JPH07110324A - Test piece and manufacture thereof - Google Patents

Abstract

PURPOSE:To enable drying quickly at a low temperature by suspending a porous carrier which has a material water-soluble and insoluble to a nonaqueous solvent adsorbed thereon into a nonaqueous solvent containing an adhesive which dissolves in a nonaqueous solvent and becomes water insoluble when dried leaving affinity to water to be applied on a support. CONSTITUTION:As an object material, which is water-soluble and will not dissolve in a nonaqueous solvent, is physiologically active substance such as enzyme or antibody is used. A porous carrier is made, for example, of silica, glass or alumina and is added to an aqueous solution of the object material so that it is mixed to have the material absorbed thereon and then, the mixture thereof is dried. As the nonaqueous solvent, for example, an ether or aromatic hydrocarbon is used and the nonaqueous solvent with the carrier having the material absorbed thereon is mixed with an additive reagent or the like used for a test piece for dry type chemical analysis. As an adhesive, a high-molecular compound which dissolves in the nonaqueous solvent while becomes insoluble to water when dried after solution but leaving affinity to water. The nonaqueous solvent containing those mentioned above is applied on a support and dried to obtain a desired test piece.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel test piece for dry chemical analysis and a method for producing the same.

[0002]

Background of the Invention So-called dry chemical analysis test strips, which are prepared by impregnating paper filters or water-absorbent carriers with detection reagents, and drying the so-called dry chemical analysis test strips, are used to detect various components in liquid samples such as urine and blood. Widely used for measurement.

A conventional general test piece is obtained by impregnating a paper filter, a water-absorbing carrier or the like with an aqueous solution of a detection reagent and drying it, and further impregnating and drying another aqueous solution of the detection reagent, if necessary. The carrier containing the detection reagent is cut into an appropriate size,
It is manufactured by attaching it to a support such as plastic using an adhesive or the like.

However, this manufacturing method has a problem in that it has many steps and the operation is complicated, and it is difficult to manufacture a test piece of a constant quality. That is, as an example, when a carrier such as a paper filter is immersed in a solution containing a detection reagent, the problem that the solution penetrates into the carrier incompletely or insufficiently, and the carrier which is a carrier Since the paper itself uses natural products as raw materials, variations in quality within the lot and between lots during production (variations due to variations in the amount of the detection reagent impregnated into the carrier) tend to occur, and the resulting test pieces The problem is that the quality of the product itself varies.

Further, in the test piece obtained by the above-mentioned method, since the carrier impregnated with the detection reagent and the support are attached using an adhesive, the chemical substance in the adhesive is the detection reagent. There was also a problem that it had a problem in that it was peeled off from the support during the measurement operation (weak mechanical strength). Therefore, there is a current need to develop a test piece that is inexpensive, has little variation, is stable, has high reliability, and has high mechanical strength.

In order to achieve such an object, many studies have been conducted so far, including shortening the manufacturing process.
For example, JP-A-62-278454 and JP-A-2-
Japanese Patent No. 6541 discloses a method of directly applying a detection reagent to a water-absorbent matrix provided on the surface of a support of a test piece. This method does not require the step of sticking paper or the like impregnated with the detection reagent to the support, but instead a step of providing a water-absorbent matrix on the surface of the support is required, so this method shortens the steps. I cannot help saying that the effect of is insufficient.

Further, Japanese Patent Application Laid-Open No. 62-119454 discloses a method for producing a test piece by directly coating a suspension of a water-soluble polymer on a support to form a reagent layer. However, even in this method, there is a problem that it is necessary to prepare a suspension having a complicated component composition, and a special device is required for production.

Further, in the above two methods, there is a problem caused by using the enzymes used for the detection reagent after dissolving them in water. That is, in order to produce a test piece by these methods, an aqueous solution containing an enzyme or the like has to be used as a test solution for preparing a test piece, and therefore, it takes time and labor such as high temperature treatment and vacuum treatment for drying water during production. Although necessary, the problem is that the enzymes are inactivated at that time and a test piece having the desired performance cannot be obtained.

Further, in Japanese Unexamined Patent Publication No. 62-263469, various test reagents dispersed in a non-aqueous solvent together with a water-absorbing powder are coated on a support and dried to give a desired test piece. A method of obtaining is disclosed. However, the test piece obtained by this method has a problem that the calibration range is narrow because the amount of the detection reagent retained is small, and the kind of the non-aqueous solvent that can be used is limited. It had problems such as being limited.

