JP2001264325A - 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 water permeable frame 2 is provided on a water impermeable support 1 and a reagent necessary for analysis is housed in the frame and, if necessary, a hydrophilic polymer layer is provided on the water impermeable support to constitute a dry analytical element having no developing layer. The hydrophilic polymer layer is a reagent layer containing the reagent necessary for analysis.

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.

In the wet method, a sample and a necessary reagent solution are placed in a container, a detection reaction is performed in the 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.

An object of the present invention is to provide an analytical instrument which can easily and quickly measure a small amount of a sample, and can also measure a colorimetric analysis when the optical density is reduced, an agglutination reaction, a coagulation reaction and the like.

[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.

Therefore, as a result of extensively examining means for securing quantitativeness without depending on the spread layer, quantitativeness can be secured by partitioning the water-impermeable sheet with a water-impermeable frame. Samples can be analyzed with sufficient accuracy by allowing the reagent to be present in the partitioned frame or by interposing the reagent layer of the dry analytical element between the frame and the water-impermeable sheet, and the analysis reaction is colorimetric. The present inventors have found that it is possible to measure even those including an agglutination reaction and a coagulation reaction in addition to analysis, and have completed the present invention.

That is, the present invention has a developing layer in which a water-impermeable support is partitioned on a water-impermeable frame, and a reagent necessary for analysis is contained in the partitioned frame. The present invention relates to a dry analytical element which is not provided and a dry analytical element as described above, wherein a hydrophilic polymer layer is provided on a water-impermeable support.

In the dry analytical element of the present invention, the supplied sample is held in a fixed amount in a partitioned frame to form a microfluid column, into which the reagent and the optional hydrophilic polymer layer are dissolved, Alternatively, the microcolumn is absorbed by the hydrophilic polymer layer to cause a detection reaction.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION As a water-impermeable support, a known water-impermeable transparent support conventionally used in a dry analytical element can be used. Specifically, it is made of polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (for example, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) and the like, and has a thickness of about 50 μm to 1 mm, preferably about 80 μm to about 80 μm. 300
A μm transparent film 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 frame side must be hydrophilic enough that the specimen can spread over the entire surface of the compartment partitioned by the frame. Therefore, the support itself is water repellent,
In the case of hydrophobicity, it is necessary to perform hydrophilic treatment at least to the extent.

It is preferable that the hydrophilic polymer layer is provided on the support, and that no other layer besides the undercoat layer and the adhesive 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 about 2 to 100 μm, preferably 5 to 50 μm.
About μm is appropriate.

The water-impermeable frame holds the supplied sample in the frame. This water impermeability is important as long as it does not allow the aqueous sample to leak out of the frame.In addition to the non-porous frame, even if there is a space such as a gap, use a water-repellent frame. It may exhibit water impermeability due to its water repellency. This frame is divided into a single frame and a partition that is divided into two or more sections. The number of sections when partitioning into two or more sections is not particularly limited, but is 2 to 1
Specific examples include about 00 sections, particularly about 4 to 25 sections. The frame partitions the upper surface of the water-impermeable support. The lower end of the frame is not limited to the upper surface of the water-impermeable support, but may extend to the lower portion. Taking a single frame as an example of a specific embodiment, there is one shown in FIGS. In the dry analysis element of FIG. 1, a water-impermeable support 1 and a water-impermeable frame 2 are integrally formed, and a dried product 4 of a reagent necessary for analysis is formed therein.
Is arranged. 5 is a sample. In the dry analytical element of FIG. 2, a reagent layer 3 which is a hydrophilic polymer layer containing a reagent necessary for analysis is provided on a water-impermeable support 1, and a frame 2 is provided thereon. I have. The dry analytical element of FIG.
A frame 2 is provided so as to surround the outer periphery of the support 1 and the reagent layer 3. The shape of the section partitioned by the frame is not particularly limited, and examples thereof include a square, a hexagon, and a circle. The size of the section is appropriately about 0.05 to 7.5 mm in diameter, preferably about 0.4 to 6 mm. The height of the frame need only be high enough to hold the specimen, and is above the support (if a hydrophilic polymer layer is provided, above it)
Height of 0.01 mm or more, preferably 0.3 to 1.0
mm is appropriate. The frame may be made of a hydrophobic material such as nylon, polyethylene, Teflon (registered trademark), or polystyrene, but is preferably subjected to a hydrophilic treatment.

