JPH03110465A - Tester - Google Patents

Abstract

PURPOSE:To eliminate deterioration of sensitivity and to prevent dispersion of quantitative value by unincorporating a compd. having amino group or carboxyl group in the molecule into a reagent layer and carrying this compd. on the other layer. CONSTITUTION:A tester 1 is formed of a laminated body having a constitution in which a reagent layer 3 is formed on the single face of a supporting body 2 and a developing layer 4 is provided on the layer 3. One part of components constituting a reagent and a hardening agent are incorporated in the layer 3. However in the components constituting the reagent, a compd. having amino group or carboxyl group as a reaction group in the molecule is not incorporated in the layer 3 but is carried on a layer 4. Thereby this compd. is not allowed to react with the hardening agent incorporated into the layer 3 but sufficiently functions. The layer 4 uniformly developes a dropped specimen on this layer 4 and functions so as to supply the specimen or the component in the specimen of nearly constant amount per unit area to the layer 3.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a test device for detecting components in a specimen, such as sugar in blood or urine.

<Prior Art> A multilayer analysis sheet for liquid sample analysis is known as a sheet that can easily quantify specific components in a specimen (Japanese Patent Publication No. 61347/1982).

This multilayer analytical sheet consists of 1 or 2 layers containing predetermined reagents in a binder, such as gelatin, on a light-transparent hydrophobic support.
The above reagent layer is coated and formed, and a liquid sample spreading layer made of a hydrophilized fabric is adhered onto the reagent layer.

According to such a multilayer analysis sheet, when a sample is dropped onto the liquid sample development layer, the sample is spread uniformly on the liquid sample development layer, and then the sample permeates into the reagent layer, allowing identification of the sample in the sample. A component (for example, glucose) reacts with a reagent to develop a color, and this light is projected from the support side, and the specific component is quantified based on the intensity of the reflected light.

In order to obtain good quantitative performance, gelatin is often used as the material for forming the reagent layer, as it has good dispersibility for reagents and color-forming substances (dyes) within the layer. , a surfactant, a hardening agent, etc.

An example of a reagent component is glucose oxyguse (
COD) enzymes such as peroxidase (POD) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium (TOO3), 4
-Contains coloring agents such as aminoantipyrine (4-AAP).

Further, the hardening agent (crosslinking agent) crosslinks gelatin, for example, N,N'-hexamethylene-1,6
-bis(1-aziridinecarboxamide) (HDt
An aziridine derivative such as J) is used.

By the way, when quantifying a specific component in a sample using such a multilayer analysis sheet, there have been cases where development such as a decrease in color sensitivity and variation in quantitative values occurs.

Therefore, the present inventor researched the cause of such development and found that the hardening agent that crosslinks gelatin
The present invention was based on the discovery that the effective amount of 4-AAP decreases because it reacts with the components in the reagent, especially the coloring agent 4-aminoantipyrine (4-AAP), resulting in crosslinking. reached.

It is also possible to anticipate a decrease in the effective amount of 4-AAP and include it in a large amount in the reagent layer. However, in this case, although a decrease in sensitivity can be prevented, the gelatin The disadvantages of insufficient crosslinking and variation in quantitative values cannot be improved.

<Problems to be Solved by the Invention> The present invention has been made in view of the above-mentioned drawbacks of the prior art, and its purpose is to provide a test device that does not reduce sensitivity and prevents variations in quantitative values. It's about doing.

Means for Solving the Problems> Such objects are achieved by the following inventions (1) to (4).

(1) A test device having two layers on a support, characterized in that the layer not adjacent to the support supports a compound having an amino group or a carboxyl group in the molecule. Test equipment.

(2) The test device according to (1) above, wherein the compound is a coloring agent.

(3) The test device according to (1) or (2) above, wherein the compound is supported on a spreading layer having a function of spreading a specimen.

(4) The test device according to (3) above, wherein the spreading layer is a woven or knitted fabric.