In order to avoid these problems, US Pat. No. 3,993,451 discloses a method of coating a detection reagent and insoluble microparticles on a support through an adhesive layer. According to this method, it is possible to improve the stability of the test piece by preparing various microparticles containing the detection reagents that cannot be mixed separately and sequentially applying them. However, this method is not necessarily a simple method because it requires a step of applying an adhesive and a step of applying fine particles coated with a detection reagent in addition to this. In addition, since this method uses fine particles without pores, there is a problem that a sufficient amount cannot be used when detecting reagents, particularly proteins such as enzymes are used for the test piece. Due to the properties of the adhesive, there are problems such as poor wetting of the test piece during use and variations in the obtained measurement results, and further improvements are desired.

Further, since the non-aqueous solvent is generally volatile and can be dried quickly at a low temperature, the non-aqueous solvent is more advantageous than water as a solvent at the time of producing the test piece, but the non-aqueous solvent like the protein is preferable. When a water-soluble substance is used in a non-aqueous solvent, precipitation or deactivation occurs, so in the previous methods, water was used to dissolve a water-soluble detection reagent such as a protein to prepare a test piece. Even in this case, there is a problem that water is required for dispersing the carrier and preparing the emulsion.

[0012]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and can be manufactured at a lower cost by a simpler operation than conventional methods, has excellent measurement sensitivity, and has a physical strength of the test piece itself. It is an object of the present invention to provide a test piece for dry chemical analysis which is extremely high even after being brought into contact with a sample aqueous solution.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a porous carrier on which a substance that is water-soluble but does not dissolve in a non-aqueous solvent is adsorbed has a property of dissolving in a non-aqueous solvent and is dried after being dissolved in a non-aqueous solvent. Chemical analysis prepared by coating a substrate with a suspension in a non-aqueous solvent containing an adhesive that has the property of being difficult to dissolve in water and water, but having an affinity for water, and drying. Is an invention of a test piece for

Further, according to the present invention, a porous carrier having a substance which is water-soluble but insoluble in a non-aqueous solvent is adsorbed, has a property of dissolving in the non-aqueous solvent, and is dried after being dissolved in the non-aqueous solvent. It is characterized in that it is prepared by suspending in a non-aqueous solvent containing an adhesive having the property of having an affinity with water, which is difficult to dissolve in water and water, and coating on a support and drying.
It is an invention of a method for producing a test piece for dry chemical analysis.

That is, according to the present invention, when the inventors of the present invention use a non-aqueous solvent to produce a test piece for dry chemical analysis, which was conventionally performed using water as a solvent, water is used as a solvent. Since it is possible to carry out the drying treatment at a lower temperature and in a shorter time compared to the case of using as the above, it is considered to be advantageous for the production of the target test piece, and in the course of earnest research, the reagents contained in the test piece Among these, reagents such as proteins such as enzymes, which have the property of causing precipitation or inactivation when added to a non-aqueous solvent, are adsorbed on a porous carrier in advance, and even when added to a non-aqueous solvent if dried. It is possible to use these substances without causing precipitation or deactivation, and it becomes possible to use these substances at sufficient concentrations, and some polymer compounds have the property of dissolving in non-aqueous solvents. Moreover, if it is dissolved in a non-aqueous solvent and then dried, it will dissolve in water. It was found that there are some that have a property that they become difficult to retain, but have an affinity for water, and as a result of further research based on these findings, by using these, a more stable and uniform test for dry chemical analysis The inventors have found that it is possible to manufacture a piece easily and inexpensively, and have completed the present invention.

The substance according to the present invention, which is water-soluble but does not dissolve in a non-aqueous solvent, is a reagent used in preparing a test piece for dry chemical analysis and has such a property. Examples thereof include, but are not particularly limited to, specifically, glucose oxidase, peroxidase, ascorbate oxidase,
Preferable examples include enzymes such as urease and cholesterol oxidase, proteins such as antibodies, and various physiologically active substances such as coenzymes such as nicotinamide adenine dinucleotide (NADH). Further, the amount of adsorption to these porous carriers is not particularly limited because it varies depending on the type of the carrier, and the measurement sensitivity of the dry chemical analysis test piece finally obtained by the method of the present invention is the same as that of the conventional test piece. It is sufficient to adsorb the same amount or more.

The porous carrier used in the present invention is not particularly limited as long as it is insoluble in water and a non-aqueous solvent and is porous, but more specifically. Specifically, a porous carrier made of a material such as silica, glass, aluminum oxide, kaolin, or microcellulose is preferably used. The average diameter of the pores of these porous carriers is not particularly limited as long as it has a size capable of adsorbing a target substance, but is usually 1,500 to 5,0.
It is appropriately selected from the range of 00 angstroms, preferably 2,000 to 4,000 angstroms. It is possible to prepare the test piece for dry chemical analysis of the present invention using a non-porous carrier, but in this case, there arise problems such as a decrease in measurement sensitivity and a long time required for measurement. Needless to say.

The shape of these porous carriers is not particularly limited and may be spherical or crushed. The average particle size is also not particularly limited, but 15
The size of 0 μm or less is preferable because the finally obtained test piece has a good appearance.

