JPH0616033B2 - Ammonia and urea analyzer - Google Patents

Description

The present invention relates to an analytical tool for measuring the concentrations of ammonia and urea in liquid samples, especially body fluids such as blood, serum, plasma, saliva.

Ammonia in the body is mainly produced by deamination of amino acids in the process of protein metabolism and decomposition of proteins ingested by diet by bacteria and the like. And in recent years, this blood ammonia concentration has been regarded as important when grasping and treating the condition of severe liver diseases with hepatic encephalopathy, especially fulminant hepatitis, cirrhosis, and congenital urea cycle enzyme deficiency and It has attracted attention as a test item that is indispensable for the diagnosis and treatment of congenital amino acid metabolism disorders.

On the other hand, urea is the main reaction product of protein metabolism in the human body, and the concentration of urea serves as an index of renal function, and toxic substances are contained in blood in proportion to the degree of urea. Middle urea is an important diagnostic index for doctors.

Thus, the measurement of ammonia and urea concentrations is extremely important from a clinical point of view.

By the way, the well-known methods for measuring ammonia in clinical chemistry include the microdiffusion method of Conway and Seligson, the ion exchange resin method, and further the deproteinization colorimetric method and recently There is an enzymatic method. or,
Conventionally, as a method for measuring urea, there is a diacetyl monooxime method in which urea and diacetyl monooxime are reacted to measure the absorption of a newly formed compound with a photometer. Further, in the modern times, urea is decomposed with urease to form ammonium carbonate, which is then decomposed into ammonia gas, which is a known Nessler reagent or Berthelo.
t) There is a method of examining the coloration degree of the colored reaction product formed in the reagent. However, while these methods are accurate, they require a large amount of sample, have to prepare a calibration curve each time, require a long time for analysis, require a special analyzer, and use poisonous substances. It has many drawbacks such as

Therefore, some measuring tools have been developed to correct these drawbacks and measure easily.

For example, there is a test paper for urea concentration measurement known as Azo Sticks (trade name) sold by Miles, Inc. of the United States. This is a small piece of filter paper impregnated with urease and a buffering agent. One drop of whole blood was applied, the reaction was waited for 1 minute, the red blood cell component in whole blood was washed off, and the color tone on the surface was shown. It is a method for roughly knowing the urea concentration in blood by visually deciding using a standard colorimetric table prepared in advance. Although this test device satisfies the requirements of simplicity and rapidity, the sample is limited to whole blood, and the hematocrit value of the whole blood is greatly affected by the measurement result, and after the blood cell component is removed by washing with water, There is a serious drawback in the precision and accuracy of the analysis, such as the immediate decrease in coloration.

Next, Japanese Examined Patent Publication No. 50-1619 discloses a urea concentration measuring composition for visually observing the coloration of a blue compound produced by the reaction of a strongly acidic ion exchange resin and paradimethylcinnamaldehyde. This is because the sample is limited to serum or plasma, the measured value is affected by the pH, buffering capacity, and color tone of the sample itself, and since urease is not used, it has poor specificity for urea and metabolites in the sample. It has the drawback that the measured value is influenced by the drug.

Further, Japanese Patent Laid-Open No. 54-151094 discloses an analyzer for ammonia and urea using whole blood or serum as a measurement sample. This is to allow the ammonia generated in the reaction system to reach the indicator through a net or a foil, and to check the concentration of ammonia or urea in the sample from the degree of color development in the indicator system. The sample soaks into the indicator system, resulting in non-uniform coloration,
Alternatively, there is a drawback that ammonia generated in the reaction system cannot uniformly color the indicator system.

Further, Japanese Patent Publication No. 56-11108 discloses that an ammonia in an aqueous solution is converted into ammonia gas in a reaction system, and the ammonia gas reaches an indicator system through a gas permeable membrane to thereby provide an indicator type developer. A technique for knowing the ammonia concentration of an aqueous solution by utilizing discoloration has been shown. However, this analysis tool uses an indicator system in which a hydrophilic gel bead is impregnated with a color developer and the gel bead is adhered to a single layer on an adhesive tape, and a gas-permeable film is used. Reproducible manufacturing is difficult.

