JPH04102059A - Test jig - Google Patents

Abstract

PURPOSE:To accurately detect and quantify a specific component in specimen even if the component is in a very small amount by reducing an area of a specimen absorbing layer smaller than an area of a reagent layer. CONSTITUTION:A test jig 1 comprises a support 2 with a reagent layer 3 placed on one of its surfaces. The support 2 is a plate as thick as 20 to 200mum while a constituent may be polyethylene terephthalate, etc. The reagent layer 3 mainly comprises coupling agent represented by gelatin in which reagent is contained (spread). Composition of the reagent is appropriately determined by a specific component to be detected (quantified) in specimen. A specimen absorbing layer 4 is placed on the layer 3. The layer 4 is constituted of a porous material and absorbs the dripped specimen and supplies an approximately constant amount of specimen per unit area to the layer 3. The area of the layer 4 is smaller than that of the layer 3. Thus when a constant amount of specimen is dripped, an amount of the specimen supplied per unit area of the layer 3 increases so that coloring intensity on a colored part increases.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention is directed to a test device that detects a specific component in a specimen by a color reaction.

<Prior art> When detecting a specific component in a specimen, such as glucose, cholesterol, or uric acid in blood, or glucose or hemoglobin in urine, a reagent that reacts with the specific component and develops a color is supported. A test device with a reagent layer is used.

The color intensity (color density) of this test device depends on the amount of the specific component in the sample, so it is possible to quantify the specific component in the sample by measuring the color intensity of the test device. can.

By the way, among such test devices, one has been developed in which a spreading layer is glued onto the reagent layer in order to spread the specimen uniformly over the entire area on the reagent layer (Japanese Patent Publication No. 61-6).
No. 1347).

In this test device, the developing layer and the reagent layer have the same planar shape and the same area, so a sample dropped onto the developing layer spreads over the entire surface of the developing layer and is simultaneously supplied to the entire surface of the reagent layer.

Therefore, the amount of specimen supplied per unit area of the reagent layer is small, and the coloring intensity per unit area is low, resulting in a decrease in measurement accuracy such as variations in measurement values.

In particular, when detecting and quantifying a specific component at low concentration, such as uric acid in blood, the difference in color intensity corresponding to the difference in concentration of the specific component is small (the measurement accuracy is significantly reduced, or
There is a problem that it becomes impossible to measure.

Therefore, in order to make the measurement accuracy as high as possible, for example B G (Bindschedler's Green
Leuc.

Ba5e) and ABTS (2,2°-azinobis(
A highly sensitive coloring agent such as 3-ethylbenzothiazolinone-6-sulfonic acid (3-ethylbenzothiazolinone-6-sulfonic acid) must be used, and the range of choices for the coloring agent to be used is narrow. In this case, the coloring agent described above has the disadvantage of lacking stability over time due to its high sensitivity (in some cases, it may be necessary to add a stabilizer to improve stability over time), but it is useful for detecting trace components. In order to make this possible, we were forced to have these shortcomings.

In order to increase the coloring intensity of the reagent layer, it is possible to increase the amount of reagent supported, but enzymes such as uricase, glucose oxidase, cholesterol oxidase, cholesterol esterase, cholinesterase, and riboprotein lipase are particularly expensive. , the cost of the test device increases, which is undesirable.

<Problems to be Solved by the Invention> The purpose of the present invention is to provide a test device that can accurately (detect and quantify) a specific component to be detected in a sample even if it is in a trace amount. .

<Means for Solving the Problems> Such objects are achieved by the following inventions (1) to (5).

(1) A test device comprising a reagent layer carrying a reagent that reacts with a specific component in a specimen to develop a color, and a specimen absorption layer placed on the reagent layer to absorb and develop the specimen, A test device characterized in that the area of the sample absorption layer is smaller than the area of the reagent layer.

(2) The area of the sample absorption layer is 1 of the area of the reagent layer.
The test device according to (1) above, which is 0 to 80%.

(3) The test device according to (1) or (2) above, wherein the planar shape of the specimen absorption layer is substantially the same as the irradiation shape of the photometric light, and the development pattern of the specimen is restricted to that shape.

(4) It has multiple analyte absorption layers for different measurement items,
The test device according to (1) or (2) above, wherein the planar shape of the specimen absorption layer is different for each measurement item, and the development pattern of the specimen is restricted to each shape.

(5) The test device according to any one of (1) to (4) above, wherein the specimen absorption layer has a thickness of 10 to 500%.

