JPH0341359A - Testing appliance - Google Patents

Abstract

PURPOSE:To shorten the reaction time of an apparatus and to enhance measuring accuracy by combining a carrier supporting a reagent, a filter and a developing layer in this order so as to set the carrier on a lower side. CONSTITUTION:A testing appliance 1 has a test piece 1a composed of a laminate constituted by arranging a reagent carrier 3 supporting a reagent on the single surface of a support 2 and mounting a filter 4 thereon and further mounting a mesh 5 being a developing layer thereon and the periphery of the test piece 1a is grasped by a holding member 8 consisting of a pair of the first and second holding member pieces 6, 7. The small space due to the bending of the fibers of the mesh 5 is formed to the contact part of the filter 4 and the mesh 5 and the roughness of the mesh 5 is pref. 20-300 fibers/inch and the filter 4 is pref. composed of a membrane filter having a pore size of 0.3-5 mum. By this method, the time. Required in analysis and measurement is shortened and highly accurate analysis and measurement become possible.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a test device for measuring and analyzing the amount of components in a specimen, such as cholesterol and 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.

Among these, in the multilayer analysis sheet for whole blood, an analysis function auxiliary layer such as a color shielding layer, a light reflection layer, or a filtration layer is formed between the reagent layer and the development layer.

According to such a multilayer analysis sheet, when a specimen (blood) is dropped onto the skin sample development layer, the specimen is uniformly spread on the liquid sample development layer, and then blood cells etc. are The sample (serum) is separated and removed, and then the sample (serum) permeates into the reagent layer, and a specific component (e.g., glucose) in the sample reacts with the reagent to develop a color.The light is emitted from the support side, and its reflection is detected. The specific component is quantified based on the intensity of light.

In this multilayer analysis sheet, the liquid sample spreading layer is made of a hydrophilic treated fabric, so the spreading speed of the sample in the lateral direction and the permeation speed in the thickness direction of the spreading layer are well balanced. is uniformly expanded.

However, since the analytical function auxiliary layer in this multilayer analysis sheet is made by dispersing fine powder such as titanium dioxide in a binder such as gelatin, the permeability of the specimen and
The disadvantage is that the permeability of oxygen in the atmosphere, which is a gas necessary for color reaction, is poor, and therefore the reaction time (time from dropping the specimen to coloration) is long.

Furthermore, there is also a multilayer analysis sheet having a structure in which a membrane filter, which functions as both the analysis function auxiliary layer and the development layer, is placed on a reagent layer.

In this case, the membrane filter has excellent shading permeability for the specimen, so the time from when the specimen is dropped until it reaches the reagent layer is short, and the membrane filter also has good oxygen permeability, so the multilayer analysis sheet described above -The reaction time is short compared to -.

However, because the membrane filter has a high permeation rate in the thickness direction, it is insufficiently spread in the lateral direction, that is, the spreadability is poor, and therefore, the entire measurement surface of the reagent layer is not uniformly colored. This method has the disadvantage of causing a decrease in measurement accuracy.

Thus, conventionally, there has been no multilayer analysis sheet that can both shorten reaction time and improve measurement accuracy.

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

〈Means for solving problems〉 Such objects are achieved by the present invention as described below.

That is, the present invention provides a test device for analyzing components in a specimen by reaction with a reagent, which comprises: a carrier supporting the reagent, a filter for filtration, and a developing layer for developing the specimen, in this order. This test device is characterized in that the test device is assembled in such a way that the top side is facing downward.

Moreover, it is preferable that the spreading layer is a test device that is a mesh.

Further, it is preferable that the test device has a small space formed by bending the fibers of the mesh at the contact portion between the filter for filtration and the mesh.

Further, the mesh roughness of the mesh is 20 to 300 lines/
Preferably, the test device is inches.

Further, the filtration filter has a pore diameter of 0.3 to 5μ.
Preferably, the test device is a Menplan filter of m.

