JPH08304382A - Manufacture of analysis element - Google Patents

Abstract

PURPOSE: To conduct quick measurement by impregnating a porous matrix with a solution which is obtained by dissolving tetrazolium salt in an organic solvent easy to evaporate, and drying the same. CONSTITUTION: This is the manufacture of an analysis element having a layer containing tetrazolium salt as a color development pigment. A tetrazolium- containing layer is manufactured by impregnating a porous matrix with a solution which is obtained by dissolving tetrazolium salt in an organic solvent easy to evaporate, and drying the same. That is, the porous matrix is impregnated with the solution which is prepared by using the organic solution easy to evaporate, and the organic solvent is quickly evaporated. Since the solvent evaporates quickly, a solute (tetrazolium salt) in the solution becomes very fine particles and remains in the porous matrix. Since the tetrazolium salt is a very fine crystal, reactivity is heightened. Methanol having the high solubility of the tetrazolium salt and a low boiling point is favorable as the organic solvent.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an analytical element for measuring components in body fluids. For more details,
The present invention relates to a method for producing an analytical element containing at least a tetrazolium salt in one layer.

[0002]

2. Description of the Related Art A tetrazolium salt-based color-developing agent can be used in a wide range of applications by combining it with an enzyme or reagent suitable for the object to be measured. And, it is often used after being processed into a dry analytical element.

In the analysis of components in body fluids, there are some substrates and color-developing agents which are unstable and gradually colored when they coexist adjacently. For example, a tetrazolium salt-based color-developing agent causes a favorable color reaction under weakly alkaline conditions, and is therefore used in combination with an alkaline buffer. However, if both are contained in the same matrix,
In dry analytical elements, the inclusion of both in separate layers often solves this problem because unwanted coloration occurs gradually (eg, US Pat. No. 4,351,899).

In JP-A-63-94995, a tetrazolium salt, enzymes and reagents are kneaded with a water-soluble polymer on a support, a porous matrix is provided on the support, and the polymer is dissolved on the support. A method is disclosed in which an alkaline buffer is dispersed in a non-existent solvent and the resulting dispersion is impregnated into a porous matrix to form a buffer layer.

[0005]

The tetrazolium salt, which is the color former (color former) used in these analytical elements, is poorly soluble in water, and the formazan produced by the color development reaction is even less soluble in water. Therefore, in a layer of the type formed by kneading a polymer and a tetrazolium salt, the polymer dissolves in water in urine or blood, and then the tetrazolium salt, which is difficult to dissolve, is slowly dissolved. It was unsuitable for automatic measuring devices in hospitals and inspection centers that had to carry out the number of measurements while holding.

Further, even in a type in which a tetrazolium salt is dissolved in water over time to form an aqueous solution, and a porous matrix is impregnated with the color former layer, large crystals of the tetrazolium salt are generated during drying, and the large crystals are formed. , It is difficult to dissolve in urine and blood containing various salts and proteins. Therefore, the reaction is slow and reliable color development cannot be expected. Therefore, it has been a problem how to make the tetrazolium salt into fine crystals in order to increase the reactivity.

[0007]

Means for Solving the Problems The above-mentioned problem is a method for producing an analytical element having a layer containing a tetrazolium salt as a coloring dye, wherein a solution obtained by dissolving a tetrazolium salt in an organic solvent that easily evaporates is porous. It was found that the problem can be solved by forming a tetrazolium salt-containing layer by impregnating into a matrix and drying.

As mentioned above, the tetrazolium salt is very insoluble in water. However, the sample serving as a sample is an aqueous solution such as urine or blood, and it takes time for the tetrazolium salt to be completely dissolved due to large particles of the tetrazolium salt or interference with coexisting polymers.

In order to solve this problem, the present inventors decided to make the particles of the tetrazolium salt in the layer as small as possible. As a method, a solution is prepared by using an organic solvent that is particularly apt to evaporate among the organic solvents for dissolving the tetrazolium salt to impregnate the porous matrix and rapidly evaporate the organic solvent. Due to the rapid evaporation of the solvent, the solute (tetrazolium salt) in the solution becomes very fine particles and remains in the porous matrix.

As described above, when water or a solvent having a large molecular weight that does not easily evaporate is used as the solvent, the crystal particles of the tetrazolium salt become huge particles and are fixed in the porous matrix. When urine or blood is dripped into the porous matrix, the tetrazolium salt giant particles are very difficult to dissolve. As a result, the measurement accuracy is reduced. Also,
As a method for forming fine crystals, it is considered that the temperature is rapidly dried by raising the drying temperature, but in general, a tetrazolium salt, which is weak against heat, may be decomposed.