<Function> According to the present invention having such a configuration, a crosslinking compound,
That is, by not including a compound having an amino group or a carboxyl group in its molecule in the reagent layer, but having it supported in another layer, particularly in the developing layer, crosslinking of the compound by the hardening agent can be prevented.

In this case, when a specimen is dropped onto the developing layer, the compound supported on the developing layer moves into the reagent layer together with the specimen and functions as a reagent.

<Example> Hereinafter, the test device of the present invention will be described in detail with reference to preferred examples shown in the accompanying drawings.

FIG. 1 is a cross-sectional front view showing an example of the configuration of the test device of the present invention. As shown in the figure, the test device 1 is a laminate in which a reagent layer 3 is formed on one side of a support 2, and a spreading layer 4 is placed on the reagent layer 3.

The support 2 has a plate shape with a thickness of about 20 to 200 mm, for example, and is made of a light-transmitting material, preferably a transparent material.

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

In addition, it is preferable that the reagent layer formation surface of the support 2 is subjected to a hydrophilic treatment (excluding PVA supports) by coating with a surfactant, etc. As a result, the support 2 of the reagent layer 3
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) a part of components constituting a reagent to be described later and a hardening agent (crosslinking agent).

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 (GO)
D) and peroxidase (POD)) and a coloring agent (
A typical example of a reagent component is a chromogen (chromogen). Note that instead of the above enzymes, cholesterol oxidase and cholesterol esterase and peroxidase, uricase and peroxidase, riboprotein lipase and glycerol oxidase and peroxidase, phospholipase D and choline oxidase and peroxidase, etc. may be used.

As a coloring agent, N-ethyl-N-(2-hydroxy-
3-sulfopropyl)-m-toluidine sodium (T
OOS), 4-aminoantipyrine (4-AAP),
Examples include orthotolidine hydrochloride.

Among the components constituting such reagents, compounds with an amino group or carboxyl group as a reactive group in the molecule (
Hereinafter, the compound (also simply referred to as a compound) is not contained in the reagent layer, but is supported on the developing layer 4, which will be described later. This ensures that the compound does not react with the hardening agent in the reagent layer 3 (and functions well).

Specific examples of such compounds in the reagents exemplified above include 4-AAP, orthotolidine hydrochloride, GOD
, POD, and reagents other than those exemplified above include orthophenylenediamine, orthotolidine, and the like. Among these, the effects of the present invention are particularly noticeable when coloring agents such as 4-AAP and orthotolidine hydrochloride are removed from the reagent layer 3 and supported on the developing layer 4.
Preferably, therefore, a configuration may be adopted in which only a coloring agent such as 4-AAP is supported on the developing layer 4, and GOD and POD are contained in the reagent layer 3 together with other reagent components.

That is, in the present invention, when there are multiple types of compounds, it is not necessarily necessary to support all of them on the spreading layer 4.

Further, for example, when the deployment layer 4 is made to carry 4-AAP,
It is preferable that the entire amount of 4-AAP is supported, but a portion of 4-AAP may be supported. Specifically, 80% or more of the amount of 4-AAP contained in the reagent layer 3 is supported on the spreading layer 4. If so, the effects of the present invention can be obtained.

The hardening agent added to the reagent layer 3 is one that crosslinks gelatin, etc., and is, for example, N,N'-hexamethylene-1.
,6-bis(1-aziridinecarboxamide) (HDU
) trimethylolpropane tri-β-aziridinylpropionate (TAZM), tetramethylolmethane tri-β-aziridinylpropionate (TAZO)
) and other aziridine derivatives.

Moreover, a pH adjuster, a surfactant, a light reflecting substance, a stabilizer, a sensitizer, etc. may be added to the reagent layer 3 as necessary.

Examples of the pH adjuster include phosphoric acid (phosphate buffer), citrate buffer, borate buffer, Tris buffer, and Gud buffer.

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 necessary when the specimen is whole blood, and includes fine particles such as titanium oxide, barium sulfate, and aluminum.

The stabilizer is a substance having reducing properties, and examples thereof include 2-mercaptobenzimidazole and its derivatives.

Examples of the sensitizer include 3-aminoquinoline hydrochloride.