On the other hand, Japanese Patent Application No. 56-177660 by the applicant of the present application discloses that urea in a sample is hydrolyzed by an enzyme in a sample hole that is kept airtight, and the generated ammonium carbonate is ammonia gas under alkaline conditions. However, there is disclosed a method and an apparatus for guiding urea to the indicator layer through a gas permeable membrane, and analyzing urea by the color change of the indicator corresponding to the pH fluctuation due to ammonia gas. This urea analysis tool has a feature that gas escapes little and that it can be analyzed without being affected by the properties of the sample, but since it also uses a gas permeable membrane, it has problems in reproducible manufacturing and its structure is Complex,
There are inevitable manufacturing disadvantages when compared to strip-shaped tools.

The present invention has been made in view of the above, and its first object is to provide a quick, simple, precise, and accurate analysis of ammonia and urea using a small amount of a sample, which does not have the drawbacks of the conventional techniques. The purpose is to provide an analytical tool that can be used. The second
It is an object of the present invention to provide an analytical tool that can accurately and accurately measure the concentration of ammonia or urea in a sample without strictly quantifying the amount of the sample. A third object is to provide an analyzer for ammonia or urea which is extremely easy to manufacture and use.

In order to achieve this object, the present inventors have developed a carrier containing a buffer system that maintains pH on the alkaline side (in the case of urea, an enzyme system and a buffer system having urease activity), and a carrier generated. An analysis comprising an indicator layer that changes color by ammonia gas, a spacer that can make the area where the ammonia gas can diffuse from the carrier to the indicator layer and the area of the indicator layer that can contact the diffused ammonia gas constant, and a cover sheet that covers the upper part of the carrier I developed a tool.

Next, the measuring mechanism of the analytical instrument according to the present invention will be described by taking the measurement of urea as an example.

First, when a sample is dropped on a carrier (4) containing urease and an alkaline buffer capable of decomposing urea in the sample in a short time, urea in the sample is immediately decomposed by urease to ammonium carbonate, and the carrier (4) The alkaline buffer causes a partial pressure of ammonia gas corresponding to the urea concentration of the sample. The generated ammonia gas is a carrier
Disperses more uniformly from the side surface of (4) and the surface facing the diffusion chamber (5). Incidentally, if the upper part of the carrier (4) is covered with a cover sheet after dropping the sample on the carrier, diffusion of ammonia from the upper part of the carrier (4) can be prevented. While the side surface is an open system, the diffusion chamber is a closed space, so there is a gradual difference in the amount of diffusion into both systems and the ammonia partial pressure at the opening.
However, the shortest distance between both systems (diffusion chamber (5) and side) and carrier
If the thickness ratio of (4) is large, the influence of the diffusion of ammonia gas from the side surface on the diffusion amount of ammonia gas into the diffusion chamber (5) can be ignored. The ammonia gas that has diffused to the diffusion chamber side reaches the indicator layer (2) and changes the color of the indicator, so that the concentration of urea in the sample can be examined by examining the degree of color change of the indicator.

Hereinafter, the analysis tool of the present invention will be described in detail with reference to the accompanying drawings.

1 to 3 show an example of the analysis tool of the present invention, in which an indicator layer (2), a spacer (3) and a carrier (4) are sequentially laminated on one end side of a support (1), The entire structure is strip-shaped. The carrier (4) is a reagent for converting ammonia in a sample into ammonia gas (in the case of ammonia analysis), or a reagent containing an enzyme system having urease activity and ammonium carbonate generated by decomposition of urea in the sample by urease. It contains a gasifying reagent (for urea analysis). The indicator layer (2) contains an indicator that changes color in response to a change in pH that fluctuates with ammonia gas, and is applied by coating and drying on the support (1).

