JPH03103197A - Selectively measuring tool of ionizable component in solution - Google Patents

Abstract

PURPOSE:To obtain the title measuring tool useful for diagnosing renopathy, having relatively simple structure, ready operation, rapidly providing accurate results, comprising an ionization reaction layer containing a specific ionizing agent, reagent layer and developing layer. CONSTITUTION:The objective measuring tool comprising an ionization reaction layer 1 containing an ionizing agent to selectively ionize a substance to be measured, a reagent layer 2 carrying a dyestuff having solubility to be related to ion concentration in a solution and a developing layer 3 of the dyestuff, wherein an amount of the dyestuff eluted from the reagent layer 2 to the developing layer 3 is changed depending upon the ion concentration produced in the ionization reaction layer 1 and reflected in coloring concentration on the surface of the developing layer 3. The ionization reaction layer 1 is preferably used as the developing layer 3 and further the ionizing agent is preferably oxygen.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Applications) The present invention relates to an instrument for selectively measuring ionizable components in a solution.

(Prior Art) Measurement of the ratio of solutions is often carried out in the fields of food crystal industry, fermentation industry, and clinical testing. For example, in the field of clinical testing, kidney disease is diagnosed by measuring Hito in urine.

Urine specific gravity indicates the amount of solute in urine and is useful as a barometer of kidney function. Further, it is known that the value shows a good correlation with the ion concentration and osmotic pressure of urine ψ. Normally, the swimming specific gravity of a healthy person is 1.016 to 1.01.
022, but if the value is smaller than the range, diabetes insipidus, acute renal failure, nephritis, etc. is suspected, while if it is larger, nephrotic syndrome, balance water loss, etc. is possible. Urine specific gravity has conventionally been measured using a pycnometer, a hydrometer (floating balance), or the like, or a urine refractometer or osmometer that correlates with urine specific gravity.

(Problem to be solved by the invention a) However, these methods using pycnometers, hydrometers, etc. not only require expensive measuring instruments, but also require the use of instruments that are difficult to break. This method had many problems, such as requiring complicated operations.

To solve these problems, we have developed a polymer that is brittle and translucent to osmotic pressure, encapsulating a substance that can produce a detectable response as a function of the specific gravity or osmolality of the liquid. It consists of at least an effective number of microcapsules having a membrane wall, and when a certain osmotic pressure gradient is generated inside and outside the capsule, the microcapsules release the encapsulated contents through the membrane wall and produce a detectable response. A composition for measuring the specific gravity or osmolarity of a test liquid resulting from the formation of a composition, and a measuring instrument using the composition (Japanese Patent Publication No. 60-46.374) (B) Weakly acidic or weakly basic polyhydric A composition for measuring the ionic strength of liquid samples comprising a polyelectrolyte and a pH-supporting substance capable of producing a detectable response to ion exchange between the electrolyte and the sample to be tested. 12,
No. 858) and (C) a test layer containing a wettable substance that exhibits a water absorption amount corresponding to the ionic strength of the aqueous solution and a water absorption indicator that causes a change in transparency by exhibiting a solubility that corresponds to the fluctuation range of the water absorption amount; Aqueous solution sample specific gravity test tool (
JP-A No. 62-95,462) is known.

However, the methods using compositions (A), (B), and (C) are not only expensive, but also
The measurement work is also complicated, and there is a problem in that sufficient accuracy cannot be obtained.

It is therefore an object of the present invention to provide a device for selectively measuring ionizable components in a solution.

(Means for Solving the Problems) The above object comprises an ionization reaction layer containing an ionizing agent for selectively ionizing the substance to be measured, and a dye whose solubility correlates with the ion concentration in the solution. Consisting of a reagent layer and a developing layer for the dye, the amount of dye eluted from the reagent layer to the developing layer changes depending on the ion concentration generated in the ionization reaction layer, thereby increasing the dye concentration on the surface of the developing layer. This is achieved by a selective measuring device for ionizable components in a solution, which is characterized in that it is configured to reflect the

The present invention also provides the above selective measurement instrument in which the ionization reaction layer also serves as the development layer. The present invention is further provided with the above-mentioned selective measuring instrument, wherein the ionizing agent is an enzyme. The present invention also provides the above-mentioned selective measuring instrument, wherein the substance to be measured is urea, and the ionizing agent is urease or urea carboxylase.

