JP2001264326A - Dry analytical element - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analytical instrument capable of simply and rapidly performing measurement with respect to a small quantity of a specimen in a clinical analysis field and capable of also measuring agglutination reaction or coagulation reaction other than colorimetric fractionation. SOLUTION: A mesh layer 1 is laminated on a water impermeable support 1 to constitute a dry analytical element having no developing layer. Or, a hydrophylic polymer layer is provided between a water impermeable layer and the mesh layer to constitute the dry analytical element having no developing layer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical instrument which can quickly and easily analyze a small amount of a sample in the field of clinical analysis and the like.

[0002]

2. Description of the Related Art Methods of diagnosing human diseases by analyzing blood, urine, etc. as specimens have been used for a long time.

[0003] This analysis method is roughly classified into a wet method and a dry method. In a dry method using a conventional dry analysis element or the like, a case where the optical density in colorimetric analysis shows a decrease, an agglutination reaction, a coagulation reaction and the like are considered. In some cases, it is not possible to perform analysis using the information.

[0004] In the wet method, a sample and a necessary reagent are put in a container, a detection reaction is performed in a solution, and measurement is performed.

[0005]

The problems of the wet method are that a large amount of sample is required and that the method is not simple and quick.
That is, about 0.1 to 0.5 ml is required for each analysis item. When analyzing a plurality of analysis items, a considerable amount of sample is required. It will be a burden. In addition, since each reagent is added to each container, it takes time and effort, and the size of the entire analyzer becomes large.

[0006] On the other hand, in the dry method, there is a case where the optical density is decreased in colorimetric analysis, or a measurement such as an agglutination reaction and a coagulation reaction cannot be performed.

[0007] An object of the present invention is to enable simple and rapid measurement with a small amount of a sample, and furthermore, a chemical reaction, an enzyme reaction, an immune reaction,
An object of the present invention is to provide an analytical instrument capable of measuring an agglutination reaction and a coagulation reaction.

[0008]

A major feature of the conventional dry analysis element lies in the spreading layer. In other words, the sample supplied to the dry analytical element is spread in a plane without substantially unevenly distributing the components contained in the sample in the developing layer, and is supplied to the layer below it at a substantially constant rate per unit area. By doing so, the stability was ensured, and the development of this spreading layer made it possible to complete the dry analytical element.

However, as a result of studying the use of a dry analytical element for colorimetric analysis with low optical density, analysis using coagulation reaction, and coagulation reaction, the present inventor has found that when a developing layer is present, light transmission is not possible. When performing reflection photometry from the support side, this developing layer functions as a reflector and diffusely reflects light, so in the case of colorimetric analysis, some of the dyes generated in the developing layer cannot be measured colorimetrically, In addition, it has been found that measurement for determining the degree of light scattering, such as turbidity measurement, cannot be performed.

[0010] Therefore, as a result of extensively examining means for ensuring quantitativeness regardless of the spread layer, quantitativeness can be ensured by combining a water-impermeable sheet with a mesh, and a reagent can be present on the mesh. By interposing the reagent layer of the dry analysis element between the mesh and the water-impermeable sheet, the sample can be analyzed with sufficient accuracy, and the analysis reaction includes agglutination reaction and coagulation reaction in addition to colorimetric analysis. The present inventors have found that the measurement can be performed even by using the method, and have completed the present invention.

That is, according to the present invention, a mesh layer is laminated on a water-impermeable support, and a dry analytical element having no spreading layer and a hydrophilic layer between the water-impermeable support and the mesh layer are provided. The present invention relates to the above-mentioned dry analytical element provided with a polymer layer.

In the dry analytical element of the present invention, the supplied sample is held in a substantially constant amount for each mesh to form a micro liquid column, in which a reagent is present, or a hydrophilic polymer is added to the micro liquid column. The layer dissolves out, or this microfluidic column is absorbed by the hydrophilic polymer layer and a detection reaction occurs.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The mesh layer is a layer that partitions a supplied aqueous sample for each mesh and holds a substantially constant amount. The shape of each mesh is not particularly limited.
Examples include a square, a hexagon, a circle, and the like.
The size of each mesh is appropriately about 0.05 to 3.0 mm in diameter, preferably about 0.2 to 1.5 mm.
The mesh has a hole diameter of about 0.05 to 7.5 mm, preferably about 0.4 to 6 mm. The thickness of the mesh layer is about 0.05 to 6.0 mm, preferably about 0.2 to 3.0 mm. Such a mesh layer may be a woven fabric, a net, a punched sheet, or the like, and in the case of a circular shape, a water-soluble substance is added to a solution of a water-insoluble material or a material in which a water-insoluble material is formed by a polymerization reaction or a crosslinking reaction. It can be manufactured by applying a suspended or emulsified liquid to form a sheet, and dissolving a water-soluble substance to form a large number of circular holes. The material of the mesh layer may be hydrophobic, such as nylon, polyethylene, Teflon (registered trademark), or polystyrene, but is preferably subjected to a hydrophilic treatment.

