JPH0518958A - Analytical element - Google Patents

Abstract

PURPOSE:To improve the simultaneous reproducibility and measurement accuracy of an analytical element by constituting an expanding layer of a specific fiber. CONSTITUTION:This analytical element is constituted of a reagent layer formed on a substrate and containing at least one kind of reagent and an expanding layer formed on the reagent layer of a fiber having such a size that can pass through an 80-mesh screen but cannot pass through a 100-mesh screen. In addition, a reagent layer containing at least one kind of reagent is provided on a substrate and an expanding layer provided on the reagent layer is formed of short fiber, having such a size that can pass through a 325-mesh screen but cannot pass through a 400-mesh screen. The expanding layer on the reagent layer formed on the substrate is composed of one layer or a plurality of layers and uniformly diffuses a fixed quantity of fluid sample into a sample layer at a fixed quantity per a fixed area. Therefore, the analytical element can be improved in simultaneous reproducibility and measurement accuracy.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical element for analyzing a specific component in a liquid sample, and more particularly to a multi-layer analytical element provided with a porous developing layer useful for a clinical test using a liquid as a sample. .

[0002]

BACKGROUND OF THE INVENTION Conventionally, as one form of a dry type analytical element, a water-absorbing reagent layer containing a color reaction reagent and a hydrophilic polymer (binder) and a porous layer are provided on a liquid-impermeable and transparent support. There has been proposed an integrated multi-layer analysis element provided with a property spreading layer. The porous spreading layer in this analytical element is
Aqueous liquid sample that has been spotted (eg, whole blood, plasma, serum,
Lymph, saliva, cerebrospinal fluid, vaginal fluid, urine, liquor, drainage, etc.) can be laterally spread without substantially unevenly distributing the components contained in the aqueous liquid sample, and the reagent can be used at a constant rate per unit area. It is a layer that has a function of diffusing into the layer (metering function). Incidentally, the porous spreading layer having such a function is described in US Pat.
9-53888, JP-A-55-164356, JP-A-57-66359, and JP-A-57-12.
5847, JP-A-57-197466 and the like.

By the way, in an analytical element provided with a porous spreading layer formed by using fibers such as cellulose, one of the improvements required is the problem of simultaneous reproducibility.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an analytical element which is excellent in simultaneous reproducibility and has high measurement accuracy. An object of the present invention is an analytical element comprising a support, on which a reagent layer containing at least one reagent is provided, and a development layer provided on the reagent layer, the development layer passing through a 80-mesh sieve. , An analysis element characterized by comprising fibers which do not pass through a 100-mesh sieve.

[0005] Further, the analytical element comprises a support, on which a reagent layer containing at least one reagent is provided, and a developing layer is provided on the reagent layer, the developing layer passing through a 325-mesh sieve, and The present invention is achieved by an analytical element characterized by being configured by using short fibers that do not pass through a mesh sieve. Hereinafter, the analytical element of the present invention will be described in more detail.

The support for the analytical element of the present invention includes:
Any type of polymer material such as cellulose acetate, polyethylene terephthalate, polycarbonate, polystyrene can be used as long as it is liquid impermeable and light transmissive. The thickness of the support is arbitrary, but it is preferably about 50 to 250 μm. One side surface of the support on the observation side can be optionally processed according to its purpose.

When the reagent layer is provided on the support, the reagent layer can be directly provided, but in some cases, a light-transmitting undercoat layer is provided to enhance the adhesiveness between the reagent layer and the support. You may do it. The reagent layer is provided to contain the reagents to be quantitatively reacted with the sample component to be analyzed and to perform the quantitative reaction. Since this reagent layer is provided by coating the support with a hydrophilic colloid substance as a binder, it is necessary to uniformly contain the reagents, unlike the conventional case in which a carrier such as a filter paper is impregnated with the reagent. And has the advantage that the content of the reagent can be freely controlled. As such a hydrophilic colloidal substance, a natural or synthetic polymer substance is preferable, and more desirably, a gelatin derivative such as gelatin or modified gelatin, polyvinyl alcohol, polyvinylpyrrolidone or the like is preferable. Among these, gelatin derivatives such as gelatin are particularly preferable. These hydrophilic colloid materials preferably have a degree of swelling of about 150-500%. The film thickness can be selected as desired, and is preferably at least about 5 μm or more.