Effects> In the present invention, when a sample is dropped onto the sample absorption layer, the sample is spread uniformly over the entire area of the sample absorption layer and is supplied to the reagent layer below the sample absorption layer.

In this case, the area where the sample is supplied to the reagent layer is restricted to the area where the sample absorption layer is located, but since the area of the sample absorption layer is smaller than the area of the reagent layer, when a certain amount of sample is dropped, The amount of specimen supplied per unit area of the reagent layer increases.

Therefore, although the area of the colored portion becomes smaller, the coloring intensity in the colored portion increases.

As a result, when detecting and quantifying a specific component in a sample, particularly a trace amount of a specific component, the difference in concentration of the specific component is expanded, so that more accurate measurement is possible.

In addition, by selecting the planar shape and area of the sample absorption layer, the shape and area of the colored portion of the reagent layer can be freely set. The color portion can be made substantially the same as the irradiation shape of the photometric light, thus making it possible to improve measurement accuracy.

Furthermore, when visually determining multiple items, by making the coloration shape different for each measurement item, identification can be facilitated and misidentification can be prevented.

<Example> Hereinafter, the test device of the present invention will be described in detail based on a preferred example shown in the accompanying drawings.

FIG. 1 is a perspective view showing an example of the configuration of the test device of the present invention. As shown in the figure, the test device 1 includes a support 2 and a reagent layer 3 disposed on one side thereof.

The support body 2 has a plate shape, for example, with a thickness of about 20 to 200 mm.

Specific constituent materials of the support 2 include polyethylene terephthalate (PET), cellulose ester (cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), bisphenol A
Examples include various resins such as polycarbonate, polymethyl methacrylate, polyvinyl chloride, polypropylene, polystyrene, polyvinyl alcohol, and glass. Further, the support 2 may be a stack of sheets made of two or more of the above materials.

When photometry is performed from the side of the support 2, the support 2 is made of a light-transmitting material, preferably a transparent material.

The surface of the support 2 on which the reagent layer is formed is preferably subjected to a hydrophilic treatment (excluding PVA supports) by applying a surfactant or the like.
Improves adhesion to

The reagent layer 3 is mainly composed of a binder typified by gelatin. Note that examples of binders other than gelatin include polyvinyl alcohol, polyvinylpyrrolidone, agarose, sodium polyvinylbenzenesulfonate, polyurethane, and polyvinylpropionate.

This reagent layer 3 contains (disperses) the following reagents.

The composition of the reagent is appropriately determined depending on the specific component to be detected (quantified) in the specimen. For example, when detecting glucose in a sample, the enzyme glucose oxidase (
COD) and peroxidase (POD), and a color former (chromogen) are the main components of the reagent.

Furthermore, when detecting uric acid in a specimen, the main components of the reagent are the enzymes uricase and peroxidase and a coloring agent.

In addition, instead of the above enzymes, cholesterol oxidase, cholesterol esterase and peroxidase,
Riboprotein lipase, glycerol oxidase and peroxidase, phospholipase D, choline oxidase and peroxidase, etc. may be used.

As a coloring agent, benzidine or its compound such as 0-tolidine, m-tolidine, benzidine, tetramethylbenzidine, 0-methylbenzidine, 0-dianisidine,
Examples include a combination of 2,7-diamitsufluorene or 4-aminoantivirine (4-AAP) and a coupling agent that causes coupling with the 4-AAP.

As the coupling agent, P-chlorophenol,
Phenol derivatives such as 2,4-dichlorophenol, 2,4-dibromophenol, 2,4.6-dolichlorophenol, naphthalene derivatives such as 4-chloro-1-naphthalene, 1,7-cyclohydronaphthalene, or N , N-
Dimethylaniline, N,N-diethyl-m-toluidine, N-ethyl-N-sulfopropyl-m-)luidine,
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOO3), 5,6,7.
8-tetrahydro-1-naphthylamine, N-ethyl-
N-(2-hydroxy-3-sulfopropyl)-3,5
-Aniline derivatives such as dimethoxyaniline and the like.

In addition, other coloring agents include 4,4°-diaminodiphenyl, 0-phenylenediamine, m-phenylenediamine, p-phenylenediamine, 2.3-tolylenediamine, 2.4-tolylenediamine, 2.5 - Various substituted phenylene diamines such as tolylene diamine and 2,6-tolylene diamine, amorphous gallic acid, phenols such as gallic acid, phloroglucinol, hydroquinone, and leucoindophenol, guaiacol, pyridine derivatives, substituted azines leucomalachite green, etc. can also be used.

