, most are present in the muscle as creatine or creatine phosphate. Creatine receives high-energy phosphoric acid from ATP, becomes creatine phosphate, and functions as an energy storage substance, and transfers high-energy phosphoric acid to ADP and returns to creatine at the time of energy consumption such as muscle contraction, or by non-enzymatic reaction Becomes Thereafter, the produced creatinine is excreted in the urine through the kidneys.

Therefore, urinary excretion of creatinine, that is, urine concentration, is used as an indicator of muscle disease or renal dysfunction. In addition, the concentration of creatinine in the blood, such as when there is renal dysfunction, may be an indicator of the disease.

As a detection method of creatinine in a body fluid, the well-known "Jaffe reaction" used the color reaction by the condensate of creatinine and a picric acid under strong alkaline conditions, and it is a wide and general detection method as a simple and economical detection method. Is being used. Moreover, this reaction is applied by the Benedict-Bhere method which uses 3, 5- dinitro benzoic acid (DNBA) instead of picric acid.

The zap reaction is mainly used as a solution system, but on the other hand, a technique in which this reaction is applied to a simple dry test piece is disclosed (Japanese Patent Laid-Open Publication No. Hei 1-29164, Japanese Patent Laid-Open Publication Hei 1-129129 US Pat. No. 5662687, etc.). However, in the case of urine, all of the methods described above can measure a certain amount of creatinine, but it is difficult to say that the degree is a good method.

That is, since urine has a wide pH range and also has a very strong buffering effect, even if an alkaline substance is added in the above-described method where strong alkalinization is a prerequisite, a lowering of the reaction pH occurs due to the buffering effect of urine, and consequently, This is because a large error is given to detection and measurement.

In order to reduce the error in the measurement of creatinine in the urine using the above method, it is necessary to add a strong alkaline substance (for example, hydroxides such as sodium hydroxide and lithium hydroxide) in a large amount to exceed the buffering capacity of urine. .

By the way, picric acid or 3,5-dinitrobenzoic acid, especially such a reaction product, is extremely unstable when adding an alkaline substance in an amount exceeding the buffering effect of urine, and also has a great influence on the coloration of the product.

When the reaction system is a liquid system, stability of picric acid and 3,5-dinitrobenzoic acid can be maintained by adding a large amount of alkaline material immediately before measurement, but in dry test pieces that are previously supported with all reagents used. This stability cannot be maintained. Therefore, it was very difficult to produce the test piece which measured creatinine highly sensitive to the urine which was not diluted, and was sensitive.

Japanese Patent Application Laid-Open No. 9-061430 attempts to solve this problem by using 3,4-dinitrobenzoic acid instead of 3,5-dinitrobenzoic acid.

However, the technology of this patent still contained this sensitivity problem.

The present invention is to overcome the disadvantages of the prior art, to provide a stable test paper that can be analyzed well creatinine with high sensitivity even in a strong body fluid of buffering action such as non-dilution urine.

As a result of diligent research to solve the above problems, it has been found that 3,5 dinitrobenzene sulfonic acid is stable even in the presence of an alkaline substance and is colored by a condensation reaction with creatinine to complete the present invention.

The present invention relates to a test paper for creatinine analysis in body fluids, including a developing layer and 3,5 dinitrobenzene sulfonic acid and a strong alkaline substance in contact with all or at least a portion of the developing layer.

In addition, the present invention includes a support for supporting the development layer in a preferred form of the test paper, wherein a reagent layer containing 3,5 dinitrobenzene sulphonic acid is provided on the support to contain a strongly alkaline substance. Provide the test strip with the development layer installed.

According to the present invention, it is possible to provide a stable dry test paper which can measure creatinine concentration in undiluted urine with high sensitivity.

1 is a measurement value of the colorimetric sensor value according to the concentration using a photo CMOS colorimetric sensor 1 minute after using a creatinine test paper prepared using a standard solution of 100 ~ 500 mg / dL concentration in a normal room temperature environment It is a graph obtained.
Figure 2 shows the value measured using a reagent for the automatic solution analysis device using a zap reaction and the test paper according to the present invention using the real urine as a sample and the photo CMOS colorimetric sensor compared with the value inverted through the calibration curve It is a graph.
Figure 3 is a graph showing the actual urine was measured as in Example 1 as a sample using 3,5-dinitro benzoic acid instead of 3,5-dinitro benzene sulfonic acid for comparison.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail. The test paper of the present invention comprises a developing layer and 3,5 dinitrobenzene sulfonic acid and a strongly alkaline substance in contact with all or at least part of the developing layer, and preferably further includes a support.

