JPH0664045B2 - Test tool - Google Patents

Description

TECHNICAL FIELD The present invention relates to a test device for measuring and analyzing a specific component in a sample, for example, the amount of sugar in blood.

<Prior Art> A multi-layer analysis sheet for liquid sample analysis is known as one that can easily perform the quantification of a specific component in a sample (Japanese Patent Publication No. 61-61347).

Among the present invention, a multilayer analysis sheet for directly measuring a colored sample such as whole blood is a light-transmitting hydrophobic support,
One or more reagent layers containing a predetermined reagent in a binder such as gelatin are applied and formed, and an analytical function auxiliary layer such as a color shielding layer and a light reflection layer is applied and formed on the reagent layers; Further, a liquid sample developing layer made of a hydrophilic treatment fabric is adhered on the analysis function auxiliary layer.

According to such a multilayer analysis sheet, when blood is dropped on the liquid sample spreading layer, the sample is uniformly spread on the liquid sample spreading layer, and then blood cells are filtered out by the analysis function assisting layer, and serum is separated. Permeation into the reagent layer through the analytical function auxiliary layer, a specific component in the serum (eg glucose) reacts with the reagent to develop a color, which is projected from the support side, depending on the intensity of the reflected light. The specific component is quantified.

The red light of the filtered blood cells is blocked by the analysis function auxiliary layer and does not pass through to the support side, so it does not affect the measurement on the support side.

However, this multilayer analysis sheet has a drawback that the reaction time (the time from the dropping of the sample to the color development) is long due to the presence of the analysis function auxiliary layer (5 to 100 μm thick) on the reagent layer. This will be described in detail below.

The principle of color development of the reagent layer in the measurement of blood glucose level is as shown in the following formula, glucose in a sample and oxygen taken from the outside air are decomposed into gluconic acid and hydrogen peroxide by an enzyme called glucose oxidase (GOD), Hydrogen peroxide is decomposed into water and an enzyme atom by an enzyme called peroxidase (POD), and this enzyme atom reacts with a chromogen to become a dye.

However, in the above-mentioned multilayer analysis sheet, O _{2 in} the atmosphere is
Since it reaches the reagent layer after passing through the analytical function auxiliary layer, the supply rate of O ₂ to the reagent layer becomes slow, and as a result, the reaction rate of the above reaction becomes slow.

Further, since the sample itself also passes through the analytical function auxiliary layer and reaches the reagent layer, it takes a long time until the sample reaches the reagent layer after being dropped and then penetrates.

<Problems to be Solved by the Invention> An object of the present invention is to solve the above-mentioned drawbacks of the prior art and to provide a test device having a short reaction time.

<Means for Solving the Problems> Such an object is achieved by the present invention described below.

That is, the present invention is a test device for analyzing components in a sample, comprising a reagent layer containing a reaction reagent on one surface of a light-transmissive support, and a spreading layer for uniformly spreading the sample. Is fixed integrally with the developing layer on the outside, and a material having light reflectivity is contained in the reagent layer adjacent to the developing layer, and in the reagent layer of the substance having light reflectivity. The test tool is characterized in that the distribution of the reagent layer in the thickness direction is denser on the developing layer side than on the light transmitting support side.

The light-reflective substance is preferably a test tool that is white fine particles.

The average particle size of the white fine particles is preferably 0.1 to 5 μm.

Further, the substance having light reflectivity is preferably a test tool which is a fibrous substance.

The content ratio of the light-reflecting substance in the reagent layer is 10
Preferably, the test device is ˜90 wt%.

<Operation> According to the present invention having such a configuration, by providing an analysis function auxiliary layer such as a light-reflecting layer separately and providing the function to the reagent layer, the dropped sample reaches the reagent layer. The time required for permeation is short, and the permeability of gases such as enzymes necessary for the color reaction is improved, and the supply thereof is smoothly performed.

As a result, the color reaction is accelerated and the time required for analysis is shortened.