The frame may be placed so that the specimen can be held in the frame. The frame may be simply adhered to the support or the hydrophilic polymer layer with an adhesive or the like, or may be simply placed. The adhesive can be selected, for example, from the aforementioned hydrophilic polymers.

The dry analytical element of the present invention basically comprises a frame, 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 conventional dry analytical element can be provided between the hydrophilic polymer layer and the support.

The size of the dry analysis 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.

In the present invention, the target analyte 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 the reagents are contained in each space in the frame 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. The drying of the reagent may be performed by heating, drying under reduced pressure, freeze-drying, etc., depending on its thermal stability. On the other hand, the reagent is not contained in advance, and is used together with the sample (before, after,
At the same time).

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

The conditions for the incubation may be the same as those for the dry analytical element.

After the reaction is completed, the side light is on the upper side, the lower side,
It may be from any of the horizontal directions, and may be either reflection photometry or transmission photometry. At that time, a prism or a mirror can be usually used.

[0025]

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.).

A 0.8 mm thick plastic sheet obtained by punching a circle having a diameter of 1 cm as a frame was adhered to the analytical element, and the analytical element was cut into a chip of 12 mm × 13 mm. No.-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
180μm thick poly undercoated with prepared gelatin
On a colorless transparent film of ethylene terephthalate,
An aqueous solution having a composition is applied and dried to form a reagent layer.
Was. 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 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.

In this way, an integrated multilayer analytical element having a polyethylene terephthalate colorless and 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 0.50 6.5 0.64 9.0 1.06 OD range = OD (total protein: 9.0 g / dl) -OD (total protein: 4.0 g / dl) = 1.06-0.5 = 0.56

On the other hand, 50 μl of serum having a total protein concentration of 4.0, 6.5, 9.0 g / dl was added to the dry analytical element 2 of Comparative Example 1.
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 is twice as large as that of Comparative Example 1.

Example 2 Dry analysis 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

A 0.8 mm thick plastic sheet obtained by punching a circle having a diameter of 1 cm as a frame was adhered to the above-mentioned analytical element, and then the analytical element was cut into 12 × 13 mm chips, which was disclosed in JP-A-57-63452. The dry analysis element 3 of Example 2 was accommodated in a slide frame described in the official gazette.

Comparative Example 2 Dry Analysis 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 over the entire surface of the reagent layer at a supply rate 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.

In this way, 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 1.010 0.325 5 1.100 0.330 25 1.201 0.320 100 1.280 0.335 500 1.374 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.

[0048]

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 cross-sectional view showing an example of the structure of a dry analysis element of the present invention.

FIG. 2 is a sectional view showing another example of the structure of the dry analysis element of the present invention.

FIG. 3 is a cross-sectional view showing another example of the structure of the dry analysis element of the present invention.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 support 2 frame 3 reagent layer 4 dried reagent 5 specimen

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yoshikazu Amano 3-11-46 Izumi, Asaka-shi, Saitama F-term in Fujisha Shin Film Co., Ltd. 2G042 AA01 BD12 BD20 CB03 DA01 DA08 FB07 FC04 FC05 2G045 AA13 AA16 AA25 CA25 CA26 CB03 DA13 DA13 DA20 DA36 DA37 DA54 DA60 FB01 FB02 FB03 FB11 FB16 FB17 HA14 HA20 2G054 AA07 AB02 AB03 AB04 AB05 AB07 CA21 CA22 CA23 CA28 CE01 EA06 EA09 FA31 GB03 GB04 GB05 GE03

Claims (3)

[Claims] 1. A dry analytical element without a developing layer, wherein a top of a water-impermeable support is partitioned by a water-impermeable frame, and a reagent necessary for analysis is contained in the frame. 2. The dry analytical element according to claim 1, wherein a hydrophilic polymer layer is provided on the water-impermeable support. 3. The dry analytical element according to claim 2, wherein the hydrophilic polymer layer is a reagent layer containing a reagent necessary for analysis.