The present invention further provides the selective 7ill determination device described above, wherein the dye is a water-soluble dye containing an ionic functional group.

(Function) According to the present invention, the basic principle is that the solubility of a certain water-soluble dye corresponds to the ion concentration in the solution. It is possible to measure ion concentration.

Furthermore, by coexisting in this system an ionizing agent that specifically ionizes a certain substance, it is possible to measure substances that can be ionized.

In the measuring device according to the method of the present invention, an ionizing agent for selectively ionizing the substance to be measured is brought into contact with the solution to be measured by bringing the ionization reaction layer into contact with the solution to be measured, and the ionizing agent generated in the drug layer is brought into contact with the solution to be measured. eluting the dye by bringing the ion into contact with a dye whose solubility correlates with the ion concentration;
This is carried out by detecting the color density in the black dye developing layer according to the amount of dye elution.

As the ionizing agent for selectively ionizing the analyte by contacting the analyte solution with the ionizing agent, any agent that can selectively ionize the analyte can be used. used. For example, if the substance to be measured is urea, urease or urea lupoxylase is used as the enzyme, and thereby the amount of urea in the solution can be determined. That is, urea is decomposed into ammonia and carbonic acid by urease or urea carboxylase. Since these decomposed products exist as ions in the solution, the amount of urea is detected as the ion concentration. 14 Specific gravity not only shows a correlation with the ion concentration in the element, but also W
It is known that there is a high correlation with the elementary quantity, so when using this method, it is possible to measure the ion concentration and urea concentration in urine at the same time, and more accurate specific gravity values can be obtained. It is.

Regarding the relationship between enzymes and substances to be measured other than urea, for example, for nicotinamide, bition, anilides, etc., amidases are used as ionizing enzymes to convert ammonium ions, and phosphate esters such as nucleotides are used. For amines such as nucleic acid bases and free amino acids, deaminases are used as ionizing enzymes to generate ammonium ions, Amino acid ions can be dissociated by the action of proteases on proteins, and fatty acid ions can be dissociated by the action of hydrases against acyl-CoA such as acetyl-CoA, succinyl-CoA, and palmitoyl-CoA.

These ionizing agents are usually used as an ionization reaction layer by being carried on a filter paper or a hanging cloth such as a woven fabric, a nonwoven fabric, or a knitted fabric. In this case, other components may be included for the ionizing agent to react most efficiently. For example, when the ionizing agent is an enzyme, this includes a buffer that maintains the optimum pH, an enzyme reaction activator, a stabilizer, and the like. For example, when using urease, dithioerythritol, glutathione, ethylenediaminetetraacetic acid (EDTA) are used as stabilizers.
) etc. are added.

When the ions generated in the ionization reaction layer come into contact with the dye, the dye is eluted according to the ion concentration.

Elution of the dye is performed within a reagent layer carrying a dye whose solubility correlates with the ion concentration in the solution.

As the dye in the present invention, any water-soluble dye whose solubility correlates with ion concentration can be used. That is, it is necessary that the solubility of the dye changes depending on the ion concentration of the solution. A dye whose solubility correlates with the ion concentration in a solution usually has an ionic functional group and is countered by an opposite ion.

For example, Basic Blue 3 has an amino group as an ionic functional group and is countered by chlorine ions.

The water-soluble pigments used in the present invention include indigo carmine, Basic Blue 3, Basic Red 9, Basic Red 29, Acid Blue-120, Acid Red 11, Acid Red 97, Crystal Violet, Brilliant Blue 01 Naphthol Green G1 Procyone, There are Ru-HB, etc.