According to the present invention having such a configuration, by combining the filter for filtration and the spreading layer, especially the mesh, the spreadability of the specimen in the lateral direction is sufficiently ensured, and the thickness of the filter is reduced. The permeability of the analyte (or its components) in the direction and the permeability of oxygen in the atmosphere are excellent.

As a result, the sample spreads sufficiently and uniformly in the reagent layer, preventing uneven coloring, improving measurement accuracy, shortening the time it takes for the sample to reach the reagent layer, and promoting oxygen supply. , the time required for analysis is reduced.

<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 an exploded perspective view showing an example of the configuration of the test device of the present invention. The test device 1 shown in the figure includes a reagent carrier 3 carrying a reagent on one side of a support 2, and a filtration filter 4 on top of the reagent carrier 3.
, has a test piece 1a made of a laminate having a mesh 5 as a spread layer placed thereon in this order;
a pair of first and second holding member pieces 6 and 7
It is held by a holding member 8 made of.

The support 2 has a plate shape with a thickness of about 20 to 500, 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, Examples include various resins such as polyvinyl alcohol, glass, and the like. Alternatively, sheets made of two or more of the above materials may be laminated.

The reagent carrier 3 may be of any type as long as it can support a reagent, but it is particularly preferable to use one made of woven or knitted fabric.

Here, woven or knitted fabrics include woven fabrics, knitted fabrics, or similar fabrics, and do not include nonwoven fabrics or filter paper.

All types of textiles that are used in practical use can be used, such as plain weave, twill weave, and satin weave.

Further, there is no particular limitation on the composition of the knitted fabric, and examples thereof include weft knitting (flat knitting), warp knitting (tricot knitting), circular knitting, flat knitting, and the like.

The reagent carrier 3 made of such a woven or knitted fabric has lower permeability to the specimen or its components and permeability to gases such as oxygen necessary for the coloring reaction, compared to a reagent layer formed by applying and drying a coating liquid, which will be described later. Are better. In particular, it easily penetrates even oily components such as cholesterol, so it is suitable for use as a test tool for detecting such components.

In addition, the reagent carrier in the present invention preferably has a regular weave or knitting method, such as a plain woven fabric or a plain knitted fabric. The reason is that the specimen permeates the reagent carrier 3 uniformly,
This is because uneven coloring can be prevented by keeping the amount of specimen per unit area of the reagent carrier constant.

Examples of fibers constituting such woven or knitted fabrics include natural fibers such as cotton, kapok, flax, hemp, ramie silk, and wool; chemical fibers such as nylon, Tetoron, rayon, acetate, vinylon, acrylic, and polyethylene terephthalate; Alternatively, a blend of two or more of these natural and chemical fibers (for example, a blend of nylon and Tetron) can be used.

Although the thickness of these fibers depends on their type, they are usually 0. It is preferably about 1 to 1.0 denier.

The reagent carrier 3 made of such a woven or knitted fabric is preferably hydrophilic. This allows the penetration of the specimen,
This is because diffusion is promoted.

In such cases, the woven or knitted fabric constituting the reagent carrier 3 is hydrophilic in itself (for example, made of fibers such as cotton, silk, nylon, etc.), or the woven or knitted fabric is hydrophilic. Examples include those that have been treated.

Hydrophilic treatment may be carried out by a simple method such as thoroughly washing a commercially available woven or knitted fabric to remove glue or other processing agents, but preferably by applying a surfactant as described below to the woven or knitted fabric. Good.

Application of a surfactant can be carried out, for example, by dipping the washed fabric in a surfactant solution (e.g. 0.05-5%) or by spraying a similar surfactant solution to drain the fabric. It is then dried.

As the type of surfactant, any of water-soluble nonionic (nonionic), cationic, anionic, and amphoteric surfactants can be used.

Particularly preferred are nonionic surfactants such as alkylaryl ethers or fatty acid esters of polyoxyethylene or polyglycerin, sorbitan esters, and the like.