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

FIG. 1 is a sectional front view showing a configuration example of a test device of the present invention. As shown in the figure, the test device 1a includes a test piece 5a composed of a laminated body in which a reagent layer 3 is formed on one surface of a support 2 and a developing layer 4 is placed on the reagent layer 3. The test piece 5a is sandwiched by a holding member 8 composed of a pair of FIG. 1 and second holding member pieces 6 and 7.

The support 2 has a plate shape with a thickness of, for example, about 20 to 200 μm, and is made of a light-transmissive 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.), bisphenol A polycarbonate, polymethylmethacrylate, polyvinyl chloride, polypropylene, polystyrene, Examples include various resins such as polyvinyl alcohol and glass. Further, of the above, a sheet in which two or more kinds of sheets are laminated may be used.

The reagent layer 3 includes, for example, gelatin, polyvinyl alcohol,
Consists of a composition in which a predetermined reagent and a substance having a light-reflecting property described later are contained (dispersed) in a binder such as polyvinylpyrrolidone, agarose, sodium polyvinylbenzenesulfonate, polyurethane, polyvinylpropionate. To be done.

As a coating method for forming a reagent layer or the like, a method used in the art, for example, a suitable method such as dip coating or extrusion coating can be selected, and if necessary, two or more layers are simultaneously formed by coating. It is also possible to obtain an arbitrary dry film thickness, for example, 1 to 50 μm, by changing the composition and coating conditions.

The reagent in the reagent layer 3 is appropriately determined depending on the component to be quantified in the sample. For example, when quantifying glucose in a sample, glycol oxidase, as a main reagent,
Includes peroxidase and chromogen.

Substances with light reflectivity (hereinafter referred to as light reflective substances) 1
Examples of 0 include white fine particles of titanium oxide, barium sulfate, aluminum, zinc oxide, or ceramics such as alumina, silica, and zirconia. In addition,
Here, white is a broad concept including not only pure white but also a light color or a metal color (for example, silver color) or the like that can ensure light reflection to some extent.

By including such white fine particles in the reagent layer, the reagent layer 3 has a component that interferes with the detection of coloration present in the sample (for example, blood cells when the sample is whole blood).
And the function of blocking the colored light from the filtered material (blood cell) side or reflecting the light from the support side.

Further, the presence of the white fine particles improves the diffusivity of the sample in the reagent layer 3, and further increases the contact area (corresponding to the surface area of the white fine particles) of the sample and the reagent per unit time.
The reagent can be effectively used, which contributes to shortening the reaction time.

The content of the white fine particles in the reagent layer 3 is 10 to 90 wt%
Is preferable, and about 25 to 60 wt% is particularly preferable. If it is less than 25% by weight, especially less than 10% by weight, the above-mentioned function may not be sufficiently fulfilled, and if it exceeds 60% by weight, particularly 90% by weight, detection of coloration may be hindered.

From the same viewpoint, it is preferable that the white fine particles have an average particle size of about 0.1 to 5 μm.

The distribution of such white fine particles in the reagent layer 3 in the thickness direction is dense on the developing layer 4 side and rough on the support 2 side. With such a distribution, the color of the reagent layer 3 tends to be dark as a whole, and the color can be easily detected. Such a distribution can be obtained by, for example, performing centrifugation before the reagent layer 3 is dried and solidified.

Other examples of the light-reflecting substance 10 include fibrous substances such as short cellulose fibers, polyethylene, polyester, polypropylene, and polyamide. When such a fibrous substance is used, the physical strength of the formed layer is increased.

Regarding the content rate of such a fibrous substance in the reagent layer 3,
For the same reason as above, it is preferably about 10 to 90 wt%, and particularly preferably about 25 to 60 wt%.

The distribution of the fibrous substance in the reagent layer 3 in the thickness direction is the same as described above.

In the present invention, the light-reflecting substance contained in the reagent layer 3 is not limited to the above white particles or fibrous substance, and may be a combination of both.

The spreading layer 4 is located on the outer surface of the test piece 5a, uniformly spreads the dropped sample on the spreading layer, and supplies a substantially constant amount of the sample or a component in the sample per unit area to the reagent layer 3. Has the effect of

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

As the non-fibrous porous material, a dispersion obtained by dispersing a porous material such as brush polymer (general membrane filter), diatomaceous earth, microcrystalline material (for example, microcrystalline cellulose (trade name of Avicel of FMC Corporation)) in a binder. ,
Examples thereof include a porous aggregate in which microspherical beads of glass or resin are point-bonded to each other.