It goes without saying that the reagents contained in the reagent layer constructed as described above depend on the analyte component to be analyzed in the sample and the analysis reaction selected for analyzing this component. When the selected analytical reaction is composed of two or more kinds of reagents, the reagents may be mixed and contained together in the same reagent layer, or two or more kinds of reagents may be formed in different layers. It may be contained. It is also possible to carry out an analysis reaction of two or more kinds of analyte components in a sample in the same reagent layer. At this time, it is desirable to select the analysis reactions so that two or more kinds of analysis reactions do not interfere with each other and do not affect each other when measuring the reaction products formed.

In the present invention, one or a plurality of spreading layers are provided above the reagent layer provided on the support. This spreading layer is provided for the following purposes. (1) A constant volume of a fluid sample is uniformly dispersed per unit area in a reagent layer. (2) When performing spectrophotometric analysis, it also has a function of performing a background action of reflecting the measurement light transmitted through the support. (3) It may have a function of removing a substance or a factor that inhibits the analysis reaction in the fluid sample, if necessary.

Therefore, the spreading layer according to the present invention may be configured so that one layer can exhibit all of the above-mentioned three functions, or each function is appropriately separated, and a separate layer is formed for each function. May be. Furthermore, of the three functions
You may make it combine the layer which exhibits two functions, and the layer which exhibits the remaining other function. The film thickness of the spreading layer according to the present invention can be arbitrarily selected according to the purpose,
Preferably about 20-600 μm, more preferably 50
˜400 μm. The porosity of the spreading layer is preferably 25 to 90%, more preferably 40 to 80%. The average pore size of the development layer is preferably 1 to
The thickness is 800 μm, more preferably 10 to 300 μm, and further preferably 20 to 300 μm.

The fiber used in the present invention means a fiber having a fibrous shape, for example, JP-A-57-197466.
The fibers disclosed in the publication, natural cellulose, and derivatives thereof, and synthetic fibers such as polyethylene, polypropylene and polyamide are listed. Natural cellulose is particularly preferable. The short fibers used in the present invention are those that can pass through a JIS standard sieve 325 mesh, and are not spherical in shape, but have a needle-like shape having a major axis and a minor axis. Unlike the material for forming the non-fibrous porous medium disclosed in JP-A-53888,
It is also different from the fibers disclosed in JP-A-57-125847. A spreading layer constructed by using such short fibers is disclosed in, for example, JP-A-49-53888.
Which has the respective features of the non-fibrous porous spreading layer disclosed in Japanese Patent Laid-Open No. 57-125847 and the respective drawbacks of the fibrous porous spreading layer disclosed in JP-A-57-125847. It has been done.

The short fibers that pass through the 325-mesh screen and do not pass through the 400-mesh screen have an average value of the length in the longitudinal direction of 1 to 200 μm, and the average of the length in the longitudinal direction / the average in the lateral direction. It is preferable to use short fibers having a value of 1.1 to 10, but the average value of the length in the longitudinal direction is 1 to 150 μm, more preferably 5 to 150 μm, and further preferably 5-1.
The average value of the length in the longitudinal direction / the length in the lateral direction is 1.1 to 5, and more preferably 1.2 to 3, more preferably 100 μm.

Such short fibers include, for example, natural cellulose and its derivatives, polyethylene, polypropylene,
It can be obtained by treating a crushed product of synthetic fibers such as polyester, polyamide and polyurethane with a sieve of 325 mesh or more. Further, for example, crystalline cellulose such as Avicel manufactured by Asahi Kasei Co., Ltd., and a crushed resin product can be used, and the crushed fiber product is not limited. It is particularly preferable to use natural cellulose.

Among these fibers and short fibers, for example, titanium dioxide, alumina, silica, zinc white (ZnO), lead white [2PbCO ₃ .Pb (OH) ₂ ] and basic lead sulfate (3
PbSO ₄ · PbO to 2PbSO ₄ · PbO), lead sulfate (PbSO ₄ ), lithopone (ZnS + BaSO ₄ ), zinc sulfide (ZnS), barium sulfate (BaSO ₄ ), particles of antimony oxide (Sb ₂ O ₃ ), etc., and others Various inorganic pigments are contained, especially 0.5 to 10%, more preferably 1 to 5% of white pigments having an average particle size of 0.01 to 50 μm, more preferably 0.1 to 30 μm. It is also possible to use the one that consists of. Fibers and short fibers containing a white pigment are obtained, for example, by applying the technique of JP-A-1-173868, and short fibers containing a white pigment are described in JP-A-1-173868. It is obtained by crushing a drawn yarn made of fibers containing a white pigment and classifying with a sieve.