When such a dye is brought into contact with a solution to be measured, the following ionic equilibrium is formed between undissolved matter and dissolved matter.

P-A”B-: P-A”10B- (I)P-
B-A": P-B-+A" (II) However, (I) is for a basic dye, and (n) is for an acidic dye.

When the ion concentration in the solution is high, the dissociation equilibrium shifts to the left, that is, toward non-ionization, while when the ion concentration in the solution is low, the dissociation equilibrium shifts to the right, that is, toward the direction where ionization tends to occur. Therefore, the lower the ion concentration, the easier the dye will dissolve in the solution, while the higher the ion concentration, the more difficult the dye will dissolve in the solution. In order to apply this principle to a measuring device in the form of a test strip, it is preferable to use a composite consisting of a reagent layer holding a dye and a developing layer.When brought into contact with a specimen sample, only the dissolved dye is eluted onto the surface of the developing layer, and the amount of dissolved dye is has a mechanism that is reflected in the color density.

The reagent layer may be of any type as long as it supports the dye and is water-permeable. Usually, the dye is directly supported on the carrier or the dye is dispersed and adsorbed on fine particles. At this time, if the dye is supported together with a water-soluble polymeric compound as a binder, the dye will be uniformly dispersed and stably supported, and will be easily dissolved when it comes into contact with an aqueous solution during use.

As the carrier, a water-permeable carrier such as filter paper, woven fabric, non-woven fabric, or knitted fabric is used. In addition, water-soluble polymer materials used as binders include polyvinyl pyrrolidone, polyvinyl alcohol, hydroxybrobyl cellulose, gelatin, etc.
used in combination.

As the fine particles, any particles can be used as long as they have an adsorption property of 6 to the dye, and the average particle size is 10 to 400μ.
m1 is preferably 50 to 150 μm. For example, there are celite, silica gel, polyamide, alumina, etc., and 1 or 2F! A combination of the above may also be used.

The amount of dye elution is determined by detecting the color density on the surface of the developing layer.

The dye spreading layer is a water-absorbing material having an appropriate thickness and drainage time, and for example, filter paper or cloth such as woven fabric, non-woven fabric, or knitted fabric is preferably used. Further, good color development may be obtained by adding a surfactant or a wetting agent to this developing layer.

Examples of surfactants or wetting agents include alkyl sulfates, alkylbenzene sulfonates, sodium dialkyl succinate sulfonates, amidosulfonates, sodium ricinoleate sulfates, α-sulfo fatty acid alkyl ester salts, monosodium α-sulfono fatty acids. Anionic surfactants such as esters and sodium dialkyl phosphates, polyoxyethylene alkylphenols, polyoxyethylene-polyoxypropylene-polyoxyethylene glycol, polyoxyethylene-9,10-octadecanediol, polyoxyethylene- Examples include nonionic surfactants such as 9,101 dihydroxystearonitrile and ethylene oxide adducts of linear secondary alcohols.

When manufacturing the measuring instrument according to the present invention, when a reagent layer is formed by dispersing a dye or fine particles adsorbed with a dye in a binder, a method may be used in which the reagent layer is coated on a support or a spreading layer. . A reagent layer is obtained by applying a solvent containing a binder in which a dye is dissolved or suspended to a carrier or a developing layer using a coating machine or a spray gun, impregnating the carrier, and then drying. The measuring instrument of the present invention can be obtained by laminating the thus obtained trial layer with a spreading layer using an adhesive if necessary. Further, the support can also be fixed using double-sided tape or adhesive. In addition, when the reagent layer is in the form of a carrier adsorbing a dye, the reagent layer and the developing layer may be sealed in a support (holder) without being bonded with a water-soluble binder or contacting.

In addition, if necessary, a solvent containing a binder in which the dye is dissolved or suspended is applied to one side of the development 1i, which has been treated with a surfactant or a wetting agent, or partially impregnated with the development layer left behind. The co-layer and the spreading layer may be formed integrally.