The punching sheet is formed by forming a large number of holes in a sheet or the like used as a support, which will be described later, and each of the holes forms a mesh. The mesh layer may be placed in a state where the specimen can be held in each mesh, and may be simply placed on the support or the hydrophilic polymer layer with an adhesive or the like, or may be simply placed. . This mesh layer may or may not restrict the development of the specimen.

It is preferable that the hydrophilic polymer layer is provided adjacent to the mesh layer, and no other layer is interposed therebetween. As the hydrophilic polymer which is a matrix for forming the hydrophilic polymer layer, various known water-soluble, swellable, and hydrophilic polymers used in dry chemistry analysis elements can be used. Specific examples include polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polymethylvinylether, copolymers of polyacrylamide and polyvinylpyrrolidone, methylcellulose derivatives, starch / acrylate graft copolymer crosslinked products, crosslinked polyacrylic acid, gelatin ( Examples thereof include, but are not limited to, acid-treated gelatin, deionized gelatin, etc., gelatin derivatives (eg, phthalated gelatin, hydroxyacrylate-grafted gelatin, etc.), carboxymethyl starch, and the like. It is preferable that the hydrophilic polymer layer is transparent in principle. The thickness (when dried) of the hydrophilic polymer layer is 2 to 10
About 0 μm, preferably about 5 to 50 μm is appropriate.

As the water-impermeable support, a known water-impermeable transparent support conventionally used for a dry analytical element can be used. More specifically, the thickness is about 50 μm made of polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (eg, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.).
A transparent film of m to 1 mm, preferably about 80 μm to about 300 μm can be used.

If necessary, a known undercoat layer or adhesive layer may be provided on the surface of the support to enhance the adhesion with the hydrophilic polymer layer. When the hydrophilic polymer layer is not provided, at least the surface of the support itself on the mesh layer side must be hydrophilic to such an extent that the sample can be spread over the entire area partitioned by the mesh. Therefore, in the case where the support itself is water repellent and hydrophobic, it is necessary to perform a hydrophilic treatment to at least the extent.

The dry analytical element of the present invention basically comprises a mesh layer, a hydrophilic polymer layer and a support, and does not include other layers. However, a functional layer, for example, a water-absorbing layer of a dry analytical element, can be provided between the hydrophilic polymer layer and the support.

Examples of the structure of the dry analytical element of the present invention are shown in FIGS.
3 is shown. The example of FIG. 1 includes the net 1 and the support 2, the example of FIG. 2 includes the net 1 and the reagent layer 3 and the support 2, and the example of FIG. 3 includes the punching sheet 1, the reagent layer 3 and the support 2.

The size of the dry analytical element is about 1 to 15 mm in diameter in the case of a circle and about 1 to 15 mm in the case of a square.
Usually, it may be about 4 to 12 mm.

This dry analytical element is used in a frame made of plastic or the like, like the conventional dry analytical element. The mesh layer of the present invention can be integrated with this frame or can be integrally formed.

In the present invention, the target test substance is not particularly limited. In addition to enzymes, lipids, inorganic ions, metabolites, proteins, etc., which are usually measured in the field of clinical tests, various globulins, immune antigens, biological antibodies such as immune antibodies,
As long as analysis methods such as drugs, hormones, tumor markers, DNA, and RNA are established, they can be analyzed.

The dry analytical element of the present invention preferably contains all reagents necessary for analysis. This reagent may be the same as a known dry analytical element. All the reagents necessary for the analysis are indispensable reagents, and other reagents are added or deleted as appropriate. In principle, all of the reagents are contained in each space of the mesh or in the hydrophilic polymer layer. As a method for containing a reagent, there are a method in which the solution is contained in a reagent solution or a hydrophilic polymer solution and applied to a support, a method in which the solution is sprayed or spotted on the support, a method using a spin coater, and the like. Reagent drying depends on the thermal stability of the reagent.
Heat drying, reduced pressure drying, freeze drying and the like are performed. On the other hand, the reagent may be added together with the specimen (at the front, back, simultaneously) without being contained in advance.