Although the thickness of the reagent layer 3 is not particularly limited, it is preferably about 1 to 100 mm, particularly about 5 to 15 mm.

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.

In the illustrated example, there is only one reagent layer 3, but a plurality of reagent layers each containing reagent components having different compositions may be laminated. In particular, H( A light reflecting layer) may be separately provided between the reagent layer 3 and the developing layer 4.

The spreading layer 4 has the function of uniformly spreading the dropped sample on the spreading layer and supplying a substantially constant amount of the sample or components in the sample per unit area to the reagent layer 3.

This spreading layer 4 is made of a non-fibrous or fibrous porous material.

Membrane filters are typical examples of non-fibrous porous materials, and other porous materials such as diatomaceous earth and microcrystalline materials (e.g., microcrystalline cellulose (commercial name Avicel of FMC Corporation)) are dispersed in a binder. Examples include porous aggregates made of microspherical beads of glass or resin bonded to each other.

Examples of the fibrous porous material include woven fabrics or knitted fabrics (these are collectively referred to as woven and knitted fabrics), nonwoven fabrics, aggregates of short fibers, etc. Among these, it is particularly preferable to use woven and knitted fabrics.

The reason is that the specimen is rapidly developed.

Such a spreading layer 4 carries the aforementioned compound having an amino group or a carboxyl group in its molecule.

A method for supporting this compound on the developing layer 4 is to impregnate the developing layer 4 with a solution (or dispersion) containing the compound,
This may then be done by drying.

Although the content of the compound in the spreading layer 4 depends on the type of the compound, it is usually preferable that the content is equal to or about 20 to 50% higher than the amount when the compound is contained in the reagent layer 3. .

Note that the spreading layer 4 is preferably subjected to a hydrophilic treatment in order to improve the spreadability of the specimen. Examples of the hydrophilic treatment include carrying a surfactant.

Such a spreading layer 4 may be adhesively fixed on the reagent layer 3, but is preferably placed on the reagent N3.

That is, it is preferable that the two layers 3.4 are in contact with each other without having substantially any bonding force by themselves.

With this configuration, the time required for the sample developed on the development layer 4 to reach and permeate the reagent layer 3 is shortened, and the permeability of gases such as oxygen necessary for color reaction is also reduced. This increases the reaction rate and reduces the time required for analysis.

In this way, the developed N4 has no binding force to the reagent layer 3 below it, so simply being placed on the reagent layer 3 will cause misalignment, and the relationship between the reagent M3 and the developed layer 4 will occur. A void is likely to be formed between the two. Therefore, when actually using the test device 1, as shown in FIG. , fixed.

This holding member 8 includes first holding member pieces 6 that fit together and hang together.
and a second holding member piece 7, and when both holding member pieces 6 and 7 are fitted, the respective clamping parts 61 and 71 press the peripheral edge of the test device 1 with a predetermined pressure. There is.

Furthermore, approximately central portions of the first and second holding member pieces 6 and 7 (inside the clamping portions 61.71) each have an opening 62.
and 72 are formed.

The opening 62 of the first retaining member piece 6 is inserted into the support body 2 by the analyzer.
It is provided as a window for measuring the intensity of coloring of the reagents 11!3 by projecting and receiving light from the side. On the other hand, the open lower portion 2 of the second holding member piece 7 is provided as a space for supplying the specimen to the deployment 14.

The shapes of these openings 62 and 72 include circular, oval, and regular polygonal shapes.

The constituent materials of the first and second holding member pieces 6.7 are not particularly limited, and examples include various resins such as polyethylene, polypropylene, vinyl chloride, polystyrene, and ABS, and metals such as aluminum and stainless steel. .

FIG. 3 is a sectional front view showing another mode of use of the test device 1. As shown in the figure, an absorbent layer 5 is further placed on the spreading layer 4 of the test device 1, and these are held between holding members 8 similar to those described above.

The absorbent layer 5 absorbs and retains excess sample from the sample supplied from the open lower lower 2, and plays the role of keeping the amount of sample supplied constant.