Examples of the fibrous porous material include woven fabrics, non-woven fabrics, aggregates of short fibers, and the like. Among them, it is particularly preferable to use the hydrophilic treatment woven fabric disclosed in Japanese Patent Publication No. 61-61347. preferable.

Such a spreading layer 4 is placed on the reagent layer 3, but the two layers 3 and 4 are in contact with each other without substantially having a binding force.

With such a configuration, the time taken for the sample spread on the spreading layer 4 to reach and permeate the reagent layer 3 is shortened, and the permeability of gas such as oxygen necessary for the color reaction is also reduced. Since it is improved, the reaction speed is increased, which contributes to shortening the time required for analysis.

As described above, since the spreading layer 4 does not have a binding force to the reagent layer 3 therebelow, a gap occurs when it is simply placed on the reagent layer 3, and the reagent layer 3 and the spreading layer 4 A gap is apt to occur between and. Therefore, as shown in FIG. 1, the periphery of the test piece 5a is clamped by the holding member 8 and the development layer 4 is pressed and fixed to the reagent layer 3.

The holding member 8 is a first holding member piece 6 that can be fitted to each other.
And the second holding material piece 7, and the holding portions 61 and 7 in the state where both holding member pieces 6 and 7 are fitted to each other.
1 presses the peripheral member of the test piece 5a with a predetermined pressure.

Further, the opening 6 is formed in each of the substantially central portions (inside the sandwiching portions 61 and 71) of the holding member pieces 6 and 7 shown in FIG.
2 and 72 are formed.

The opening 62 of the first holding member piece 6 is made into a support 2 by an analyzer.
It is provided as a window for measuring the intensity of coloration of the reagent layer 3 by projecting and receiving light from the side. Meanwhile, the second
The opening 72 of the holding member piece 7 is provided as a space for supplying the sample to the spreading layer 4.

The shapes of these openings 62 and 72 include a circle, an ellipse, a regular polygon, and the like.

FIG. 3 is a perspective view showing another configuration example of the holding member.
The first holding member piece 6 shown in the figure has a structure in which an annular convex portion 63 that surrounds the opening 62 is formed inside the holding portion 61 (on the side in contact with the support body 2).

The convex portion 63 is an annular shape formed along the peripheral edge portion corresponding to the circular opening 62, and the top portion thereof is preferably flat.

Further, unlike the illustrated example, two or more annular convex portions having different diameters may be concentrically provided.

The height of the convex portion 63 is preferably about 0.05 to 0.3 mm.

Although not shown, the second holding member piece 7 can also be formed in the same manner as the convex portion 63.

In this way, by providing the annular convex portion on one or both of the first and second holding member pieces 6 and 7, it is possible to reliably prevent the development layer 4 from deviating from the reagent layer 3 and floating. It is possible to bond each part of the development layer 4 (especially within the range of the opening 72) to the reagent layer 3 with a uniform pressure. As a result, it is possible to prevent unevenness in coloration and reduce measurement errors.

When forming the annular protrusions on both the first and second holding member pieces 6 and 7, the outer diameters of them should be different, and one of them should be shaped so as to fit inside the other. Is preferred.

The constituent material of the first and second holding member pieces 6 and 7 is not particularly limited, and for example, various resins such as polyethylene, polypropylene, vinyl chloride, polystyrene and ABS, or metals such as aluminum and stainless steel. Is mentioned.

Further, in FIGS. 1 and 3, the first holding member piece 6 and the second holding member piece 7 are shown as separate members, but in the present invention, both holding member pieces are Connected,
Alternatively, it may be integrally formed. For example, one side of both holding member pieces 6 and 7 may be connected to each other, and the connecting portion may be made flexible so that both holding member pieces rotate about the connecting portion.

FIG. 2 is a sectional front view showing another configuration example of the test device of the present invention. The test tool 1b shown in the figure is the same as the test tool 1a in that the test piece 5b has a plurality of reagent layers.
Other configurations are the same as the above.