Next, one embodiment of the aIl1 fixing device according to the present invention will be described with reference to the drawings.

FIG. 1(A) shows a first embodiment of the measuring instrument used in the present invention, which basically consists of an ionization reaction layer 1
A spreading layer 3 is provided adjacent to the spreading layer 3, and a reagent layer 2 is further formed adjacent to the spreading layer 3, and the reagent layer 2 includes:
If necessary, the support 5 is attached via a double-sided adhesive tape 4 or an adhesive layer (not shown).

FIG. 1(B) is a modification of FIG. 1(A), in which an ionization reaction layer 1, a spreading layer 3, and a reagent layer 2 are laminated, or any two of them are formed integrally. It is made by fixing the holding part 6 to a support body 5.

FIG. 2(A) shows a second embodiment of the measuring instrument used in the present invention, in which a reagent layer 12 is formed adjacent to an ionization reaction layer/deployment layer 17. If necessary, the support 15 is attached to the reagent layer 12 using double-sided adhesive tape 14.
Alternatively, it may be attached via an adhesive layer (not shown) or the like.

FIG. 2(B) is a modification of FIG. 2(A), in which a laminate of an ionization reaction layer/deployment layer 17 and a reagent layer 12 is fixed to a support l5 provided with a holding part l6. It is what it is.

FIG. 3 shows a third embodiment of the measuring instrument used in the present invention, in which a reagent layer 22 is provided adjacent to the ionization reaction layer 21, and further, a reagent layer 22 is provided adjacent to the reagent layer 22. A transparent film 28 is attached to a support body 25 having a holding part 26 and an opening part 29.
It is fixed to .

In the measuring instrument according to the present invention, the substance to be measured is selectively dissociated into ions by the action of the ionizing agent by being immersed in the solution to be measured, and the dissociated ions are brought into contact with the dye, and the dye is It dissociates into ions, forms an ionic equilibrium between the undissolved matter and the dissolved matter, and the color density changes depending on the degree of dissociation and appears in the development layer. It is measured by projecting and measuring the reverse absorbance. It can also be measured by comparison with a standard color density table. Furthermore, in the case of the measuring instrument shown in FIG. 3, the ionization reaction layer 21 is
When a constant solution such as urine is dropped onto the reagent layer 22, the urea in the urine is dissociated into ions by the ionizing agent, and the urea in the urine is dissociated into ions by the ionizing agent.
to form the ionic equilibrium and elute the dye, forming the developing layer 2.
3, the coloring density of the developing layer 23 changes in accordance with the ion concentration of the dye. By projecting light from the lower opening 29 and measuring the reflected absorbance,
Even if the urine is colored, the color density can be measured without being affected by the coloring components.

Incidentally, Fig. 4 shows the solubility curves for indigo carmine, Fig. 5 shows the solubility curves for Basic Blue 3, and Fig. 6 shows the solubility curves for Basic Red 2.

As is clear from the examples below, such absorbance is
Since there is a correlation with the concentration and ratio of the solution to be measured, the concentration and ratio can be measured by measuring the absorbance. Further, the absorbance is correlated with the ion concentration, that is, the osmotic pressure, so the osmotic pressure can be measured by measuring the absorbance. Furthermore, detection can also be carried out colorimetrically.

(Example) Next, the present invention will be described in further detail by giving examples.

Example 1 Filter paper (Advantech No. 51A) was mixed with 0.25% urease (inactive 140 units/■) and 0.1% ED.
TA, 1% Triton X-100 (1'riton
X-100) (alkylaryl polyether nonionic surfactant manufactured by Rohm & No.
After immersing the sample in an aqueous solution containing % dithioerythritol (DTE), the sample was dried at 40° C. for 40 minutes to undergo an ionization reaction.

On the other hand, filter paper (Advantech No. 202) was added at 0.5%.
After impregnating it by immersing it in ethanol containing Triton X-100, it was dried at 60° C. for 30 minutes to obtain a spread layer.