Concentrations of these porous carriers (which are water-soluble but have already adsorbed substances which are not soluble in non-aqueous solvents) in the non-aqueous solvent are as follows:
Or, depending on the amount of water-soluble substance which is adsorbed on the carrier but does not dissolve in the non-aqueous solvent, etc., it is usually 1 to 50%,
Preferably, it is appropriately selected from the range of 5 to 20%.

As a method for adsorbing a substance that is water-soluble but is not soluble in a non-aqueous solvent on a porous carrier, the porous carrier is added to an aqueous solution containing the desired substance, mixed, and dried as it is. It suffices to centrifuge to remove excess solution and then dry. Further, as a drying method after adsorption, a freeze-drying method is preferably mentioned.

During the above adsorption reaction, for example, cellulose ether, polyvinyl acetate, polyamides such as Gan.
Copolymer of maleic anhydride and methyl vinyl ether such as trez139 (trade name of GAF), Paogen pp-15
Polyethylene glycol such as (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.),
It is desirable that a hydrophilic polymer such as polyvinylpyrrolidone or polyvinyl alcohol is allowed to coexist, since the obtained porous carrier is easily wet with the sample aqueous solution and the storage stability of the adsorbed substance is good.

The porous carrier on which the target substance is adsorbed can be stored at room temperature until it is used, but it is desirable to store it by cooling in consideration of stability. Also,
Needless to say, a chromogen (dye) or the like may be adsorbed on such a porous carrier at the same time.

Further, it is possible to prepare several kinds of porous carriers in which the adsorption amounts of these substances are changed in advance, and appropriately mix them at the time of producing a desired test piece to adjust the measurement sensitivity, reaction rate and the like. is there.

Further, when there are a plurality of reagents to be adsorbed on the porous carrier in order to prepare a test piece for dry chemical analysis,
Needless to say, these may be adsorbed on the same carrier, or the test pieces may be prepared by appropriately mixing carriers adsorbing these separately.

Furthermore, it is also possible to sequentially adsorb several kinds of substances onto the porous carrier to control and adjust the reaction status and reaction sequence on the porous carrier to some extent. That is, for example, for the purpose of eliminating the influence of ascorbic acid in the sample, a treating agent such as ascorbic acid oxidase may be adsorbed again on the porous carrier on which the target detection reagent is adsorbed. By forming a plurality of reaction layers on the porous carrier by such a method, it is possible to obtain high measurement sensitivity with a small amount of reagent.

The non-aqueous solvent according to the present invention may be any organic solvent having no hydrogen bond between molecules and capable of dissolving the adhesive according to the present invention. The non-aqueous solvent is immiscible with water, such as ethers such as dimethyl ether and diethyl ether, aromatic hydrocarbons such as benzene, toluene and xylene, and halogenated hydrocarbons such as dichloromethane, dibromomethane and chloroform. Some of them are miscible with water, such as acetone, tetrahydrofuran, 1,4-dioxane, etc., and any of them can be used in the present invention. Generally, non-aqueous solvents that are immiscible with water do not have the property of absorbing moisture in the air, and because the original moisture content is low,
If a target test piece is prepared using this, there is an advantage that the substance previously adsorbed on the porous carrier is less likely to be eluted. In particular, halogenated hydrocarbons having a small molecular weight, such as dichloromethane, dibromomethane, and chloroform, have a low boiling point and a high specific gravity, and thus are more preferable as a solvent for suspending the porous carrier according to the present invention. However, the preferred solvent naturally varies depending on the material of the support used when preparing the test piece. That is, it is preferable that the support is not completely dissolved but has the property of swelling (softening) the support because the porous carrier can be strongly immobilized on the support. Therefore,
For example, when using a support made of polyvinyl chloride,
As the non-aqueous solvent, for example, acetone or tetrahydrofuran is more preferable than the halogenated hydrocarbon.

Used in preparing a desired test piece,
The non-aqueous solvent in which the porous carrier adsorbing the target substance is suspended contains various other substances such as a chromogen (coloring reagent) such as ascorbate oxidase (AOD),
Reagents that are usually added when preparing test pieces for dry chemical analysis, such as substances used to reduce the influence of coexisting substances in the sample solution such as iodate on the measured values, are further mixed. Needless to say, the concentrations in these non-aqueous solvents may be appropriately selected from the range of concentrations used for these reagents that are usually used when preparing test pieces for dry chemical analysis.