This absorption layer 5 has an opening 5 having a shape substantially similar to that of the open lower lower part 2.
1 is formed.

Examples of the constituent material of the absorbent layer 5 include woven fabrics, nonwoven fabrics, knitted fabrics, filter paper, porous water-absorbing polymers, glass fibers, and the like.

When the sample is dropped from the open lower 2, the sample is spread out uniformly on the spreader N4. At this time, if there is an absorbing layer 5 (see FIG. 3), excess sample is absorbed and retained in this absorbing layer 15.

The specimen that has permeated the spreading layer 4 moves to the reagent layer 3 below it. At this time, the compound (for example, 4-AAP) supported on the reagent layer 3 is eluted into the sample and transferred to the reagent layer 3 together with the sample.

As a result, all the components constituting the reagent gather in the reagent N3, which functions as a reagent. Therefore, if a specific component to be detected is present in the sample, it will develop a color due to a predetermined reaction.

Although the test device of the present invention has been described above with reference to the illustrated structural example, the present invention is not limited thereto. In particular, the layer supporting the compound is not limited to the spreading layer 4,
The light reflecting layer, the absorbing layer 5, the protective layer, and other layers provided for various purposes or layers provided exclusively for supporting compounds, etc., outside the reagent layer 3 (on the opposite side from the support 2) Any layer may be used as long as it is present in the above layers, and one or more of these layers (layers other than the reagent layer 3) may support the compound.

The test device of the present invention is used for testing specimens (e.g. whole blood,
It can be applied not only to the analysis of sugar content in body fluids such as serum, urine, and saliva, but also to the analysis of uric acid, GOT, GPT, etc., and can also be applied to other fields such as the analysis of food and environmental samples. is also possible.

Experimental Example> (Example of the present invention) A solution having the composition shown in Table 1 below was coated on a transparent support made of a 115-thick PET film that had been subjected to a hydrophilic treatment and dried, resulting in a dry film thickness of approximately 8 mm. A reagent layer was formed.

Next, this was cut into a size of 1.5ca+X1.5cm, and the reagent layer was impregnated with a solution having the composition shown in Table 2 below, and then dried (40°C, 60 minutes) on a knitted fabric.
A test device with the configuration shown in Fig. 3 was obtained by placing a spreadable layer of the same size made of woven fabric, further placing an absorbent layer made of woven fabric on top of it, and placing these in close contact with each other by sandwiching them between holding members (mounts). I got an A.

Table 1 Reagent layer coating solution composition 20% gelatin aqueous solution 1M phosphate buffer (pH 6) Surfactant (0.25% Aerosol-OT aqueous solution)
Crosslinking agent (HDU) N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m- Toluidine sodium glucose oxidase peroxidase Titanium oxide content mj mj O, 5ml 0mg 0mg 0mg 0mg 0.4g Table 2 Component 4- Impregnating liquid composition of aminoantipyrine distilled water spreading layer Content: 25 mg 0 mj (Comparative Example 1) A solution having the composition shown in Table 3 below was coated on the same support as in the present invention example, dried, and the reagent of the dried membrane summary 8- formed a layer.

Next, this was cut into a size of 1.5cm x 1.5c+a, and a spreadable layer of the same size made of a knitted fabric of the same material as the example of the present invention was placed on the reagent layer, and a similar absorbent layer was placed on top of it. were mounted, and these were sandwiched and brought into close contact with a holding member (mount) to obtain a test device B having the configuration shown in FIG. 3.

Table 3 Reagent layer coating solution composition Component content 20% gelatin aqueous solution 1M phosphate buffer (pH 6) Surfactant (0.25% Aerosol-OT aqueous solution)
Crosslinking agent (HDU) 4-aminoantipyrine N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m- Toluidine sodium glucose oxidase peroxidase titanium oxide mj 111 0, 5ml 0mg 0mg 0mg 0mg 0mg o, 4g (comparison Example 2) Test device C was obtained in the same manner as in Comparative Example 1, except that a reagent layer was formed using a coating liquid having the composition shown in Table 4 below.