Other methods for making woven or knitted fabrics hydrophilic include an aqueous solution of a hydrophilic polymer such as polyvinyl alcohol, or an aqueous solution of a hydrophilic polymer containing fine powder such as titanium oxide or barium sulfate, or a wetting agent such as glycerin or polyethylene glycol. It is also possible to wet the woven or knitted fabric at night and then dry it.

In this case, the concentration of the hydrophilic polymer aqueous solution is 0.1 to 5
．． It is preferable to set it to about 0 wt%.

The thickness of the reagent carrier 3 is preferably 50 to 500 μm, more preferably 100 to 400 μm. Thickness is 50μ
If it is less than m, the amount of supported reagent will be small and the degree of coloration will be reduced. In addition, if the thickness exceeds 500, if the amount of sample is small, the sample may not permeate the entire thickness of the reagent carrier 3, or it may take time to permeate, resulting in uniform color development and rapid reaction. It becomes difficult.

Such a reagent carrier 3 supports a reagent to be described later.

This reagent is appropriately determined depending on the component to be quantified in the specimen. For example, when quantifying glucose in a specimen, the reagents include glucose oxidase, peroxidase, and a chromogen. In addition, when quantifying total cholesterol in a sample, cholesterol esterase, cholesterol oxidase,
Contains peroxidases and chromogens.

The method for supporting the reagent on the reagent carrier 3 includes, for example, immersing the reagent carrier 3 in a liquid containing a reagent (for example, 0.1 to 10% chromogen), or spraying a similar liquid onto the reagent carrier. It is preferable to wet the material 3 and then dry it.

Further, the amount of the reagent in the reagent carrier 3 varies depending on the type of reagent, and the preferable range is illustrated in Table 1 below.

Table: Such a reagent carrier 3 may simply be placed on the support 2 (having no binding force with the support), but
It may be bonded or integrated with the support 2 (having a bonding force).

Other structural examples of the reagent carrier 3 include, for example, gelatin, polyvinyl alcohol, polyvinylpyrrolidone, agarose, sodium polyvinylbenzenesulfonate,
Binders such as polyurecne and polyvinylpropionate (
Examples include a reagent layer composed of a composition containing the same reagent as described above in a binder).

This reagent layer is formed by an appropriate method such as dip coating or extrusion coating.
This is carried out by applying a coating solution of the above composition onto the surface of the support 2 and then drying it. In this case, the dry film thickness of the reagent layer is preferably about 0.5 to 50 μm.

In addition, in the illustrated example, the reagent carrier 3 has only one layer, but
It may also be a stack of a plurality of reagent carriers each containing a different reagent composition.

The filtration filter (hereinafter simply referred to as a filter) 4 is for filtering out, for example, blood cells in blood, solids in urine, etc., and may be of any type as long as it has such a function, but in particular , it is preferable to use a membrane filter. The reason for this is that membrane filters have excellent filtration functions, do not adsorb specific components in the filtrate, do not elute filter components, and are inert themselves.

Also, among membrane filters, the pore diameter is 0.
It is more preferable to use one with a diameter of 3 to 5 μm. This is because if the pore diameter is less than O, g, or m, the filtration rate will be slow, and if the pore diameter is more than 5, the filtration ability (for example, for blood cells) will be reduced.

Further, the thickness of the filter 4, particularly the membrane filter, is preferably 50 to 300 tffi.

Such a filter 4 itself has excellent water absorption properties, that is, excellent permeability to samples and permeability to gas (oxygen).

Therefore, the reaction rate can be reduced compared to conventional gelatin-based filtration and light shielding layers.

A mesh (or net) 5 placed on such a filter 4 is used to uniformly spread the dropped sample. The mesh 5 is a sheet-like member having regularly arranged meshes, and its form includes a woven or knitted fabric of fibers, an integrally molded product, a processed product, and the like.

In addition, examples of the constituent material of the mesh 5 include Tetron, nylon, rayon, polypropylene, polyvinyl chloride, polyester, and the like.

Moreover, the diameter of the fibers of the mesh 5 is preferably about 30 to 500 fibers.