The adhesive to be added for the purpose of adhering the porous carrier to the support according to the present invention has a property of dissolving in a non-aqueous solvent and dissolves in water when dissolved after drying in a non-aqueous solvent. As long as it is a high molecular compound having a property that it becomes difficult to retain the affinity for water, it can be dissolved in a non-aqueous solvent to be used and has a property that the physical strength of the obtained test piece can be increased to some extent. Although not particularly limited, for example, G
Copolymer of maleic anhydride and methyl vinyl ether such as Antrez139 (trade name of GAF), Paogen pp
-15 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.) and the like are preferable. The concentration of these adhesives used is not particularly limited, depending on the type of non-aqueous solvent, the type of adhesive used, and the concentration of the porous carrier to be added, but is usually 1 to 10% as the concentration in the non-aqueous solvent, preferably Is appropriately selected from the range of 2 to 5%. In addition, a polymer compound such as polystyrene, polyvinyl chloride, ethyl cellulose, polyvinyl butyral, polyacrylic acid ester, and vinyl acetate resin may be further added to the solution containing the adhesive as described above.

Further, hydrophilic polymers such as cellulose ether, polyvinyl acetate, polyamides, polyvinylpyrrolidone and polyvinyl alcohol may be further added to the non-aqueous solvent containing the adhesive as described above.
By adding such a hydrophilic polymer, it is possible to prevent the dye or the like from flowing out of the test piece during or after the reaction between the test piece obtained and the sample solution.

When the test piece of the present invention is prepared by using a copolymer of maleic anhydride and methyl vinyl ether as an adhesive, it is desirable to carry out the drying step as quickly as possible. That is, when a solution containing the polymer and a porous carrier on which a target substance is adsorbed is applied onto a support and dried, a test piece obtained when the solution is a water or alcohol solution, or A test piece obtained by using an ether-based non-aqueous solvent solution but drying it for 10 hours or more has a property that it is easily wet with water but the immobilized porous carrier is easily peeled off. On the other hand, using a non-aqueous solvent to prepare a solution containing the polymer and a porous carrier to which the target substance is adsorbed, after applying this on a support, quickly in about 30 seconds When the test piece is prepared by drying, the obtained test piece has a property that it is easily wetted by water, but the immobilized porous carrier is difficult to be peeled off. Will have. Further, in the case of preparing a test piece under such conditions, it is not necessary to further add a hydrophilic polymer or polymer compound other than this, and the production time of the test piece can be shortened, which is an advantage. Also occurs. The copolymer of maleic anhydride and methyl vinyl ether is used by dissolving it in a ketone or ether non-aqueous solvent at a concentration of usually 1 to 25%, preferably 2 to 10%.

Further, in a non-aqueous solvent in which a porous carrier is suspended, for example, polyoxyethylene sorbitan monolaurate, sorbitan monolaurate, polyoxyethylene octyl phenyl ether and the like surfactants usually used in this field. May be added in a concentration range of usually 0.1 to 10%, preferably 0.5 to 2%. Since the test piece obtained by doing in this way becomes easier to get wet with water,
It becomes possible to improve the reactivity with the sample solution.

Further, as the support according to the present invention, as long as it does not affect the reaction of the test piece according to the present invention with the sample solution, it is usually a support for preparing a test piece for dry chemical analysis in this field. It can be used without particular limitation as long as it is used as a body. More specifically,
For example, plastics such as polyvinyl chloride, polystyrene and polypropylene, metal plates (or foils), paper, ceramics, glass and the like can be used.

The test piece for dry chemical analysis of the present invention may be prepared, for example, as follows. That is, a non-aqueous solvent containing a porous carrier and an adhesive having adsorbed a target substance prepared as described above, and further a chromogen, a polymer compound, a hydrophilic polymer, a surfactant, etc., if necessary. Then, the test piece for dry chemical analysis of the present invention can be obtained by coating on a support and drying.

A non-aqueous solvent containing the desired reagents is applied,
Drying may be carried out by a method usually used when preparing a test piece for dry chemical analysis, for example, a method of dropping a certain amount of the test piece on a support and drying it, or a belt-like form on the support. A method of coating and drying, a method of spraying and drying the non-aqueous solvent on a support, a method of removing the solvent from the non-aqueous solvent to obtain a film-like material, and affixing it to the support to prepare a test piece. It is enough if you use such as. In addition, the coating may be carried out two or more times, or the composition of the first coating and the composition of the second coating or later may be appropriately changed to form two reaction layers on the support. You may form more than one layer.

By thus forming two or more reaction layers, it is possible to reduce the influence of interfering substances in the sample solution. That is, for example, if ascorbic acid oxidase is contained in the upper layer, the influence of ascorbic acid in the sample solution can be removed in advance, and thus the influence on the intended color development reaction in the lower layer can be reduced. Further, by forming the reaction layer, it is possible to sequentially carry out continuous reactions. Furthermore, several kinds of non-aqueous solvent solutions having different compositions may be lined up and coated on a support and dried to prepare a test piece capable of simultaneously measuring several kinds of items.