The supply amount of the sample may be about 5 to 100 μl, usually about 5 to 60 μl.

Incubation conditions may be the same as those for a conventional dry analytical element.

The side light after the completion of the reaction may be from the side of the support, the side of the mesh layer, or the lateral direction, and may be any of reflection photometry and transmission photometry. At that time, a prism or a mirror can be usually used.

[0027]

EXAMPLES Example 1 Preparation of Dry Analysis Element 1 for Quantifying Total Protein
180μm thick polyethylene undercoated with gelatin
On a colorless transparent film of lentephthalate, the following composition
Was applied and dried to form a reagent layer. AA-NVP-MA copolymer 24.9 g / m²  Surfactant 5.4 g / m²  Glycerin 3.4 g / m²  Copper sulfate 12.5g / m²  L (+) tartaric acid 7.3g / m²  LiOH 13.4 g / m²  (+) Sodium hydrogen tartrate 0.9 g / m²

Here, the AA-NVP-MA copolymer is
Acrylamide-vinylpyrrolidone-metallialcohol is shown.

The surfactant is polyoxy (2-hydroxy) propylene nonyl phenyl ether (Surfac)
(Tant 10G, manufactured by Ohlin Co., Ltd.).

The analytical element was cut into a chip of 12 mm × 13 mm, and a fiber diameter of 0.3 mm and a diameter of 0.8 mm were placed on the chip.
mm × 0.8 mm polyethylene mesh
The chip cut into a size of 13 mm was placed in a slide frame (described in JP-A-57-63542) to obtain a dry analytical element 1 for quantifying total protein according to the present invention.

COMPARATIVE EXAMPLE 1 Dry Analysis Element 2 for Quantifying Total Protein
Preparation Polyethylene with a thickness of 180 μm undercoated with gelatin
On a colorless transparent film of lentephthalate, the following composition
Was applied and dried to form a reagent layer. AA-NVP-MA copolymer 24.9 g / m²  Surfactant 5.4 g / m²  Glycerin 3.4 g / m²  Copper sulfate 12.5g / m²  L (+) tartaric acid 7.3g / m²  LiOH 13.4 g / m²  (+) Sodium hydrogen tartrate 0.9 g / m²

Here, the AA-NVP-MA copolymer is
Acrylamide-vinylpyrrolidone-metallialcohol is shown.

The surfactant is polyoxy (2-hydroxy) propylene nonyl phenyl ether (Surfac)
(Tant 10G, manufactured by Ohlin Co., Ltd.).

Next, water was supplied over the entire surface of the reagent layer at a supply amount of about 30 g / m ² to wet the surface, and then a tricot knitted fabric in which a polyethylene terephthalate spun yarn equivalent to 50 denier was knitted with 36 gauge was lightly lightened. The layers were laminated under pressure and dried.

Thus, an integrated multilayer analytical element having a polyethylene terephthalate colorless transparent film, a reagent layer and a developing layer in this order was produced.

The above-mentioned integrated multilayer chemical analysis element is 12 mm
The chip was cut into a chip of 13 mm square and placed in a slide frame (described in JP-A-57-63542) to prepare a dry analytical element 2 for quantifying total protein according to the present invention.

Measurement Example 1 In the dry analytical element 1 of Example 1, the total protein concentration was 4.
50 μl of 0, 6.5, 9.0 g / dl serum was spotted.
After incubating each analytical element at 37 ° C for 5 minutes, 5
Reflection photometry was performed from the support side at 40 nm.

At this time, a white plate made of Teflon was disposed on the reagent layer side of the dry analytical element 1 as a reflecting plate. The calibration curve is shown below. Total protein concentration [g / dl] OD (540 nm) 4.0 0.65 6.5 1.02 9.0 1.25 OD range = OD (total protein: 9.0 g / dl) -OD (total protein: 4.0 g / dl) = 1.25-0.65 = 0.60

On the other hand, in the dry analytical element 2 of Comparative Example 1, 50 μl of serum having a total protein concentration of 4.0, 6.5, 9.0 g / dl was used.
I got 1 point. After incubating each analytical element at 37 ° C. for 5 minutes, reflection photometry was performed at 540 nm from the support side.

The calibration curve is shown below. Total protein concentration [g / dl] OD (540 nm) 4.0 0.92 6.5 1.07 9.0 1.19 OD range = OD (total protein: 9.0 g / dl) -OD (total protein: 4.0 g / dl) = 1.19−0.92 = 0.27

As is clear from the above results, the OD range of Example 1 can be twice as large as that of Comparative Example 1.