The method for forming a porous spreading layer using the above fibers or short fibers is a water-permeable material such as gelatin, agarose, gum arabic, carboxymethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. as a binder (adhesive). When a water-swellable polymer is used, it can be produced by the method disclosed in JP-A-57-125847. When a water-impermeable polymer that does not swell with water is used as an adhesive, it is disclosed in JP-A-57-19.
It can be manufactured by the method disclosed in Japanese Patent No. 7466.

The water-swelling hydrophobic polymer that can be used as an adhesive is, for example, vinyl chloride-vinyl acetate copolymer, vinyl chloride-vinylidene chloride copolymer, vinyl chloride-acrylonitrile copolymer, acrylic ester. -Acrylonitrile copolymer, acrylic acid ester-vinylidene chloride copolymer, acrylic acid ester-styrene copolymer, methacrylic acid ester-acrylonitrile copolymer, methacrylic acid ester-vinylidene chloride copolymer, methacrylic acid ester-styrene copolymer Polymer, nylon-silicone resin, nitrocellulose-polyamide resin, polyvinyl fluoride, vinylidene chloride-acrylonitrile copolymer, butadiene-acrylonitrile copolymer, polyamide resin, polyvinyl butyral,
Cellulose derivative (cellulose acetate butyrate,
Cellulose diacetate, cellulose triacetate,
Cellulose propionate, nitrocellulose, etc.), styrene-butadiene copolymer, polyester resin, chlorovinyl ether-acrylic acid ester copolymer, amino resin, adhesives such as various synthetic rubber thermoplastic resins, KAISHO 55-90859, page 9 to 1
Polymers disclosed on page 3 of JP-A-57-19
Polymers disclosed on pages 5 to 7 of 7466, and reactive hydrophobic polymers described in JP-B-2-19906 can be used.

The binder may be used in a widely selected amount, but the substantial part of the void volume formed by the fibers or short fibers is 0.05 to 0.5% with respect to the fibers or short fibers.
50% by weight, more preferably 0.05 to 30% by weight can be used. The spreading layer of the present invention can be manufactured using various methods. For example, a spreading layer can be formed by dispersing fibers or short fibers in a liquid carrier that does not dissolve, then adding the binder to adjust the fibers or short fibers, and then coating and drying.

Dispersions useful for forming the spreading layer must be stable for a sufficient time. Many methods can be used alone or in combination to produce such a stable dispersion. For example, one useful method is to add a surfactant to the liquid carrier to facilitate dispersion and stabilization in the fiber or staple fiber dispersion.

Examples of typical surfactants that can be used include cationic or anionic surfactants, Triton X-100 (Rohm and Haas Co., Octylphenoxypolyethoxyethanol), Surfactant 1
There are nonionic surfactants such as 0G (Nonylphenoxypolyglycidol, manufactured by Olein Co.). Although these surfactants can be used in widely selected amounts, it is preferable to use 0.005 to 30% by weight, more preferably 0.05 to 20% by weight, based on the fibers and short fibers. .

As another method, there are ultrasonic treatment of the above dispersion liquid, physical mixing, physical stirring treatment, and pH adjustment.
It is effective to combine these with the above method.
The liquid carrier of the fiber or short fiber dispersion can be an aqueous liquid. It should be noted that other liquid carriers such as various organic liquids in which the fibers and short fibers are insoluble in the carrier can be used. Representative liquid carriers other than water include water-miscible organic solvents, aqueous mixtures of water and water-miscible organic solvents, and suitable water-immiscible organic solvents. Water-miscible organic solvents include lower alcohols (where the alkyl group has 1 carbon
~ 4 alcohols), acetone and tetrahydrofuran. Water-immiscible solvents include lower alkyl esters such as ethyl acetate, halogenated organic solvents such as halogenated hydrocarbons (eg chloroform, methyl chloride and carbon tetrachloride, etc.), aromatic hydrocarbons (eg benzene, toluene and There are solvents such as xylene) and aliphatic hydrocarbons (eg hexane, decalin etc.).