Furthermore, filter paper (Advantech No. 462) was added to 4%
After being impregnated by immersing it in an aqueous solution of Basic Blue 3, it was dried at 60° C. for 30 minutes, cut into appropriate sizes, and the pieces were used as a reagent layer. The obtained ionization reaction layer, reagent layer, and development layer were sequentially enclosed in a holder to obtain a measuring instrument.

Example 2 An ionization reaction layer was formed in the same manner as in Example 1, and 2
A dye suspension consisting of chloroform containing 2% Basic Blue 3 and 2% Polyvinylpyrrolidone K-3000 was sprayed and dried at room temperature for 30 minutes to form a reagent layer.

The resulting ionization reaction layer and the development layer formed with the reagent layer (
(with the reagent layer facing down) were sequentially sealed in a holder to obtain a measuring instrument.

Example 3 Filter paper (Advantech No. 202) was treated with 0.25% urease (specific activity 140 units/mg), O. 1%E
After being impregnated by immersion in an aqueous solution containing DTA, 1% Triton X-100, and dithioerythritol (DTE), it was dried at 40° C. for 40 minutes to form an ionization reaction layer and developing layer.

On the other hand, a dye suspension consisting of chloroform containing 2% Basic Blue 3 and 2% polyvinylpyrrolidone K-3000 was sprayed onto the double-sided tape and dried for 30 minutes at room temperature to form a reagent layer. The reagent layer side of the double-sided tape provided with the obtained reagent layer was adhered to the ionization reaction layer/deployment layer, cut into an appropriate size, and adhered to one end of a polystyrene film support to obtain a measuring instrument. Ta.

Example 4 A dye solution consisting of methanol containing 1% Basic Blue 3 and 1% Polyvinylpyrrolidone K-3000 in N'M was sprayed on a tape and dried for 30 minutes at room temperature to form a reagent layer.

The reagent layer side of the double-sided tape provided with the obtained reagent layer was adhered to the ionization reaction layer/deployment layer obtained in the same manner as in Example 3, cut into an appropriate size, and adhered to one end of the support. and obtained measuring instruments.

Example 5 3 g of Celite was stirred in 3 ml of a 3% Basic Blue 3 aqueous solution for 8 hours, filtered, and dried in a desiccator to prepare dye-adsorbing fine particles. A suspension of dye-adsorbing microparticles made of chloroform containing 2% dye-adsorbing microspheres and 2% polyvinylpyrrolidone K-3000 was added to the back side of the ionization reaction layer and development layer obtained in the same manner as in Example 3. Spray and dry at room temperature for 30 minutes to form a reagent layer.

The obtained ionization reaction layer/deployment layer provided with the reagent layer was cut to an appropriate size and adhered to one end of a polystyrene film support with double-sided tape to obtain a measuring instrument.

Example 6 A nonwoven fabric (Seiren SK-706) was treated with 0.25% urease (specific activity 140 units/mg) and 0.1% ED.
TA11% Triton X-1oo and 1% dithioerythritol (DTE) were impregnated by immersion in an aqueous solution containing 1% dithioerythritol (DTE) and dried at 40'C for 40 minutes to form an ionization reaction layer and developing layer. Next, 2% Basic Blue 3
A dye suspension thicker than chloroform containing 2% polyvinylhyloridone K-3000 was sprayed onto the double-sided tape and dried for 30 minutes at room temperature to form a reagent layer.

The reagent layer side of the double-sided tape provided with the reagent layer was adhered to the ionization reaction layer and spreading layer obtained by the method of Example 3, cut to an appropriate size, and then attached to a polyethylene film using double-sided tape. It was glued to one end of the support to obtain a measuring instrument.

[Evaluation of Test Sample] Figure 7 shows the relationship between the change in reflection absorbance on the surface of the developing layer and time at a wavelength of 608 nm for 3 minutes after dropping water (standard solution with specific gravity 1.000) in Example 2. The measurement was performed using MCPD-200 manufactured by Otsuka Electronics.