In addition, a hardening agent (crosslinking agent) may be added to the reagent layer 3 as necessary.
, a pH adjuster, a surfactant, a light-reflecting substance, a stabilizer, a sensitizer, an oxidizing agent, etc. may be added.

The hardening agent crosslinks gelatin, etc., and includes, for example, N,N'-hexamethylene-1°6-bis(1-aziridinecarboxamide) (HDU), trimethylolbroban tri-β-aziridinylbrobio Nate (TA
ZM), tetramethylolmethane tri-β-aziridinylpropionate (TAZO), and other aziridine derivatives.

As a pH adjuster, phosphate buffer, citrate buffer,
Examples include borate buffer, Tris buffer, Gud buffer, and the like.

Examples of the surfactant include sodium di-2-ethylhexylsulfosuccinate (Aerosol-OT), sodium dioctylsulfosuccinate, Triton x-100, sodium lauryl sulfate, sodium cholate, and the like.

The light-reflecting substance is used to eliminate the influence of red blood cells on color density measurement when the sample is whole blood, and includes fine particles such as titanium oxide, barium sulfate, aluminum, and various ceramics.

Among the stabilizers, those for improving color stability after coloring include a copolymer of methyl vinyl ether and maleic anhydride, or a half ethyl ester thereof.

In addition, as stabilizers for preventing changes over time during storage of test devices, Japanese Patent Publication No. 56-432'38, Japanese Patent Application No.
1-238148 (especially 2-mercaptobenzimidazole) and the like.

The sensitizer is one that enhances peroxidase activity in hemoglobin, and includes quinoline and its derivatives, such as quinine, cinchonine, 6-medoxyquinoline, quinaldine, 8-amino-6-medoxyquinoline,
Examples include 2-quinolitool, inquinoline, benzo(f)quinoline, 3-aminoquinoline, and the like.

The oxidizing agent is one that can eliminate the influence of reducing substances such as ascorbic acid present in the specimen, and preferably oxyacids or salts thereof and metal salts can be used.

As the oxygen acid or its salt, MXOlMXOa, M
XO4, M3 Hz , specifically, HCl20. NaC
nO4,.

KCj20. HBr0%NaBr0. K-Bro.

1 (IOlNaIO, KIOlHCfiO.

N a CROa, K CI20-HB
r OsNaBrOm, K B r Os, HIO,,
N a I Os, K103, HCff04, NaCn
O4, KCj20. , HB r 04, N a Br r
O4, K B r Oa, HIO4, NaIO4, K
There are IO4, Naa H2I08, K, H2IO, etc.

Metal salts include ferric chloride, cuprous chloride, copper sulfate,
Examples include copper acetate, mercury acetate, bismuth acetate, and lead acetate.

Although the thickness of the reagent layer 3 is not particularly limited, it is preferably about 1 to 100p, particularly about 5 to 20p.

The reagent layer 3 is preferably formed by a coating method, and for example, dip coating, extrusion coating, etc. can be applied. Moreover, two or more layers can be formed simultaneously if necessary.

Moreover, the reagent layer 3 in the test device of the present invention may be one in which the above-mentioned reagent is supported on a carrier.

In this case, as the carrier, the same carrier as the specimen absorption layer 4 described later can be used.

The reagent layer 3 having such a structure is manufactured by impregnating a carrier with a liquid containing a reagent and drying the impregnated liquid.

In the illustrated example, there is only one reagent layer 3, but a plurality of reagent layers each containing reagent components of different compositions may be laminated. In particular, a layer in which the light-reflecting substance is dispersed (light-reflecting layer) may be separately provided on the surface of the reagent layer 3.

A sample absorption layer 4 is provided on the reagent layer 3. The sample absorption layer 4 is preferably made of a porous material described later, absorbs the dropped sample, spreads the sample uniformly within the entire area of the sample absorption layer 4, and spreads the sample per unit area. It has a function of supplying a substantially constant amount of specimen to the reagent layer 3.

The sample absorption layer 4 is preferably made of a non-fibrous or fibrous porous material.

Typical examples of non-fibrous porous materials include filter paper and membrane filters, as well as dispersions of porous materials such as diatomaceous earth and microcrystalline materials dispersed in a binder, and microspherical beads of glass or resin that are bonded to each other. Examples include glued porous aggregates.

Furthermore, examples of the fibrous porous material include woven or knitted fabrics, nonwoven fabrics, short fiber aggregates, and the like.

Examples of the fiber material include natural fibers such as cotton, silk, and wool, and resins such as glass and nylon.