On the other hand, the spacer (3) has a through hole (6) for forming a diffusion chamber (5) between the indicator layer (2) and the carrier (4) near one end, and the portion supports It is releasably adhered to the body (1). Further, an adhesive (7) is applied to the upper surface near the end-resistant portion, and plays the role of a cover sheet for covering the carrier (4) immediately after dropping the sample. For that purpose spacers (3)
As the material, a soft plastic film or sheet is preferably used. Further, the spacer (3) functions to stop the reaction in the indicator layer (2) after a predetermined reaction time has elapsed by removing it together with the carrier (4). Since the upper and lower parts of the through hole (6) of the spacer (3) are adhered to the carrier (4) and the indicator layer (2), respectively, the contact area between the carrier (4) and the diffusion chamber (5) and It is easy to accurately reproduce the contact area between the indicator layer (2) and the diffusion chamber (5) and the distance between these two surfaces (2) and (4).

By the way, the reagent (reagent for gasifying ammonia in the sample) contained in the carrier (4) is preferably an alkaline buffer which can keep the pH of the sample alkaline. This alkaline buffer may be anything that is solid at room temperature, such as boric acid and borax, sodium carbonate and sodium bicarbonate,
Examples include Tris buffer. PH of alkaline buffer
Is higher, ammonia gasification is more active,
If it is too high, proteins and amino acids will be decomposed and extra ammonia will be produced as a by-product. In the case of urea analysis, urease present in the same system becomes unstable at high pH. So in the case of ammonia analysis, pH 7.5 to 11, especially
It is preferably around 10.5, and in the case of urea analysis, a pH of around 7.5 to 10 and especially around 8.5.

Any material can be used as the material of the carrier (4) as long as it can retain a dry residue when it is impregnated in a solution such as filter paper or nonwoven fabric and the solvent is dried. If desired, it is advantageous to add a wetting agent or a surfactant to the carrier so that the dropped sample can permeate well. As the material of the support, plastics such as polystyrene, polyethylene and polyester are preferable from the viewpoint of workability and economy.

Next, as the indicator system, various chemicals or a group of chemicals that change their colors in response to changes in pH are used. Better results are also obtained by using a trace amount of pH adjuster.
And, preferably, an acid which is solid at room temperature such as malonic acid, phthalic acid, tartaric acid or a mixture of these acids and a salt of the acid is used as a pH adjuster, if desired, and various kinds having a discoloration region on the acidic side are used. Use different pH indicators alone or in combination.
Examples of single use include bromcresol purple, chlorophenol red, and bromcresol green. Examples of combined use include a combination of bromcresol green, bromxylenol blue, and paraxylenol blue, a combination of chlorophenol red, bromphenol red, phenol red, cresolphthalein, phenolphthalein, methyl red, dimethylaminoazobenzene, bromthymol blue. , A combination of thymol blue [a universal indicator of Bogen] and the like. In the case of a pH indicator group that changes color with a single indicator or a similar hue, it is effective for measuring the concentration of the substance to be measured using a light reflectometer, and the pH indicator can be set by setting it to a predetermined wavelength. The degree of discoloration can be measured without individual differences. On the other hand, among the pH indicators used in combination, for example, those that discolor over a wide range of pH typified by the above-mentioned universal indicator of Bogen or Kolthoff's universal indicator, etc. Is especially advantageous.
That is, the universal indicator changes its color depending on the pH, such as red-orange-yellow-green-blue, which is convenient for the naked eye to judge.

These pH indicators are blended with natural and synthetic film-forming polymers as binders and coated on a support. Suitable binders are water-insoluble film-forming agents such as cellulose acetate, polyvinyl butyral and ethyl cellulose. It is a polymer and is effective for uniform color change and uniform application of pH indicator. Furthermore, it is also preferable to add a wetting agent such as polyoxyethylene alkyl ether, polyethylene glycol, glycerin or a surfactant as a substance effectively acting for uniform color change.