Such mesh 5 is preferably subjected to a hydrophilic treatment in order to improve the spreadability of the specimen. This hydrophilic treatment may be performed by the same method as above, such as washing or applying a surfactant.

The coarseness of the mesh is determined by the conditions of filter 4 (type,
Although the suitable range varies depending on the conditions of the specimen (type, drop amount, viscosity, etc.), the form of the mesh, the constituent materials, the fiber diameter, the presence or absence of hydrophilic treatment, etc., it is usually 20 to 300.
Preferably, it is books/inch (= mesh). In particular, in the case of hydrophilized mesh, 20 to 300
The optimum number of threads/inch is 20-50 threads/inch for untreated mesh.

When the coarseness of the mesh is less than 20 lines/inch,
A small space 9, which will be described later, becomes larger, and when the sample is blood, the lateral spreadability of the sample becomes worse. Moreover, if it exceeds 300 lines/inch, the small space 9 described later becomes small, and when the specimen is blood, the development speed becomes slow.

In addition, in the present invention, two or more meshes similar to those described above can also be installed.

These filters 4 and mesh 5 each have poor specimen spreadability when used alone, but by combining them, both the horizontal spreadability of specimens and the permeability (filtration speed) in the filter thickness direction are excellent. It becomes something.

The principle is presumed to be as follows.

As shown in FIG. 2, the mesh 5 bends the fibers 51 at the intersections of the fibers 51, so the filter 4
A large number of small spaces 9 are formed between the two. mesh 5
When a sample is dropped on top, the sample instantly enters the small space 9, moves to the adjacent small space 9 by capillary action, spreads radially laterally across the interface between the filter 4 and the mesh 5, and is developed. Here, if the volume of the small space 9 is too large because the mesh 5 is too coarse or the mesh 5 and the filter 4 are separated from each other, the expandability will be poor.

In addition, since the development of the specimen is performed by moving the specimen in the small space 9, in order to obtain good deployability,
It is necessary for the specimen to be retained at the interface between the filter 4 and the mesh 5 for a certain amount of time, but if the water absorption of the filter 4 is too good, the retention time of the specimen on the filter 4 will be shortened, and the expandability of the filter 4 itself will be reduced. However, since the project will be left to the

On the other hand, if the water absorption of the filter 4 is poor, although the sample developability is excellent, the filtration speed of the filter will be slow.
It takes a long time for the reagent to reach the reagent carrier 3, and it takes a long time for color development.

In the end, by setting the balance between the horizontal spread speed of the specimen on the filter 4 and the filtration speed of the filter 4 to a suitable range, it is possible to improve the spreadability and shorten the time required for the sample to reach the reagent carrier. As such, a combination of the above-mentioned filter 4 and mesh 5 is preferable.

As shown in FIG. 1, the test piece 1a is held around its periphery by a holding member 8, and the laminated mesh 5, filter 4, and reagent carrier 3 are pressed and fixed to the support 2.

This holding member 8 is a first holding member piece 6 that fits into each other.
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 piece 1a with a predetermined pressure. There is.

Further, openings 62 and 72 are formed at approximately the center portions of the first and second holding member pieces 6 and 7 (inside the holding portions 61 and 71), respectively.

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 reagent carrier 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 test piece 1a.

The shapes of these openings 62 and 72 are 1. Round,
Examples include an ellipse and a regular polygon.

Furthermore, a grip portion 63 for gripping and manipulating the test device 1 is formed on the first holding member 6 .

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, vinyl chloride, polystyrene, and ABS, or metals such as aluminum and stainless steel. can be mentioned.

Furthermore, in FIG. 1, the first holding member piece 6 and the second holding member piece 7 are shown as being constructed separately, but in the present invention, both the holding member pieces are connected or It may be formed integrally. For example, both holding member pieces 6.
7 may be connected to each other, and by making this connecting portion flexible, both holding member pieces may be configured to rotate around the connecting portion.