As a result of diligent research on many organic solvents, it has been found that methanol is preferable as an organic solvent having a high solubility of a tetrazolium salt and a low boiling point.

The type of porous matrix may be one used in this industry, such as filter paper, cloth,
Membrane filters can be used.

The type of tetrazolium salt that can be used in the present invention may be one used in this industry,
3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl) -4,4'-diyl] bis [2- (p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride] and , 3- (p-iodophenyl) -5-phenyl-2H-tetrazolium chloride, 3- (4,5-dimethyl-2-thiazolyl) -2H-tetrazolium bromide, 3,3 '-(4,4-biphenylene) -Bis (2
5-diphenyl) -2H-tetrazolium chloride,
3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis (2,5-diphenyl) -2H-tetrazolium chloride, and 3,3'-(3,3'-dimethoxy-4) , 4'-biphenylene) -bis [2,5-bis (p-nitrophenyl) -2H-tetrazolium chloride] and the like can be used. None of these tetrazolium salts are practically insoluble in water but well in methanol.

As mentioned above, the tetrazolium salt develops well under alkaline conditions, so the tetrazolium salt-containing layer according to the present invention is also combined with the weakly alkaline buffering agent-containing layer. The reason for making it weakly alkaline is that if it is made strongly alkaline, other reagents (such as enzymes) are deactivated.

[0015]

[Examples] Examples produced by two-stage impregnation are shown below, but it is needless to say that the present invention is not limited to this and can be processed into an integrated multilayer analytical element having a tetrazolium salt provided in a separate layer. . ○ Example: Preparation of analytical element for measuring β-hydroxybutyric acid according to the present invention The following components were mixed and the filter paper manufactured by Toyo Roshi Kaisha, Ltd. (No. 6) was used.
5) was cut into 100 mm × 100 mm pieces and impregnated, and dried at 40 ° C. for 60 minutes in the dark. (Formulation) 0.2m Phosphate buffer solution (pH 8.0) 100ml NAD ⁺ 200mg 1-methoxy PMS 2mg β-hydroxybutyrate dehydrogenase 2000U Next, the following components were mixed and impregnated into the previously prepared filter paper, and 4
It was dried at 0 ° C. for 10 minutes in the dark. (Formulation) NTB * 200 mg Methanol 100 ml *; 3,3 '-[3,3'-dimethoxy- (1,1'-
Biphenyl) -4,4'-diyl] -bis [2- (p-
Abbreviation of [nitrophenyl) -5-phenyl-2H-tetrazolium chloride] The obtained reagent layer is cut into 5 mm x 5 mm, and attached to the tip part of a 5 mm x 60 mm white polyethylene terephthalate film with a double-sided tape. Comparative Example: Preparation of Analytical Element for Measuring β-Hydroxybutyric Acid by Prior Art In the same manner as in Example except that the component of methanol was replaced with purified water, an analytical element for measuring β-hydroxybutyric acid was prepared. It was made. ○ Measurement The analytical element according to the present invention was dipped in an aqueous solution of β-hydroxybutyric acid prepared to each concentration shown on the horizontal axis of Fig. 1 to remove excess water, and then a color difference meter (manufactured by Nippon Denshoku Co., Σ90). Was used to measure the reflectance at 600 nm after 3 minutes. Further, the time courses of the analysis element according to the present invention and the analysis element of the comparative example were compared. The β-hydroxybutyric acid aqueous solution used was 0.5 mM, and the reflectance at 600 nm was measured every hour. The graph is shown in FIG. The horizontal axis is time (minutes). -Results It can be seen that the solvent in which the tetrazolium salt is dissolved is methanol, and a good calibration curve is drawn, and more rapid measurement is performed than that in water.

[0016]

EFFECTS OF THE INVENTION According to the present invention, the tetrazolium salt is rapidly dissolved by the aqueous solution of the sample, so that the rapid measurement can be performed.

[0017]

[Brief description of drawings]

FIG. 1 is a graph showing a calibration curve of the output value of a color difference meter with respect to the β-hydroxybutyric acid concentration. FIG. 2 is a graph showing a time course comparing a solution of a tetrazolium salt dissolved in water and a solution of the tetrazolium salt dissolved by the method according to the present invention.

Claims (2)

[Claims] 1. A method for producing an analytical element having a layer containing a tetrazolium salt as a color-forming agent, which comprises impregnating a porous matrix with a solution obtained by dissolving a tetrazolium salt in an organic solvent that easily evaporates and drying the solution. A method for producing an analytical element, which comprises producing a tetrazolium salt-containing layer. 2. The organic solvent used is methanol.
The method according to claim 1.