In the test piece 5b, the reagent layer 3 is formed on one surface of the support 2, a reagent layer 9 containing reagents having different compositions is formed on the reagent layer 3, and the developing layer 4 is placed on the reagent layer 9. It has been placed.

Regarding the support 2, the reagent layer 3 and the development layer 4,
It is similar to that described above.

According to the test tool 1b having such a configuration, it is possible to detect and analyze two or more specific components in the sample. For example, sugar can be detected in the reagent layer 3 and bilirubin can be detected in the reagent layer 9. In this case, the main reagents in the reagent layer 9 are:
It is a diazonium salt.

Further, when forming a plurality of reagent layers, as described above, it is not limited to the detection of a specific component different for each reagent layer 3, 9. For example, from the viewpoint of preventing oxidation and deterioration of reagents, 1
The component of the reagent necessary for detecting the specific component of is divided into two, one of which is contained in the reagent layer 3 and the other of which is contained in the reagent layer 9, so that the sample permeates the reagent layer 9 and permeates into the reagent layer 3. In this case, the reagents in both reagent layers 3 and 9 may be mixed or reacted with each other to thereby have a function of detecting the specific component.

According to such a test tool 1b, the reagent layer adjacent to the spreading layer 4, that is, the reagent layer 9, contains the light-reflecting substance.

Also in this case, the kind, content rate, distribution, etc. of the photoreactive substance are the same as described above.

Although the test device of the present invention has been described above with reference to some configuration examples, the present invention is not limited thereto, and various modifications within a range not changing the gist thereof,
Application examples are possible.

For example, the test piece may be provided with a layer for other purposes in addition to the reagent layer and the spreading layer.

Further, unlike the above-described respective structural examples, the reagent layer 3 or 9 and the spreading layer 4 may be adhered.

In this case, the adhesion between the reagent layer 3 or 9 and the developing layer 4 is performed by interposing an adhesive layer made of a composition having an adhesive function such as gelatin between the reagent layer 3 or 9 and the developing layer 4 to form both layers. It may be carried out by a method of adhering, or a method of press-bonding the spreading layer 4 on the reagent layer 3 or 9 in a wet state and adhering both layers with gelatin or the like in the reagent layer.

Further, the application of the test device of the present invention, a sample (for example, whole blood,
The present invention can be applied not only to analysis of components such as serum, urine, saliva and the like) but also to analysis of food and environmental samples.

<Experimental Example> (Inventive Example) An aqueous solution having the composition shown in Table 1 below was applied onto a transparent support made of a PET film having a thickness of 0.1 mm and dried to form a reagent layer having a dry film thickness of about 10 μm. . Before the reagent layer was dried, centrifugation was performed to unevenly distribute titanium oxide on the surface of the coating film, so that the distribution of titanium oxide in the reagent layer was dense on the developing layer side and rough on the support side.

Then cut this into a size of 1.5 cm x 1.5 cm,
A spreading layer of the same size made of a knitted fabric impregnated with a surfactant is placed on the reagent layer, and these are held by a pair of convex holding member pieces (made of polypropylene) having the structure shown in FIG. A test tool A was obtained by sandwiching the members (mount) and closely contacting each other.

Comparative Example A solution having the composition shown in Table 2 below was applied onto a support similar to that of the present invention and dried to form a reagent layer having a dry film thickness of about 10 μm.

Then, a solution having the composition shown in Table 3 below was applied onto the reagent layer and dried to form a light-reflecting layer having a dry film thickness of about 5 μm and having a filtering function.

After that, this was cut into a size of 1.5 cm × 1.5 cm, a spreading layer similar to that of the example of the present invention was placed on the light reflecting layer, and these were sandwiched by similar holding members and closely adhered to the test tool. B was obtained.

The test tools A and B were colored using a sample containing glucose (human whole blood), and the reaction rate was examined.
The detailed procedure is as follows.

First, glucose was added to physiological saline to a concentration of 5000 mg / d and dissolved, and heat-treated at 37 ° C. for 2 hours,
The isomerized product was used as a glucose stock solution.