Example 7 Specific gravity 1.00 for the measuring instruments created in Examples 1 to 6
A J4 aqueous solution of 0 to 1.030 was added dropwise, and the reverse absorbance was measured. Table 1 shows the results.

As shown in Table 1, the coloration correlated with each city was obtained. In addition, indigo carmine, basic red 2
9, a similar correlation between specific gravity and black color was obtained.

Furthermore, when a urine sample was actually used and the results were compared with the results measured using the method of the present invention and a refraction type urine hydrometer URIVET (manufactured by Nippon Kogaku Co., Ltd.), the graph shown in FIG. 8 was obtained.

(Effects of the Invention) As described above, the present invention provides an ionization reaction layer that Ah-fis an ionizing agent for selectively ionizing a substance to be measured, and a method in which the solubility is correlated with the ion concentration in the solution. It consists of a reagent layer supporting a dye and a developing layer for the dye,
The method is characterized in that the amount of dye eluted from the reagent layer to the developing layer changes in accordance with the ion concentration generated in the ionization reaction layer, and is reflected in the color density of the surface of the developing layer. Since it is a selective measurement device for ionized ionizable components in solutions, it can measure both substances that do not dissociate into ions in water, as well as substances that can be ionized by an ionizing agent. When the substance to be measured is a component of living organisms, in many cases there are enzymes that specifically decompose and ionize it, so it can be used in a wide range of applications. The ion concentration is then detected using a system that uses a dye whose solubility correlates with the ion concentration in the solution, so the reagent composition and structure are relatively simple, and quick and accurate results can be obtained with easy operation. In particular, according to the test method using urease as an ionizing agent, the amount of urea and the amount of ions can be measured simultaneously, making it possible to accurately measure the urine specific gravity.
This can contribute to the diagnosis of kidney disease in the field of clinical testing. In addition, when measuring the specific gravity of urine, the amount of urea in the urine can be measured by using urease to ionize urea, but this system is also affected by the ion concentration in the urine. It becomes possible to detect both the amount of urea and the amount of ions. Since urine specific gravity has a deep correlation with the amount of urea and the amount of ions, it is possible to measure specific gravity values more accurately than conventional methods that detect only ion concentration or osmotic pressure.

[Brief explanation of drawings]

Figures 1 (A) to (B), Figures 2 (A) to (B), and Figure 3 are cross-sectional views showing each embodiment of the measuring instrument according to the present invention, and Figures 4 to 6 are absorbances of each dye. FIG. 7 is a graph showing the change in reflection absorbance on the surface of the developed layer, and FIG. 8 is a graph showing the relationship between absorbance and specific gravity. 1, 2l...Ionization reaction layer, 2,12.22...Reagent layer, 3.23...Development layer, 4,14...Both table, 5. 15. 25...Support, 17...Ionization reaction layer and development 1-. Figure 1 (A) 5 Figure 2 (A) Patent distributor Terumo Corporation Doji

Claims (5)

[Claims] (1) An ionization reaction layer containing an ionizing agent for selectively ionizing the analyte, a reagent layer supporting a dye whose solubility correlates with the ion concentration in the solution, and a developing layer for the dye. The method is characterized in that the amount of dye eluted from the reagent layer to the developing layer changes depending on the ion concentration generated in the ionization reaction layer, and is reflected in the color density on the surface of the developing layer. An instrument for selectively measuring ionizable components in solutions. (2) The device for selectively measuring ionizable components according to claim 1, wherein the ionization reaction layer also serves as the development layer. (3) The device for selectively measuring ionizable components according to claim 1 or 2, wherein the ionizing agent is an enzyme. (4) Claims 1 to 4, wherein the substance to be measured is urea, and the ionizing agent is urease or urea carboxylase.
3. The device for selectively measuring ionizable components in a solution according to any one of 3. (5) The device for selectively measuring ionizable components in a solution according to any one of claims 1 to 4, wherein the dye is a water-soluble dye having an ionic functional group.