Example 2 The dry analytical element 3 for HCG measurement
Production: 180 μm colorless and transparent polyethylene terephthalate (support
Apply the reagent solution on the carrier
And dried to form a reagent layer. Carboxymethyl starch 5.8 g / m²  Anti-HCG antibody-sensitized latex (2.5 μm particle size) 1.6 g / m²  Bovine serum albumin 1.6 g / m²

The analysis element was cut into chips of 12 × 13 mm, and a fiber diameter of 0.5 mm and a diameter of 1.5 m
m × 1.5 mm polyethylene mesh is also 12 ×
A chip cut to 13 mm was placed, and then
In the frame of the slide described in JP-A-7-63452,
The dry analytical element 3 of Example 2 was used.

Comparative Example 2 The dry analytical element 4 for HCG measurement
Production: 180 μm colorless and transparent polyethylene terephthalate (support
Apply the reagent solution on the carrier
And dried to form a reagent layer. Carboxymethyl starch 5.8 g / m²  Anti-HCG antibody-sensitized latex (2.5 μm particle size) 1.6 g / m²  Bovine serum albumin 1.6 g / m²

Next, water was supplied to the entire surface of the reagent layer at a supply amount of about 30 g / m ² to wet the surface, and then a tricot knitted fabric in which a polyethylene terephthalate spun yarn equivalent to 50 denier was knitted with 36 gauge was lightly lightened. The layers were laminated under pressure and dried.

Thus, an integrated multilayer analytical element having a colorless and transparent film of polyethylene terephthalate, a reagent layer and a developing layer in this order was produced. Next, this analytical element was cut into chips of 12 × 13 mm.
A dry analytical element 4 of Comparative Example 2 was accommodated in a slide frame described in Japanese Patent No. 3452.

Measurement Example 2 HCG Measurement SIGMA was used as a dry analytical element in Example 2 and Comparative Example 2.
Manufactured by Human Chorionic Gonado
The tropin (HCG) is diluted with 100 mM phosphate buffer (pH 7.4), and 0.5, 25, 100, 50
A series of 0 IU / mL was spotted at 50 μL. After incubating each analytical element at 37 ° C. for 5 minutes, the reflection density was measured from the support side at 650 nm. At this time, a black plate was used as a color plate for absorbing the light transmitted through the reagent layer during the reaction among the incident light from the support side.

The calibration curve is shown below. Example 2 Comparative Example 2 HCG concentration [IU / mL] OD (650 nm) OD (650 nm) 0 1.110 0.325 5 1.170 0.330 25 1.268 0.320 100 1.346 0.335 500 1.437 0.320

As shown in the above calibration curve, it was shown that the dry analysis element 3 of Example 2 can perform quantitative measurement of HCG.

[0050]

The dry analytical element of the present invention has both advantages of the wet method and the dry method, and can perform measurement quickly and easily using a small amount of a sample. It has the advantage that agglutination and coagulation reactions can also be measured.

[Brief description of the drawings]

FIG. 1 is a perspective view of an example of a dry analysis element of the present invention using a net for a mesh layer.

FIG. 2 is a perspective view of an example of a dry analytical element of the present invention using a net for a mesh layer and a net for a mesh layer further provided with a reagent layer.

FIG. 3 is a perspective view of an example of a dry analysis element of the present invention using a punching sheet for a mesh layer and a net for a mesh layer provided with a reagent layer.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Mesh layer 2 ... Support 3 ... Reagent layer (hydrophilic polymer layer)

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshikazu Amano 3-11-46 Izumi, Asaka-shi, Saitama F-term in Fujisha Shin Film Co., Ltd. (reference) 2G042 AA01 BD12 BD20 CB03 DA01 DA08 FA11 FB07 FC04 FC05 FC07 2G045 AA13 AA16 AA25 CA25 CA26 CB03 DA13 DA14 DA20 DA36 DA37 DA54 DA60 FB01 FB02 FB03 FB11 FB16 FB17 HA14 HA20 2G054 AA07 AB02 AB03 AB04 AB05 AB07 CA21 CA22 CA23 CA28 CA30 CE01 EA06 EA09 FA31 GB03 GB04 GB05 A03 4

Claims (3)

[Claims] 1. A dry analytical element in which a mesh layer is laminated on a water-impermeable support. 2. The dry analytical element according to claim 1, wherein a hydrophilic polymer layer is provided between the water-impermeable support and the mesh layer. 3. The dry analytical element according to claim 2, wherein the hydrophilic polymer layer is a reagent layer containing a reagent necessary for analysis.