Further, as a drying method after applying a non-aqueous solvent solution containing a porous carrier, an adhesive or the like, on which a target substance is adsorbed, onto a support, a test piece having a target performance can be obtained. It is not particularly limited as long as it is a method, for example, 50
A method of drying in an air or nitrogen gas stream at a temperature of ℃ or less is desirable because it can shorten the production time. A drying method by heating with infrared rays or the like may be used. The drying time is not particularly limited as long as it is appropriately set depending on the composition of the non-aqueous solvent solution and the conditions such as the speed and temperature of the air stream during drying.
Usually within 10 minutes, preferably within 5 minutes, more preferably 30 minutes
Within seconds is desirable. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0037]

【Example】

Example 1 Production of glucose measuring test piece using tetrahydrofuran (a) Production of test piece 1.03 mg / ml peroxidase (manufactured by Wako Pure Chemical Industries, Ltd.,
Horseradish), 2.9 mg / ml glucose oxidase (Wako Pure Chemical Industries, Ltd., Aspergillus genus) and G
100 m containing 1% of Antrez 139 (trade name of GAF, a copolymer of maleic anhydride and methyl vinyl ether)
To 10 ml of M phosphate buffer (pH 5.4), 3 g of P-3000-200
(Manufactured by Sigma, porous glass having pores with an average of 3,000 Å) was immersed for 1 hour. Next, this was centrifuged to remove the supernatant, and the porous glass was freeze-dried. 120 mg of the obtained porous glass (freeze-dried product), 10 μl of polyoxyethylene sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1 ml of tetrahydrofuran (10 mM tetramethylbenzidine and Gantre)
Contains 5% of z139. ) Was mixed and 7 μl of this was applied to a polyvinyl chloride support (6 × 110 mm) (0.4 mm thickness), and then dried with warm air at 45 ° C. for 30 seconds to prepare a glucose test piece. (b) Practical test of glucose measurement test piece (operating method) The test piece obtained in (a) above was immersed in water or an aqueous glucose solution having a predetermined concentration, and the change in reflection absorbance at 760 nm was measured [measuring device: Pretester RM-4
Type 05 (manufactured by Wako Pure Chemical Industries, Ltd.) is used]. (Result) 10 mg / g of the glucose measurement test piece obtained in (a) above
It was found that when it was immersed in a glucose solution of dl, it turned blue within 30 seconds. Further, using a glucose solution of 0, 2.5, 5.0 or 10 mg / dl, the detection limit of the test piece obtained in the above (a) was compared with the commercially available test piece for glucose measurement (Wako Pure Chemical Industries, Ltd.
As a result of comparison with Pretest 3A manufactured by Co., Ltd., it was found that the detection sensitivity was equal to or higher than that of a commercial product.
Although the commercially available test piece is easily wet and rubbed, the test paper portion is easily peeled off, but the detection reagent-immobilized surface of the test piece obtained by the method of the present invention is firmly bound to the support and is wet. It did not come off even after rubbing. Also 2,000, 200 or 50
A glucose measurement test piece was prepared by performing the same operation using the same reagent as the above (a) except that a porous glass having pores of the following respective average angstroms was used, and the measurement sensitivity of each (a) was obtained. It was compared with that of the test piece obtained in. As a result, the measurement sensitivity of the test piece prepared using the porous glass having an average pore size of 2,000 angstroms is 1/2 to 1/3 of that of the test piece obtained in (a) above, and the average pore size of 200 angstroms. It is 1/5 to 1/10 of the test piece obtained in the above (a), and the average of 50
That of a test piece prepared using a porous glass having pores of angstrom or less is 1/10 of that of the test piece obtained in (a) above.
It was below. In addition, the drying condition of the test piece in the above (a) was replaced with "drying with warm air at 45 ° C for 30 seconds" to "room temperature (about 25
℃) left for 3 hours to dry ”. The obtained test piece showed the same measurement sensitivity as that obtained in the above (a), but when it was taken out after being immersed in the glucose solution for about 30 seconds, the detection reagent-immobilized surface of the test piece was easily separated from the support. It peeled off.

Example 2 Test Piece for Glucose Measurement Using Acetone (a) Production of Test Piece The same operation as in (a) of Example 1 was repeated except that the same volume of acetone was used instead of tetrahydrofuran. Then, a test piece for glucose measurement was manufactured. (b) Practical test of glucose measurement test piece (operating method) The same operation was performed using the same reagents as in Example 1 (b). (Results) The detection sensitivity equivalent to that of the glucose measurement test piece obtained in (a) of Example 1 was obtained. Further, the detection reagent-immobilized surface of the test piece obtained above was firmly bound to the support and was not peeled off even when it was rubbed after being wet.