In the measurement, first, the sample is dropped on the carrier (4) and immediately the upper part of the carrier is covered with the spacer adhesive section. In the carrier (4), an ammonia gas partial pressure corresponding to the concentration of ammonia or urea in the sample is generated, and the ammonia gas reaches the indicator layer (2) through the diffusion chamber (5). Ammonia gas ultimately causes a color change in the indicator that corresponds to the concentration of ammonia or urea in the sample. Water and blood cells in the sample are carriers.
Since it is held in (4), the degree of discoloration of the indicator layer depends only on the ammonia gas that has passed through the diffusion chamber. Then,
After removing the spacer (3) and the carrier (4), the degree of discoloration is compared with a color sample when viewed from the direction of FIG. 2A, or a calibration curve between the light reflectance and the concentration of the substance to be measured is prepared in advance. By making it in advance, the concentration of the substance to be measured in the sample can be easily known by the light reflectometer.

Next, examples will be given for better understanding of the present invention, but the present invention is not limited to the examples.

Example 1 Ammonia Analyzer A] Production of Ammonia Analyzer (1) Preparation (a) Carrier 15 cm square filter paper [# 1026 manufactured by Toyo Roshi Kaisha, Ltd.] was impregnated with a solution having the following composition and dried at 50 ° C. After that, cut into 1 cm squares.

0.5 M carbonate buffer (pH 10.5) 25 ml triton X-100 0.1 g (b) Coating solution for forming indicator layer A solution having the following composition is used as a coating solution for forming an indicator layer.

Phenolphthalein 20mg methyl red 40mg dimethylaminoazobenzene 60mg bromothymol blue 80mg glycerin 1g thymol blue 100mg ethyl cellulose 8g ethyl alcohol 50ml acetone 50ml purified water 2ml (c) Support and indicator layer 10 cm wide white polyester sheet [Toray Industries, Ltd.] , Thickness 0.
2mm] 10mm to 20mm from one end of the above (b)
The coating solution prepared in 1. is applied to have a coating thickness of 0.25 mm, dried at 50 ° C., and then cut in a width of 1 cm in the direction perpendicular to the coating direction.

(D) Spacer 10 mm x 80 mm polyester sheet [Toray Industries, Inc., thickness: 0.
1mm] 6mm diameter centered at 20mm from one end
Make a through hole and attach a 10mm x 30mm double-sided tape to the part 3cm from the other end.

(2) Assemble (a), (d) and (c) in this order as shown in FIG.

B] Measurement Ammonia aqueous solution 30 μ was dropped on the carrier, and the carrier was immediately covered with the adhesive portion of the spacer. After 10 minutes, the spacer and the carrier were removed, and the color tone of the indicator layer was visually observed. .

Example 2 Urea Analyzer A] Production of Urea Analyzer (1) Preparation (a) A 15 cm square filter paper [# 1026 manufactured by Toyo Roshi Kaisha, Ltd.] was impregnated with a solution having the following composition and dried at 50 ° C. Cut into 1 cm squares.

Urease 3000U 1.0M Tris buffer (pH 8.5) 25 ml Polyethylene glycol 1 g (b) Coating solution for forming indicator layer The solution having the following composition is used as the coating solution for forming the indicator layer.

Bromcresol green 40 mg Glycerin 0.4 g Cellulose acetate 4 g Acetone 20 ml Ethanol 20 ml 1.0 M citrate buffer (pH 3.0) 0.3 ml (c) Support and support drug layer Same as in Example 1 (d) Spacer Example 1 Same as (2) Assembly Same as in Example B] Measurement (1) 30 μm of urea aqueous solution is dropped on the carrier, and the carrier is immediately covered with the adhesive portion of the spacer. After 10 minutes, the carrier and the spacer were removed, and the light reflectance of the indicator layer was measured with a light reflectance meter (measurement wavelength: 620 nm).

(2) Measurement (change in sample amount) Table 3 shows the results of measurement in the same manner as in (1) above, except that the amount of the urea aqueous solution (40 mg / dl) dropped on the carrier (4) was changed.

As is clear from this table, in this example, the amount of sample liquid is 20
In the range of up to 60 μ, almost equal values are obtained, and the concentration of urea in the sample can be measured precisely and accurately without strict quantification of the sample amount. This is because the side surface of the carrier (4) is open.