As shown in the figure (the area of this sample absorption layer 40 is smaller than the area of the reagent layer 3. As a result, when a certain amount of sample is dropped, the amount of sample supplied per unit area of the reagent layer 3 is This increases the coloring intensity in the colored portion.

For this reason, the present invention particularly focuses on trace amounts of specific components in a specimen, such as uric acid, glucose, GOT, and GOT in blood.
PT, bilirubin, creatinine, urinary glucose,
It is effective for application to test devices for detecting urobilinogen and bilirubin.

Specifically, 1 to 50 mg, especially 2 to 50 mg per 1 dj of specimen.
20 mg uric acid or 10-500 mg, especially 20-2
One that can detect 50 mg of glucose is preferable.

The area of the sample absorption layer 4 is preferably about 10 to 80%, particularly about 10 to 40%, of the area of the reagent layer 4. If it is less than 10%, the sample may not be retained sufficiently and may overflow from the sample absorption layer 4 before being absorbed into the reagent layer 3. If it exceeds 80%, the color intensity may decrease. This is because the increase will be insufficient.

The planar shape of the specimen absorption layer 4 is preferably a shape suitable for optically measuring color intensity. In the illustrated example, the shape is circular to match the shape of the light beam for optically measuring color intensity. This results in
Accurate photometry is possible with the smallest area.

Further, the present invention can also be applied to a so-called multi-item test device that has a plurality of reagent layers and detects pressures of different specific components in a specimen in each reagent layer. That is, the same sample absorption layer 4 as described above is installed on each reagent layer of this multi-item test device, but in this case, the planar shape of each sample absorption layer can be made different for each measurement item.

This makes it easy to identify and prevent misidentification when visually determining multiple items.

The thickness of the specimen absorption layer 4 is not particularly limited, but is 10 to 50 mm.
It is preferably about 0 JIJl, especially about 50 to 200. If the thickness is less than 10 mm, the sample cannot be retained, and the sample may overflow outside the sample absorption layer 4 before being absorbed into the reagent layer 3. This is because if it exceeds 1, the time required for the sample to be absorbed into the reagent layer 3 becomes longer, the determination time becomes longer, and problems such as evaporation of the sample occur.

Although such a sample absorption layer 4 may simply be placed on the reagent layer 3, it is preferable that it is adhesively fixed to the reagent layer 3. In this case, the specimen absorption layer 4 can be stacked on the wet reagent layer 3, and both layers 3.4 can be bonded together by the binder in the reagent layer 3. Moreover, the reagent layer 3 and the specimen absorption layer 4 can also be bonded together via a separately prepared adhesive.

In addition, when the reagent layer 3 is a carrier impregnated with a reagent as described above and is not adhesively fixed to the support 2,
If the sample absorption layer 4 is not adhesively fixed to the reagent 13, it is preferable to have a structure in which they are fixed by a holding member 5 called a holder or mount, as shown in FIG.

This holding member 5 includes first holding member pieces 6 that fit together and hang together.
and a second holding member piece 7, and in a state where both holding member pieces 6 and 7 are fitted, the holding part 7 of the second holding member piece 7
1 presses the peripheral edge of the sample absorption layer 4 onto the reagent layer 3 with a predetermined pressure to fix them.

Furthermore, openings 65 and 75 are formed in substantially central portions of the first and second holding member pieces 6 and 7, respectively.

The opening 65 of the first holding member piece 6 is opened to the support body 2 by the analyzer.
It is provided as a window for measuring the coloring intensity of the reagent layer 3 by projecting and receiving light from the side. On the other hand, the second
The holding member piece 70 open lower portion 5 is provided as a space for supplying the sample to the sample absorption layer 4 .

The shapes of these openings 65 and 75 include a circle, an oval, a regular polygon, and the like.

The constituent materials of the first and second holding member pieces 6.7 are not particularly limited, and include various resins such as polyethylene, polypropylene, polyvinyl chloride, polystyrene, ABS resin, etc.
Alternatively, metals such as aluminum and stainless steel can be used.

Although the test device of the present invention has been described above with reference to the configuration example shown in the drawings, the present invention is not limited thereto. In particular, the layer structure of the test paper may be arbitrary, and may include a light-reflecting layer, a protective layer, an oxidizing agent-containing layer, and other layers provided for various purposes.