The analytical element of the present invention may have various functional layers, reagent layers and members, for example, US Pat.
No. 58, the reagent layer, the filtration layer, the reflection layer, the undercoat layer,
Radiation blocking layer described in US Pat. No. 4,042,335, barrier layer described in US Pat. No. 4,066,403, registration layer described in US Pat. No. 4,144,306, migration prevention layer described in US Pat. No. 4,166,093. U.S. Pat. No. 4,127
Scintillation layer described in Japanese Patent Publication No. 499, JP-A-55
The scavenger layer described in Japanese Patent No. 90859 and the destructible pod-shaped member described in U.S. Pat. No. 4,111,079 can be arbitrarily combined to form an analytical element.

In the analytical element having the spreading layer of the present invention, the smoothing of the surface of the spreading layer is increased by carrying out calendering treatment by a method of passing between a pair of pressure rollers, and a preferable effect in optical reflection is obtained. It is possible to obtain. The analysis element of the present invention can be obtained, for example, by a dip coating method, an air knife method, a curtain coating method or US Pat.
It can be coated by various coating methods such as extrusion coating using a hopper as described in US Pat. No. 294, and if desired two or more layers can be coated.
It is also possible to apply simultaneously by the method described in No. 1 and British Patent No. 837095.

The analytical element of the present invention can achieve the object by supplying a fluid sample from the developing layer side and observing from the transparent support side. The amount of the fluid sample applied to the analytical element of the present invention can be arbitrarily determined, but is preferably about 5
˜50 μl, more preferably about 5-20 μl. It is usually preferred to apply about 10 μl of fluid sample.

The analytical reaction used in the analytical element of the present invention can be arbitrarily determined depending on its purpose. For example, biological reactions such as whole blood, plasma, serum, lymph, saliva, spinal fluid, vaginal fluid, and urine. Used for analysis of components in fluid samples.
By appropriately selecting an analytical reagent, for example, glucose, urea nitrogen, ammonia, uric acid, cholesterol,
It can be readily configured for use in components such as triglycerides, creatine, creatinine, bilirubin as well as many other analyses.

The analytical element of the present invention can be applied not only to the field of clinical chemistry but also to other fields of chemical analysis, and has the function of retaining a constant fluid within a constant membrane area. It is also possible to combine with other functional layers. Hereinafter, the present invention will be described more specifically with reference to examples.

[0026]

【Example】

Example 1 A reagent layer having the following composition was provided on a transparent subbed polyethylene terephthalate support having a film thickness of 180 μm. [Reagent layer] Glucose oxidase 45000 U / m ² peroxidase 67000 U / m ² gelatin 14.0 g / m ² 1,7-dihydroxynaphthalene 0.84 g / m ² 4-aminoantibilin hydrochloride 0.81 g / m ² dimedone 0 .19g / m ² potassium phosphate buffer (pH 6.1) 2.82 g / m ² sodium triisopropyl naphthalene sulfonate 0.22 g / m ² 1,2-bis (vinylsulfonyl) ethane 0.10 g / m ² above On the reagent layer, a solution of N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 80:20) in n-butanol was applied so as to have a dry film thickness of about 2 μm to form an adhesive layer.

On the other hand, powder filter paper D (40-
(100 mesh) was classified by a sieve, that is, a fiber F1 that passed through a 80 mesh sieve and did not pass through a 100 mesh sieve was prepared. Then, a dispersion liquid for developing layer constitution as shown in Table 1 below was prepared. Table 1 Fiber material Liquid carrier Binder Surfactant dispersion 1 F1 100g Xylene 308g Note 25g Triton X100 10g Dispersion 2 Filter paper powder D100g Same as above Same as above Note Styrene-glycidyl methacrylate copolymer (90:10) And in this way The resulting dispersion 1 was applied onto the adhesive layer and dried to prepare the analytical element 1 according to the present invention. or,
Dispersion 2 was applied onto the adhesive layer and dried to prepare analytical element 1 as a comparative example.

10 μl of human serum having various glucose concentrations was spotted on the analytical element of the present invention and the analytical element of the comparative example, incubated at 37 ° C. for 7 minutes, and then 546.
The reflection density was measured using a nm filter, and a calibration curve was prepared in advance from this reflection density and the glucose concentration value of human serum tested by Unikit Glucose-E (Chugai Pharmaceutical Co., Ltd.).