Example 3 Test Piece for Glucose Measurement Using Polystyrene and Paogen (a) Production of Test Piece 1.03 mg / ml peroxidase (manufactured by Wako Pure Chemical Industries, Ltd.,
Horseradish derived), 2.9 mg / ml glucose oxidase (Wako Pure Chemical Industries, Ltd., Aspergillus origin), polystyrene (average molecular weight: 45,000, manufactured by Aldrich) 4% and Paogen pp-15 (Daiichi Kogyo) Pharmaceutical Co., Ltd.) 2
% In 100 mM phosphate buffer (pH 5.4) containing 3 g of P
-3000-200 (manufactured by Sigma, porous glass having pores with an average of 3,000 Å) was immersed for 1 hour. Next, this was centrifuged to remove the supernatant, and the porous glass was freeze-dried. 120 mg of the obtained porous glass
(Lyophilized product), 10 μl of polyoxyethylene sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1 ml of dichloromethane [10 mM tetramethylbenzidine, polystyrene (average molecular weight: 45,000, manufactured by Aldrich) 4%
Also contains 2% of Paogen pp-15 (Daiichi Kogyo Seiyaku Co., Ltd.). ] Was mixed, and 7 μl of this was applied to a polyvinyl chloride support (6 × 110 mm) (0.4 mm thickness), and then dried with warm air at 45 ° C. for 30 seconds to prepare a test piece for measuring glucose. (b) Practical test of glucose measurement test piece (operating method) The same operation was performed using the same reagents as in Example 1 (b). (Results) The detection sensitivity equivalent to that of the glucose measurement test piece obtained in (a) of Example 1 was obtained. Further, the detection reagent-immobilized surface of the test piece obtained above was firmly bound to the support and was not peeled off even when it was rubbed after being wet.

Example 4 Test Piece for Glucose Measurement Using Carboxyl Group-Containing Vinyl Chloride Resin (a) Preparation of Test Piece 1.03 mg / ml of peroxidase (manufactured by Wako Pure Chemical Industries, Ltd.,
Horseradish derived), 2.9 mg / ml glucose oxidase (Wako Pure Chemical Industries, Ltd., Aspergillus derived), low molecular weight polyvinyl chloride (viscosity: 0.68, Aldrich)
5% and a vinyl chloride resin containing a carboxyl group (manufactured by Aldrich) 5% in 100 mM phosphate buffer (pH 5.4) 1
In 0 ml, 3 g of P-3000-200 (Sigma, 3,000 on average)
Porous glass with Angstrom holes. ) Was immersed for 1 hour. Next, this was centrifuged to remove the supernatant, and the porous glass was freeze-dried. Obtained porous glass 120 mg (lyophilized product), 10 μl of polyoxyethylene sorbitan monolaurate (Wako Pure Chemical Industries, Ltd.)
And 10 ml of tetramethylbenzidine, 4% of polystyrene (average molecular weight: 45,000, manufactured by Aldrich) and 2% of Paogen pp-15 (Daiichi Kogyo Seiyaku Co., Ltd.). ] Was mixed and 7 μl of this was applied to a polyvinyl chloride support (6 × 110 mm) (0.4 mm thickness) and then dried with warm air at 45 ° C. for 5 minutes to prepare a test piece for measuring glucose. (b) Practical test of glucose measurement test piece (operating method) The same operation was performed using the same reagents as in Example 1 (b). (Results) The detection sensitivity equivalent to that of the glucose measurement test piece obtained in (a) of Example 1 was obtained. Further, the detection reagent-immobilized surface of the test piece obtained above was firmly bound to the support and was not peeled off even when it was rubbed after being wet.

Example 5 Test piece for glucose measurement using silica gel (a) Production of test piece 1.03 mg / ml peroxidase (manufactured by Wako Pure Chemical Industries, Ltd.,
Horseradish), 2.9 mg / ml glucose oxidase (Wako Pure Chemical Industries, Ltd., Aspergillus genus) and G
100 m containing 1% of Antrez 139 (trade name of GAF, a copolymer of maleic anhydride and methyl vinyl ether)
3 g of silica gel (Wakogel C-300, manufactured by Wako Pure Chemical Industries, Ltd.) was immersed in 10 ml of M phosphate buffer (pH 5.4) for 1 hour. Next, this was centrifuged to remove the supernatant, and silica gel was lyophilized. 120 mg of the obtained silica gel (freeze-dried product) and 1 ml of the specified organic solvent
[Sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.) 5%, 3.8 mg / ml 4-aminoantipyrine, 3.8 mg / ml
Contains N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), 5% Gantrez 139 and a predetermined polymer compound in a predetermined concentration. ], And mix this 7μ
l is coated on a polyvinyl chloride support (6 x 110 mm) (0.4 m
m thickness), and then dried with warm air at 45 ° C. for 5 minutes to produce a glucose measurement test piece. Table 1 shows the organic solvent and the polymer compound (and the concentration thereof used) used in the production.