The developing layer absorbs test objects such as urine to form a liquid reaction system. Examples of the material include porous materials such as filter paper, woven fabric, nonwoven fabric, glass fiber, nitrocellulose, and hydrophilic polymer. Moreover, a strip shape, a rod shape, etc. are mentioned as a shape. In such a structure, since the fiber material derived from nature, such as cellulose like filter paper etc., may hydrolyze and deteriorate under strong alkali conditions, chemical synthetic fiber cloth etc. are preferable.

When the physical strength of the developing layer is weak, the test paper of the present invention may have a support for supporting the developing layer. The support may be a shape that can be integrated with the developing layer, or may support only a part of the developing layer. As a raw material of a support body, the plastic resin normally used when producing dry test paper, for example, polyethylene terephthalate, polyvinyl chloride (henceforth "PVC"), etc. are mentioned. When a light-transmitting material is used for a support body, when using a test paper, it becomes possible to observe and measure a color from a support body side.

The 3,5 dinitrobenzene sulfonic acid and the strongly alkaline substance are in contact with all or at least part of the developing layer as described above. Here, "contact" does not mean that 3,5 dinitrobenzene sulfonic acid and a strongly alkaline substance are in contact with the surface of the development layer, but also in contact with the material constituting the development layer inside the development layer. Include. In addition, the said substance may indirectly contact with a developing layer, for example, may be in contact with the state encapsulated in a microcapsule. In addition, 3,5 dinitrobenzene sulfonic acid and a strongly alkaline substance may be supported on the developing layer so as to be in contact therewith.

As a preferable form of the test paper of this invention, the support layer in which the layer containing 3,5 dinitrobenzene sulfonic acid was formed on the surface, and the developing layer in which the layer containing strong alkaline substance was formed on the surface are 3,5 dinitrobenzene sulfonic acid Test papers may be deposited repeatedly over several orders, dividing the layer and the strongly alkaline material layer in order to stabilize. By repeatedly depositing in this way, the stability of 3,5 dinitrobenzene sulfonic acid can be improved further. At this time, when using a non-porous material such as a plastic material as the support, in order to fix 3,5 dinitrobenzene sulfonic acid on the support, a binder may be used as the reagent layer on the support. In addition, the strongly alkaline substance may be impregnated into the developing layer. Although the site | part which exists in the test paper of 3,5 dinitrobenzene sulfonic acid and a strongly alkaline substance may be contrary to the above description, it is necessary to make a reaction environment strong alkali. Among the developing layer and the supporting layer, it is preferable to first include a hydroxide on the developing layer side in which the object to be inspected is in contact with. In addition, a strong alkaline substance may be contained in both a reagent layer and a developing layer in order to strengthen strong alkaline conditions.

As a strong alkaline substance, what is necessary is just to be able to induce a strong alkaline environment, when melt | dissolving in a sample, and it is especially preferable to be able to maintain reaction pH to 12-13 in test paper when urine is absorbed to test paper. Specific examples thereof include alkali metal hydroxides and lithium hydroxide is preferred.

The test paper of the present invention can be produced by the same method as the conventional test paper, except that 3,5 dinitrobenzene sulfonic acid is used as a substance to be colored by condensation reaction with creatinine. For example, a highly viscous coating solution is prepared by dissolving or dispersing a binder and 3,5 dinitrobenzene sulfonic acid in a solvent, coating the coating solution on a surface of a support to a constant thickness, and drying the reagent layer. Examples of the binder include inert and hydrophilic polymers such as methyl cellulose. It can be prepared by impregnating a support and a strong alkaline solution obtained as described above. Such a method may use a material having the same size as the final product, but may be used by cutting a laminated material using a large material to an appropriate size.

Although the reagent may be repeatedly deposited and dried as a test paper, it is also possible to fix a small piece of this by fixing it to the end of a support part made of PVC or the like.