Table 4 Reagent layer coating solution composition 20% gelatin aqueous solution 1M phosphate buffer (pH 6) Surfactant (0.25% Aerosol-OT aqueous solution)
Crosslinking agent (HDU) 4-aminoantipyrine N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m- Toluidine sodium glucose oxidase peroxidase Titanium oxide content mj mj O, 5ml 0mg 20mg 0mg 0mg 0mg 0.4g The above test devices A%B and C were colored using a sample (serum) containing glucose, and the state of coloring was investigated. The detailed procedure is as follows.

First, glucose was dissolved in physiological saline to a concentration of 5000 mg/dl, and heat treated at 37° C. for 2 hours to isomerize the solution, which was used as a glucose stock solution.

Next, the glucose concentration of a commercially available standard serum (Bio-Rad, Liphocheck Biochemical Control Serum I) was measured using a commercially available measurement kit (Glucose Test Wako, Wako Tsumugi Co., Ltd.), and based on this measurement value, the standard Add glucose stock solution to serum, glucose concentration is 100.20 respectively.
0 and 400 rrrg/dj,
Three types of serum samples No. 1.2 and 3 were prepared.

Prepare 10 test devices A, B, and C, and drop 10μβ of each of the three serum samples N001 to 3 onto the spreading layer of each test device. After 1 minute, place the sample on the support side. Light was projected and received, and the intensity of reflected light at a wavelength of 565 nm was measured.

The measurement results thus obtained are shown in Tables 5, 6 and 7 below.

Note that the numerical values in each table indicate blood sugar levels (mg/dg).

Table 5 (Example of the present invention) No, 1 No, 2 No, 3 Average value .5 6.7 36g, 8 5.29 1.4 366, 5±8.5 2.3 Table 6 (Comparative Example 1) No. 1 No. 2 Average value X Standard deviation SD Coefficient of variation CV (%) Intermediate Upper middle believer (%) 87.9 4.2 4.8 87.5±6,5 7.4 136,4 7.5 5.5 130,5±12.5 9.6 226,1 9. 30 4.1 226±15 6.6 Table 7 (Comparative Example 2) No, 1 No, 2 No, 3 Average value X Standard deviation SD Coefficient of variation CV (%) Above median value Above median value (%) 103.7 7.17 6.9 104, 5±11.5 11.0 199, 9 11.73 5.9 197±19 9.6 359.9 23, 99 6.7 362±36 9.9 Shown in Table 5 As can be seen, the sensitivity is high in the example of the present invention, and there is little variation in the obtained data.

On the other hand, as shown in Table 6, in Comparative Example 1, the sensitivity was low and the obtained data varied.

In addition, as shown in Table 7, in Comparative Example 2, a large amount of 4-aminoantipyrine was added compared to Comparative Example 1, so
Although no decrease in sensitivity was observed, there was considerable variation in the obtained data. Further, during the manufacture of Test Device C, when the reagent layer was observed through a magnifying glass, it was found that dura mater defects had occurred.

<Effects of the Invention> As described above, according to the test device of the present invention, it is possible to maintain high sensitivity and prevent variations in quantitative values, so that more accurate detection results can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a cross-sectional front view showing an example of the configuration of the test device of the present invention. FIGS. 2 and 3 are cross-sectional front views each showing an example of a configuration in which the test device of the present invention is loaded into a holding member. Explanation of symbols 1...Test device 2...Support 3...Reagent layer 4...Development layer 5...Absorption layer 51...Opening 6...First holding member piece 7...・Second holding member piece 61.71... Holding part 62.72... Opening 8... Holding member 1G, 1

Claims (4)

[Claims] (1) A test device having at least two layers on a support, characterized in that the layer not adjacent to the support supports a compound having an amino group or a carboxyl group in the molecule. . (2) The test device according to claim 1, wherein the compound is a coloring agent. (3) The test device according to claim 1 or 2, wherein the compound is supported on a spreading layer having a function of spreading the specimen. (4) The test device according to claim 3, wherein the spreading layer is a woven or knitted fabric.