[Table 1] * 1: Made by Wako Pure Chemical Industries, Ltd. * 2: Made by Aldrich. * 3: manufactured by Daicel Chemical Industries, Ltd. * 4: manufactured by Daicel Chemical Industries, Ltd. * 5: manufactured by Daicel Chemical Industries, Ltd.
* 6: Made by Chuo Science Co., Ltd. * 7: Hoechst Synthesis Co., Ltd.
Made. (b) Practical test of glucose measuring test piece When the test piece obtained in (a) above was reacted with a glucose solution of 50 to 2000 mg / dl, a change in color tone depending on the glucose concentration was observed. Further, the detection reagent-immobilized surface of the test piece obtained above is firmly bound to the support,
It did not come off even when rubbed after getting wet.

Example 6 Glucose Measuring Test Piece Containing Ascorbate Oxidase (a) Preparation of Test Piece-1 2.5 mg / ml ascorbate oxidase (Wako Pure Chemical Industries, Ltd.
Ltd., from pumpkin), 4 mg / ml peroxidase (Wako Pure Chemical Industries, Ltd., horseradish origin), 40 mg / ml glucose oxidase (Wako Pure Chemical Industries, Ltd., Aspergillus genus) and Gantrez 169 (GAF
Company name, copolymer of maleic anhydride and methyl vinyl ether) 100 mM phosphate buffer (pH 5.4) containing 1% 10 ml
Then, 3 g of P-3000-200 (manufactured by Sigma, porous glass having pores with an average of 3,000 Å) was immersed for 1 hour. Next, this was centrifuged to remove the supernatant, and the porous glass was freeze-dried. 120 mg of the obtained porous glass (freeze-dried product), 10 μl of polyoxyethylene sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.) and a mixed solvent (acetone: tetrahydrofuran = 1: 1)
4) 1 ml [containing 10 mM tetramethylbenzidine, 4% polystyrene (average molecular weight: 45,000, manufactured by Aldrich) and 5% Gantrez 169]. ] Was mixed and 7 μl of this was applied to a polyvinyl chloride support (6 × 110 mm) (0.4 mm thickness) and then dried with warm air at 45 ° C. for 5 minutes to prepare a test piece for measuring glucose. (b) Production of test piece-2 4 mg / ml peroxidase (manufactured by Wako Pure Chemical Industries, Ltd., derived from horseradish), 40 mg / ml glucose oxidase (manufactured by Wako Pure Chemical Industries, Ltd., derived from Aspergillus) and G
100m containing 1% of Antrez 169 (trade name of GAF, a copolymer of maleic anhydride and methyl vinyl ether)
To 10 ml of M phosphate buffer (pH 5.4), 3 g of P-3000-200
(Manufactured by Sigma, porous glass having pores with an average of 3,000 Å) was immersed for 1 hour. Then, this was centrifuged to remove the supernatant, containing 2.5 mg / ml ascorbate oxidase and 1% Gantrez 169.
After further immersing in 10 ml of 100 mM phosphate buffer (pH 5.4) for 1 hour, the mixture was centrifuged again to remove the supernatant, and the porous glass was freeze-dried. 120 mg of the obtained porous glass
(Lyophilized product), 10 μl of polyoxyethylene sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1 ml of mixed solvent (acetone: tetrahydrofuran = 1: 4) [tetramethylbenzidine 10 mM, polystyrene (average molecular weight) : 45,000, Aldrich) 4% and Gantr
Contains 5% ez 169. ], And 7 μl of this was applied to a polyvinyl chloride support (6 × 110 mm) (0.4 mm thickness) and then dried with warm air at 45 ° C. for 5 minutes to prepare a glucose measurement test piece. (c) Manufacture of test piece -3 Using the same reagent as in (a) above, except that the porous glass was immersed in a solution obtained by removing only ascorbate oxidase from the porous glass immersion solution in (a) above. A glucose measuring test piece was similarly produced by the method. (d) Practical test of glucose measurement test piece Using a 400 mg / dl glucose aqueous solution containing a predetermined concentration of ascorbic acid as a sample, the test pieces obtained in (a), (b) and (c) above are used for these samples. Of each test piece after immersion in
The change in reflection absorbance at 760 nm was measured by the pretester RM-
It was measured using a 405 type (manufactured by Wako Pure Chemical Industries, Ltd.).
The obtained results are shown in FIGS. 1 shows the results obtained for the test piece obtained in (a) above, FIG. 2 shows the results obtained for the test piece obtained in (b) above, and FIG. 3 shows the results obtained for the test piece obtained in (c) above. In each figure, the solid line (---) shows the results obtained with the glucose solution without ascorbic acid added, and the dotted line (----) shows the glucose solution containing 0.1% ascorbic acid as the sample. The obtained results are shown respectively. As is clear from the results of FIGS. 1 to 3, the reflection absorbance value obtained by using a glucose solution containing 0.1% ascorbic acid 1 minute after the reaction was started as a sample and a glucose solution without addition of ascorbic acid as a sample were obtained. The closest value was the value measured by the test piece obtained in (b) above. As a result, the test piece obtained in (b) above, that is, 2
It can be seen that the test piece prepared by using the porous carrier having the reaction layer of the layer formed thereon and having ascorbic acid oxidase held thereon is the least affected by ascorbic acid.