Example 3 Measurement of Urea Concentration in Saliva The urea concentration in human saliva was measured using the urea analyzer manufactured in Example 2, a light reflectometer, and the calibration values obtained from Table 2, and the correlation with the urease indophenol method was performed. I checked. The results are shown below.

Number of samples 48 Correlation coefficient 0.982 Equation of regression line y = 0.99x + 1.7 In the equation of regression line, x is a value measured by the urease indophenol method, and y is a value measured by the urea analyzer of the present invention. The measurement results are shown in FIG. 7 for easier understanding. From this result, it is understood that the present invention can measure the urea concentration in the sample by the standard curve in the aqueous solution without affecting the properties of the sample (influence by hematocrit value, viscosity, pH).

The preferred embodiment of the analysis tool of the present invention has been described above.
Various modifications can be considered within the scope of the technical idea of the present invention. First, in FIG. 4, a through hole (8) similar to the through hole (6) of the spacer (3) is provided near the end of the support (1), and both through holes are formed.
(6) ・ (8) forms a diffusion chamber (5), and an indicator carrier (9) separately coated with an indicator layer (2) is adhered so as to cover the lower part of the through hole (8). . The diffusion room
Since (5) becomes deeper, the reaction will be somewhat slower, but more stable measurement values can be obtained.

5 and 6 show another example in which the re-diffusion prevention sheet (10) is provided on the support (1) in addition to the spacer (3). This re-diffusion prevention sheet (10) is a carrier (4) at the time of measurement.
And after removing the spacer (3), covering the indicator layer (2) to prevent re-diffusion of ammonia gas from the indicator layer (2),
It serves to prevent discoloration of the indicator layer (2) and is convenient when it is not possible to measure immediately after the reaction such as mass measurement.

As described above, the analysis tool of the present invention is provided with an absorptive carrier that receives a sample to generate ammonia gas and an indicator layer that changes color due to ammonia gas via a diffusion chamber provided in a spacer. Therefore, the area and distance between the two can be made constant by the thickness of the spacer and the size of the through hole, and the reproducibility is extremely excellent because no permeable membrane is used. In addition, since the whole is strip-shaped, it is extremely easy to manufacture and use, and since the absorbent carrier is covered with the cover sheet, the evaporation of ammonia gas is small, and the side surface of the absorbent carrier is open, so that it is within a certain range. If it is present, it is characterized by being able to perform rapid, simple, precise, and accurate concentration analysis of ammonia and urea using a small amount of sample without being affected by the amount of sample.

[Brief description of drawings]

1 to 3 show one example of the analysis tool according to the present invention. FIG. 1 is a perspective view showing a state in which a part of a spacer is peeled from a support, and FIG. 2 is an X- line in FIG. X-ray sectional view,
FIG. 3 is an exploded perspective view. 4 to 6 are cross-sectional views showing other examples different from each other, and FIG. 7 is a correlation diagram of the measurement carried out in Example 3, in which the abscissa is the measurement value by the urease indophenol method and the ordinate is the ordinate. With the measured value with this analysis tool,
All units are (mg / dl). 1 ... Support 2 ... Indicator layer 3 ... Spacer 4 ... Carrier 5 ... Diffusion chamber 6.8 ... Through hole 7 ... Adhesive 9 ... Indicator carrier 10 ... Re-diffusion prevention sheet

Claims (1)

[Claims] 1. Detecting ammonia in a sample or ammonia gas applied directly or indirectly on a carrier or a support containing an alkaline buffer that gasifies ammonia generated by decomposition of urea in the sample. Indicator layer and a spacer positioned between the carrier and the indicator layer, and the spacer has a constant area of the indicator layer which can contact the diffused ammonia gas and the area where the ammonia gas can diffuse from the carrier to the indicator layer side. Ammonia characterized in that it is formed as a cover sheet that covers the upper surface of the carrier by providing through holes that can be formed, and the spacer is detachably fixed to the indicator layer, and an adhesive layer is provided at the extended end of the spacer. And urea analyzer.