Also, a structure in which an annular convex portion surrounding the opening 62, 72 is formed on the inside of the holding portion 61 and/or 71 of the holding member 8 (the side that contacts the support 2 or the mesh 5) (not shown) can be mentioned.

In this manner, by providing the annular convex portion on one or both of the first and second holding member pieces 6.7, the mutual misalignment of the support 2, reagent carrier 3, filter 4, and mesh 5 can be prevented. It is possible to reliably prevent floating or floating, and to join them with uniform pressure. As a result, uneven coloring can be prevented from occurring and measurement errors can be reduced.

Note that when annular convex portions are formed on both the first and second holding member pieces 6 and 7, their inner and outer diameters are different, and one is shaped so that it fits inside the other. is preferred.

Note that the test device of the present invention is not limited to one in which the test piece is held between holding members as in the illustrated example of the structure, but also in the case of a test piece alone or a test piece attached to a strip-shaped support. It may be in any form, such as a

The test device of the present invention is used for specimens (e.g., whole blood, serum,
It can be applied not only to the analysis of cholesterol and sugar content in body fluids such as 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. Applications are also possible.

<Experimental example> (Example of the present invention) The reagent carrier, filter, and mesh shown below were placed in this order on a transparent support made of PET film with a thickness of 0.1 mm, and these were held with a pair of protrusions. A test device A was obtained by holding the test device A by holding it between a holding member (mount) made of a member piece (made of polypropylene) and bringing it into close contact.

The sizes of the support, reagent carrier, filter, and mesh were all 1.5 cm x 1.5 cm.

[Reagent carrier] Material 20, knitted fabric using LD nylon thread Thickness: 240 μm Manufacturing method - The above knitted fabric piece was impregnated with a liquid containing a reagent and a surfactant and having the composition shown in Table 2 below. was dried at 40°C for 60 minutes to obtain a reagent carrier.

3 Table [Filter] Material: Menplan filter (FM manufactured by Fuji Photo Film Co., Ltd. Pore diameter: 3 pJIl Thickness = 140 units 300) (Comparative example 1) On the same support as the present invention example, a layer for forming a reagent layer was added. A solution having the composition shown in the upper column of Table 3 below is applied as a coating liquid, dried,
A reagent layer with a dry thickness of 6 to 8 μm was formed.

Next, on this reagent layer, an aqueous dispersion (lower column of Table 3) in which 1 part of dry gelatin was mixed with 8 parts of fine titanium dioxide powder (weight ratio) was applied and dried to give a dry film thickness of 8 to 10%. Formed the filtration and light shielding layer of the door.

Thereafter, a coating solution made of gelatin for adhesion was applied, and in an undried state, a developing layer made of a hydrophilized fabric shown below was layered.

Next, a load of about 50 gf/am2 was applied for 5 minutes, and this was dried to bond and integrate the spreading layer and the filtration and light shielding layer.

Thereafter, this was cut into a size of 1.5 cm x 1.5 cm to obtain a test device B.

[Development Layer] Material, Nilat Thickness: 240 μm Table (Comparative Example 2) A test device C similar to the inventive example was obtained except that no mesh was provided.

Each of the above test devices A, B, and C was colored using a sample containing cholesterol (human whole blood), and the reaction rate and color development were examined.

The detailed procedure is as follows.

First, low-density LDL (LDL) containing cholesterol is isolated from human plasma, and LDL with known cholesterol concentration is extracted.
A D La stock solution was obtained and used as a stock solution.

Next, the total cholesterol concentration of human whole blood was measured using a commercially available measurement kit (Determiner TC"555°, manufactured by Kyowa Medex).
Based on this measurement value, a cholesterol stock solution is added to human whole blood, and the cholesterol concentration is 50%.
, 100.200.300.400 and 500 mg
/di! How to add 6 kinds of blood sample No.
, 1.2.3.4.5 and 6 were adjusted.

Prepare six test devices A, B, and C, and drop 20 uf2 of each of the six blood samples N011 to 6 onto the test piece of each test device, and emit and receive light on the support side. Then, the intensity of the reflected light with a wavelength of 560 nm is reduced to 10
Measured at second intervals.