The test device of the present invention is used for testing specimens (e.g. whole blood,
BUN
, creatinine, calcium, oxalic acid, cholesterol, triglyceride (neutral fat), free fatty acids, ketone bodies, bilirubin, urobilinogen, protein, nitrite, ascorbic acid, hemoglobin, myoglobin, white blood cells, GOT, GPT, ALP, γ - It is applicable to the detection of GT, etc., and furthermore, it is possible to apply it to other fields such as analysis of food and environmental samples.

<Experiment example> Experimental examples of the present invention will be described below.

(Example of the present invention) A solution having the composition shown in Table 1 below is coated on a transparent support made of a PET film having a thickness of 120 mm and has been subjected to a hydrophilic treatment, and dried to form a reagent layer with a dry film thickness of about 1 Op. After forming,
This was cut into a size of 9 mm x 13+ nm.

Next, as a sample absorption layer, a small piece of nylon fiber knitted fabric (thickness: 150 mm) cut into a circle with a diameter of 6 mm was placed approximately in the center on the reagent layer, and these pieces were placed in a holder as shown in Figure 2. Fixed.

In the test device thus obtained, the ratio of the area of the sample absorption layer to the area of the reagent layer was 24%.

Table 1 Reagent layer coating solution composition Ingredient content 20% gelatin aqueous solution 2ml
50mM borate buffer (pH 8,5)
1m1O025% TritonX-100 aqueous solution (surfactant) 0.5mj4-AAP (color former)
30mg TOO3 (coupling agent) 40mg uricase
20 mg peroxidase 20 mg (comparative example) The same procedure as the inventive example was used except that a developing layer of the same size as the reagent layer was placed on the reagent layer. Note that this spreading layer was made of the same material as the specimen absorbing layer in the example of the present invention.

A coloring test was conducted using each test device of the above-mentioned invention examples and comparative examples.

Three types of serum samples adjusted to have uric acid concentrations of 2.10 and 20 mg/dp were dropped in an amount of 10με each onto the specimen absorption layer and development layer of each test device, and after 6 minutes, the support side The intensity of the reflected light with a wavelength of 565 nm was measured using an analyzer (manufactured by Otsuka Electronics, M
CPD-200 model).

The results are shown in Table 2 below.

Note that the numerical values in Tables 2 and 3 below indicate logarithmically transformed values of relative reflection intensity.

Table 2 As shown in Table 2 below, in the test device of the present invention, an increase in reflection intensity was confirmed as the uric acid concentration increased, and the difference in reflection intensity at low and high concentrations was also observed in the test device of the comparative example. It is expanding comparatively.

<Effects of the Invention> According to the test device of the present invention, specific components in a sample can be detected with high accuracy (
It can be detected and quantified, and even trace amounts of specific components can be measured with high precision using a small amount of sample.

As a result, the range of choices for reagent compositions, especially the type of coloring agent, has been expanded, making it possible to use less toxic reagents, eliminating the need to increase the amount of expensive enzymes supported, and improving stability over time. It is possible to improve other properties of the test device, etc., and it is also advantageous in terms of manufacturing cost.

Furthermore, by selecting the planar shape and area of the sample absorption layer, the shape and area of the colored portion of the reagent layer can be freely set, resulting in the following advantages.

First, since the color development shape can be made to match the irradiation shape of the photometric light, highly accurate photometry can be performed with a minimum area.

Second, when measuring multiple items by visual judgment, by making the colored shape different for each item, identification can be facilitated and misidentification can be prevented.

65. 75...opening Chill

[Brief explanation of the drawing]

FIG. 1 is a perspective view showing an example of the configuration of the test device of the present invention. FIG. 2 is a cross-sectional side view showing an example of the configuration of the test device of the present invention.

Claims (5)

[Claims] (1) A test device comprising a reagent layer carrying a reagent that reacts with a specific component in a specimen to develop a color, and a specimen absorption layer placed on the reagent layer to absorb and develop the specimen, A test device characterized in that the area of the sample absorption layer is smaller than the area of the reagent layer. (2) The area of the sample absorption layer is 1 of the area of the reagent layer.
The test device according to claim 1, which is 0 to 80%. (3) The test device according to claim 1 or 2, wherein the planar shape of the specimen absorption layer is substantially the same as the irradiation shape of the photometric light, and the developed pattern of the specimen is restricted to that shape. (4) It has multiple analyte absorption layers for different measurement items,
3. The test device according to claim 1, wherein the planar shape of the specimen absorption layer differs for each measurement item, and the development pattern of the specimen is regulated to each shape. (5) The test device according to any one of claims 1 to 4, wherein the specimen absorption layer has a thickness of 10 to 500 μm.