When using the test paper of the present invention, the color change of the reagent layer is read by dropping or soaking a sample (urine or the like) on the developing layer using a visual or photo CMOS colorimetric sensor, a reflectance measuring device or the like.

Since the test paper of the present invention has the same structure as described above, when the sample paper is absorbed into the test paper, the pH in the sample solution can be kept high and the condensation reaction between creatinine and 3,5 dinitrobenzene sulfonic acid contained in the sample can be maintained. Can stabilize color. As a result, it has high analytical sensitivity and can easily quantify creatinine.

<Production Example>

EMBODIMENT OF THE INVENTION Below, this invention is demonstrated further more concretely.

After mixing the following coating liquid components, it coats on a filter paper and dries at 70 degreeC for 10 minutes.

Coating Composition of Reagent Layer

An aqueous solution containing 4.5 g of lithium hydroxide, 100.0 g of distilled water, 1.5 g of gelatin, and 2 mL of 0.1% Triton X 100 was immersed in a filter paper and dried at 70 ° C. for 10 minutes.

After immersing again in a solution containing 6.61 g of 3,5 dinitrobenzene sulfonic acid in 100mL of ethanol and dried for 10 minutes at 50 ℃.

After drying, double-sided tape is bonded to one side and cut into rolls of 5 mm length. Using double-sided tape, adhere to the white PVC film and cut it to obtain a test bar with 5 × 5 mm creatinine test paper.

&Lt; Example 1 >

The test paper was produced as in the preparation example. This test paper measured creatinine in urine. Samples were prepared by adding creatinine to human urine at a concentration of 100 to 500 mg / dl. The standard solution was quantified using a reagent for commercially available solution-based automated analysis apparatus using a zap reaction. The color value of the test paper was digitized using a photo CMOS colorimetric sensor, which was used to correlate the color change according to creatinine concentration.

The amount of the sample dropped on the developing layer of the test paper was 100 μl, so that the test paper was sufficiently wetted, and the excess sample was removed by using a hygroscopic paper.

One minute after using a creatinine test paper prepared using a standard solution in a concentration range of 100 to 500 mg / dL in a normal room temperature environment, the test paper was measured using a photo CMOS colorimetric sensor.

This is shown in FIG. In the regression analysis, the value of the correlation coefficient square was 0.984, indicating good correlation.

The amount of creatinine excreted in the urine varies, but there is generally a range of up to 300 mg / dl, and the calibration curve obtained by the present invention shows linearity up to 500 mg / dl, which is sufficient for the determination of creatinine in the urine. It has a measuring range.

<Example 2>

The actual urine was used as a sample for the solution-based automatic analysis device using the zap reaction and the test paper according to the present invention, and the value measured using the photo CMOS colorimetric sensor was compared with the inverted value through the calibration curve. The correlation of these values is shown in FIG. 2. Thus, both showed good correlation with actual urine.

&Lt; Comparative Example 1 &

For comparison, 3,5 dinitrobenzene sulfonic acid instead of 3,5 dinitrobenzoic acid was used as a sample to measure the actual urine as in Example 1, and the comparison results were shown in FIG. 3.

In this result, the slope difference is larger when 3,5 dinitrobenzene sulfonic acid is used than when using 3,5 dinitrobenzoic acid, and the change in concentration is large. Do. This is because the urine had a strong buffering capacity that the pH at the time of reaction was not maintained, and thus the accurate measurement could not be performed on the test paper of the comparative example.

Claims (6)

A test paper for measuring creatinine in a body fluid comprising a developing layer and 3,5 dinitrobenzene sulfonic acid and a strongly alkaline substance in contact with all or at least part of the developing layer.
The method according to claim 1,
Test paper further comprises a support for supporting the development layer.
The method according to claim 2,
And a reagent layer containing 3,5 dinitrobenzene sulfonic acid is provided on the support, and a developing layer containing strongly alkaline substance is provided on the reagent layer.
The method according to claim 1,
Test paper, characterized in that the body fluid is urine.
The method according to claim 1,
The test paper, characterized in that the strongly alkaline substance is a substance capable of maintaining a reaction pH of 11 to 14 when the test paper is infiltrated with urine
The method according to claim 5,
Test paper, characterized in that the strongly alkaline substance is lithium hydroxide.

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