Note that the light is projected onto the support from two directions at an angle of 45° to the support surface, and the light is received in a direction perpendicular to the support surface.The light projection surface (measurement surface) is a circle with a diameter of 5 mm. It was inside.

In addition, for measurement, an instant multi-measurement spectrometer MC manufactured by Tenba Denshi Co., Ltd.
PD-200 was used.

The measurement results obtained in this way are shown in Figure 3 (example of the present invention).
and shown in the graph of FIG. 4 (Comparative Example 1). The graphs in FIGS. 3 and 4 show changes over time in logarithmically converted values of the relative reflection intensity after coloring with respect to the reflection light intensity before blood drop, which was measured in advance.

As shown in FIG. 3, in the test device A of the present invention example, the difference in total cholesterol concentration among blood samples N011-6 was recognized from about 10 seconds onwards, and clearly appeared at 30 seconds.

On the other hand, as shown in FIG.
Therefore, it is estimated that the difference in total cholesterol concentration between blood samples Nos. 1 to 6 is small even after 80 to 90 seconds, and a clear difference does not appear until about 10 minutes or more have elapsed.

In this way, the test device A of the present invention is different from the test device B of Comparative Example 1.
It can be seen that the coloration corresponding to the total cholesterol concentration appears earlier than in the conventional method, and it is possible to analyze the total cholesterol amount in a shorter time.

Next, the color development of the reagent carrier was visually observed from the support side of each test device A, B, and C.

The results are as follows.

In the test device A of the present invention, the metal area within the measurement window was colored, and the colored portion was uniformly colored without any color unevenness.

The same was true for Test Device B of Comparative Example 1.

On the other hand, in Test Device C of Comparative Example 2, the shape of the colored portion was not circular, but was band-like or spotted around the measurement window. In addition, the central portion was often uncolored. Moreover, color unevenness occurred in the colored portion. Therefore, measurement of reflected absorbance was not possible.

For this reason, although the reliability of the measured values shown in Figures 3 and 4 is high, there is a high possibility that Comparative Example 2 contains a considerable amount of error, and it is likely that actual blood was measured using Test Device C. It is considered that it is not possible to measure the total cholesterol concentration in the blood.

<Effects of the Invention> As described above, according to the test device of the present invention, by combining a filtration filter and a spreading layer, especially a mesh, the time required for analysis and measurement can be shortened, and highly accurate analysis and analysis can be achieved. Measurement becomes possible.

[Brief explanation of drawings]

FIG. 1 is an exploded perspective view showing an example of the configuration of the test device of the present invention. FIG. 2 is an enlarged cross-sectional side view showing the boundary between the filter and the mesh in the test device of the present invention. Figures 3 and 4 show the coloring states (relative reflection intensities) of the test devices of the present invention example and comparative example 1 in the experimental examples, respectively.
It is a graph showing the change over time. Explanation of symbols 1...Test device 1a...Test piece 2...Support 3...Reagent carrier 4...Filter 5...Mesh 51...Fiber 6...First holding member Piece 2 7... Second holding member piece 61.71... Clamping part 62.72... Opening 63... Gripping part 8... Holding member 9... Small space

Claims (5)

[Claims] (1) A test device for analyzing components in a specimen by reaction with a reagent, comprising: a carrier supporting the reagent, a filter for filtration, and a developing layer for developing the specimen in this order, with the carrier facing downward. A test device characterized by being combined in such a manner that (2) The test device according to claim 1, wherein the spreading layer is a mesh. (3) The test device according to claim 2, wherein a small space is formed by bending fibers of the mesh at the contact portion between the filtration filter and the mesh. (4) The coarseness of the mesh is 20 to 300 lines/
The test device according to claim 2 or 3, which is inch. (5) The filtration filter has a pore diameter of 0.3 to 5μ.
The test device according to any one of claims 1 to 4, which is a Menplan filter of m.