Next, the glucose concentration of whole human blood was measured by a commercially available measurement kit (Glycose Test Wako manufactured by Wako Pure Chemical Industries, Ltd.), and based on this measurement value, the glucose whole solution was added to whole human blood, and the glucose concentration was 100 and 200, respectively. , 300, 400 and 500 mg / d, and 5 blood sample Nos. 1, 2, 3, 4 and 5 were adjusted.

Five pieces of the above-mentioned test tools A and B were prepared, and the above-mentioned five blood samples N were respectively placed on the spreading layers of these test tools.
o. 1 to 5 drops of 10 μm each are projected on the support side,
After receiving light, the intensity of reflected light having a wavelength of 565 nm was measured at intervals of 30 seconds.

The light was projected onto the support in two directions at an angle of 45 ° with respect to the surface of the support, and the light was received in the direction perpendicular to the surface of the support.

In addition, for measurement, an instantaneous multi-measurement spectroscope MC manufactured by Otsuka Electronics Co., Ltd.
PD-200 was used.

The measurement results thus obtained are shown in FIG. 4 (example of the present invention).
And the graph of FIG. 5 (comparative example). The graphs of FIGS. 4 and 5 show changes with time of a value obtained by logarithmically converting the relative reflection intensity after coloring with respect to the reflection light intensity before blood dropping which was measured in advance.

As shown in FIG. 4, in the test device A of the example of the present invention, the blood sample No. Difference in glucose concentration between 1 and 5 is 30
It is recognized from about second, and it appears clearly in 60 seconds.

On the other hand, as shown in FIG. 5, in the test device B of the comparative example, the relative reflection intensity was high overall (the color was dark), but the blood sample No. The difference in glucose concentration between 1 to 5 is still small in 60 seconds and appears in about 90 to 120 seconds.

As described above, the test device A of the present invention shows a coloration according to the glucose concentration earlier than the test device B of the comparative example, and it is understood that the sugar amount analysis can be performed in a shorter time.

As the light-reflecting substance, short cellulose fibers (200
Test piece produced in the same manner as the above-mentioned inventive examples and comparative examples except that a reagent layer containing 35 wt% (about 300 mesh) of 35 wt% in the layer (in a distribution in which the developing layer side is dense and the support side is rough) is formed. When C and D were measured in the same manner as above, the same results as above were obtained, and the test tool C of the present invention was analyzed in a shorter time than the test tool D of the comparative example. It was confirmed that it was possible.

<Effects of the Invention> As described above, according to the test device of the present invention, by separately providing the analysis function auxiliary layer such as the light reflection layer and imparting the function to the reagent layer, The permeation and color reaction are promoted, thus reducing the time required for analysis.

In addition, the distribution of the light-reflecting substance in the reagent layer in the thickness direction is
By making the rotation layer side denser than the support side, a darker coloration is obtained and the coloration can be easily detected, so that the measurement accuracy is improved.

[Brief description of drawings]

FIG. 1 and FIG. 2 are cross-sectional front views each showing a configuration example of the test device of the present invention. FIG. 3 is a perspective view showing a configuration example of a holding member used in the test device of the present invention. FIG. 4 and FIG. 5 show the coloration state (relative reflection intensity) of the test devices of the present invention example and the comparative example in the experimental example.
FIG. Explanation of reference numerals 1a, 1b ... Test tool 2 ... Support body 3 ... Reagent layer 4 ... Development layer 5a, 5b ... Test piece 6 ... First holding member piece 7 ... Second holding member piece 61, 71 ... Clamping part 62, 72 ... Opening 63 ... Convex part 8 ... Holding member 9 ... Reagent layer 10 ... Light-reflecting substance

Claims (1)

[Claims] 1. A test device for analyzing components in a sample, comprising a reagent layer containing a reaction reagent and a spreading layer for uniformly spreading the sample on one surface of a light-transmitting support. And a reagent which is fixed integrally with the spreading layer on the outside and which has a light-reflecting substance in a reagent layer adjacent to the spreading layer, wherein the reagent having the light-reflecting substance in the reagent layer The test tool, wherein the layer distribution in the thickness direction is denser on the developing layer side than on the light transmissive support side.