Furthermore, the normal value (85 mg
/ Dl) and an abnormal value (260 mg / dl) were spotted each 18 times, the same operation as above was carried out, the glucose concentration value was calculated by applying it to the calibration curve, and the coefficient of variation CV (%)
I asked. The results are shown in Table 2. Table 2 Normal value Abnormal value X (mg / dl) CV (%) X (mg / dl) CV (%) Analytical element 1 85 1.88 260 1.57 of the present invention Analytical element 1 85 4.45 260 4.23 From Table 2, it can be seen that the present invention uses the filter paper powder D similarly to the comparative example, but the CV value is much smaller than that of the comparative example, and It can be seen that the reproducibility is excellent and the measurement accuracy is high.

[Example 2] A reagent layer having the following composition (dry film thickness of the first reagent layer of 23 µm, dry film thickness of the second reagent layer of 15 µm) was formed on a transparent subbed polyethylene terephthalate support having a film thickness of 180 µm. Was set up. [First Reagent Layer] Gelatin 18.9 g / m ² glutamate dehydrogenase 19000 U / m ² diaphorase 1900 U / m ² 3,3 ′-(3,3′-dimethoxy-4,4′-biphenylene ) -Bis [2,5-diphenyltetrazolium chloride] 0.94 g / m ² 1,2-bis (vinylsulfonyl) ethane 0.1 g / m ² Triton X-100 0.1 g / m ² [second reagent layer] trishydroxymethylaminomethane 3.87 g / m ² HCl tris (hydroxymethyl) aminomethane 0.83 g / m ² Triton ^{X-100 0.1 g / m 2} N- vinylpyrrolidone - vinyl acetate copolymer (molar ratio 80 / 20) 1.35 g / m ² On the other hand, powder filter paper E (200 mesh or more) manufactured by Toyo Roshi Kaisha, Ltd. is divided by a 325 mesh sieve, and a short fiber F4 that passes through the 325 mesh sieve. Got Although the short fibers F4 were further classified, that is, passed through a 325 mesh sieve,
Short-fiber powder F3 that did not pass through a 400-mesh sieve was obtained.

A dispersion having the composition shown in Table 3 was prepared using the short fibers F3 and F4. Table 3 Dispersion 3 Dispersion 4 Short fiber F3 100g-Short fiber F4-100g Xylene 308g Same as left binder (Note) 25g Same as left Triton X100 10g Same as left alanine 4.8g Same as left Oxidized nicotinamide nucleotide 8.8g Same as left α-ketoglutarate 0 .3 g Same as on the left Note Styrene-glycidyl methacrylate copolymer (90:10) Then, the dispersion liquid 3 was applied onto the reagent layer and dried to prepare the analytical element 2 according to the present invention, and the dispersion liquid 4 was added. An analytical element 2 as a comparative example was prepared by coating and drying on the reagent layer.

To the analytical element 2 of the present invention and the analytical element 2 of the comparative example, 10 μl of human serum having various GPT activities was added dropwise, followed by incubation at 37 ° C., and 2 minutes and 4 minutes after the addition.
The reflection density after the measurement was measured with a reflection spectrophotometer through a filter of 546 nm, and a calibration curve was prepared in advance from the difference in the reflection density and the GPT activity value of human serum tested by the standard method.

Furthermore, normal values (25 carmen units) and abnormal values (98 carmen units) of human serum were dropped 18 times each, and the same operation was carried out to obtain the difference in reflection density, which was prepared in advance. The standard deviation and the coefficient of variation CV were calculated by fitting the results to a calibration curve, and the results were shown in Table 4.
Shown in. Table 4 Normal value Abnormal value X (Carmen unit) CV (%) X (Carmen unit) CV (%) Analytical element 2 25 1.21 98 98 1.55 of the present invention Analytical element 2 25 4.45 98 5 .34 As is clear from Table 4, the analytical element of the present invention showed good simultaneous reproducibility, and moreover, the coloring within the coloring region was uniform. On the other hand, the analytical element of the comparative example was inferior in simultaneous reproducibility, and was also inferior in uniformity of color development in the coloring region.

Claims (2)

[Claims] 1. An analytical element comprising a support, on which a reagent layer containing at least one reagent is provided, and a development layer provided thereon, the development layer passing through a 80-mesh screen, An analysis element comprising a fiber that does not pass through a mesh screen. 2. An analytical element comprising a support, on which a reagent layer containing at least one reagent is provided, and a development layer provided thereon, the development layer passing through a 325 mesh sieve, An analytical element comprising a short fiber that does not pass through a mesh sieve.