[0043]

As described above, the present invention is characterized in that the test piece can be produced without using water, which was essential in the conventional method for producing a test piece for dry chemical analysis. It is an invention, and by using the present invention, an effect that a stable and uniform test piece for dry chemical analysis having high measurement sensitivity and high physical strength can be produced more simply and inexpensively than the conventional method It is a great invention that contributes to the industry.

[Brief description of drawings]

FIG. 1 is a graph showing a change in reflection absorbance at 760 nm after a glucose measurement test piece obtained in Example 6 (a) was reacted with a sample.

FIG. 2 is a graph showing a change in reflection absorbance at 760 nm after the glucose measurement test piece obtained in Example 6 (b) was reacted with a sample.

FIG. 3 is a graph showing the change in reflection absorbance at 760 nm after the glucose measurement test piece obtained in Example 6 (c) was reacted with a sample.

[Explanation of symbols]

In FIGS. 1 to 3, the solid line (-) shows the results obtained by using the glucose solution without ascorbic acid added, and the dotted line (----) shows the glucose solution containing 0.1% ascorbic acid. The results obtained as samples are shown respectively.

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[Procedure amendment]

[Submission date] October 20, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

The substance according to the present invention, which is water-soluble but does not dissolve in a non-aqueous solvent, is a reagent used in preparing a test piece for dry chemical analysis and has such a property. Examples thereof include, but are not particularly limited to, more specifically, for example, glucose oxidase, peroxidase, ascorbate oxidase ,
Preferable examples include enzymes such as urease and cholesterol oxidase, proteins such as antibodies, and various physiologically active substances such as coenzymes such as nicotinamide adenine dinucleotide (NADH). Further, the amount of adsorption to these porous carriers is not particularly limited because it varies depending on the type of the carrier, and the measurement sensitivity of the dry chemical analysis test piece finally obtained by the method of the present invention is the same as that of the conventional test piece. It is sufficient to adsorb the same amount or more.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

During the above adsorption reaction, for example, cellulose ether, polyvinyl acetate, polyamides such as Gan.
A copolymer of maleic anhydride and methyl vinyl ether such as trez139 (trade name of GAF), Paogen pp-
If a hydrophilic polymer such as polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, etc. such as 15 ( manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.) is allowed to coexist, the obtained porous carrier is easily wetted by the sample aqueous solution and is adsorbed. It is desirable because the storage stability of the substance is also improved.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

Furthermore, it is also possible to sequentially adsorb several kinds of substances onto the porous carrier to control and adjust the reaction status and reaction sequence on the porous carrier to some extent. That is, for example, for the purpose of removing the influence of ascorbic acid in a sample, ascorbic
A treating agent such as acid oxidase may be adsorbed again on the porous carrier on which the target detection reagent is adsorbed. By forming a plurality of reaction layers on the porous carrier by such a method, it is possible to obtain high measurement sensitivity with a small amount of reagent.

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Claims (5)

[Claims] 1. A porous carrier on which a substance that is water-soluble but does not dissolve in a non-aqueous solvent is adsorbed, has a property of dissolving in a non-aqueous solvent, and is dissolved in a non-aqueous solvent and then dried to form water. A test piece for dry chemical analysis, which is prepared by applying a suspension of a non-aqueous solvent containing an adhesive having a property that it becomes difficult to dissolve but retains an affinity for water on a support and drying the suspension. 2. The test piece according to claim 1, wherein the porous carrier has pores of 1,500 to 5,000 angstroms. 3. The test piece according to claim 1, wherein the substance that is water-soluble but does not dissolve in a non-aqueous solvent is a protein. 4. A porous carrier, which is adsorbed with a substance which is water-soluble but is not soluble in a non-aqueous solvent, has a property of dissolving in a non-aqueous solvent, and when dissolved in a non-aqueous solvent and dried, it becomes water-soluble. Test for dry chemical analysis, characterized by being prepared by suspending in a non-aqueous solvent containing an adhesive having a property of becoming difficult to dissolve but having an affinity for water, coating on a support, and drying. Piece manufacturing method. 5. The method according to claim 4, wherein the substance that is water-soluble but is insoluble in a non-aqueous solvent is a protein.