JPH08159932A - Sampling instrument, measuring method using it and measuring kit - Google Patents

Abstract

PURPOSE: To provide a sampling instrument, a measuring method and a measuring kit by which a substance, as an object to be measured, having a low molecular weight in a liquid to be inspected, in which substances having various molecular weights are mixed can be separated and detected simply. CONSTITUTION: The sampling instrument is composed of a water-insoluble and water-permeable porous film 1 and a water-impermeable film 2 which is bonded to one face of the film so at to be strippable. The porous film 1 is featured so as to comprise a pore diameter through which an object to be measured in a liquid to be inspected, can permeate but through which a substance having a molecular weight larger than it cannot permeate substantially. In the sampling instrument, the water-nonpermeable film is stripped and removed from the porous film after a sample has been sampled, and the substance as the object to be measured is detected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample collecting tool for separating and detecting a low molecular weight substance to be measured in a test liquid in which substances having various molecular weights are mixed. More specifically, the present invention comprises a water-insoluble and water-permeable porous membrane having a specific pore size and a water-impermeable membrane releasably bonded to one surface of the membrane, which comprises a porous membrane after sample collection. The present invention relates to a sample collecting tool for peeling and removing a water-impermeable film to detect a substance to be measured, a method for detecting and measuring a substance to be measured in a test liquid using the sample collecting tool, and a measurement kit therefor.

[0002] The present invention is particularly useful in the case of qualitatively or quantitatively detecting the glycosaminoglycan in the gingival crevicular fluid present in the periodontal pocket by separating it from the high molecular weight mucin.

[0003]

2. Description of the Related Art As a method for separating low-molecular substances dissolved in body fluid as a diagnostic marker for various diseases, gel filtration, ethanol fractionation, electrophoresis, quaternary ammonium salt fractionation The law is known. However, not only are these methods cumbersome and time consuming,
Since a certain amount of specimen sample is required, it is practically impossible to measure the specimen by these methods when a sample of about 10 μl can be obtained by one sampling such as gingival crevicular fluid. It is possible.

As a method for diagnosing periodontal disease, measurement of gingivitis index by macroscopic observation by a dentist, measurement of periodontal pocket depth by periodontal probe, measurement of alveolar bone resorption by radiograph, periodontal examination Pathogenic bacteria ( Porphyromo
nas ginginvalis , Prevotella intermedia , Actinobaci
llus actinomycetemcomitans ) and the method of measuring the quantity. However, in these methods, there are individual differences in dentist judgment criteria, it takes a long time for measurement, the diagnostic cost is high, and whether the inflammatory site of periodontal disease is in the active phase or the stationary phase can be judged. There are problems such as being difficult to do.

Recently, inflammatory products in the gingival crevicular fluid have been attracting attention as a dynamic marker showing the activity of periodontal disease. When periodontal disease occurs, glycosaminoglycan is said to increase as an inflammatory product in the gingival crevicular fluid, and whether the symptoms of periodontal disease patients are in the active phase by detecting this qualitatively or quantitatively. Alternatively, an attempt has been made to diagnose whether the patient is in the stationary phase and use it for subsequent treatment policy.

For example, JM CARTER and PH STAPLE
(J. Dent. Res., 71, 1992) SP LONG and G.
J. HARRAP (J. Dent. Res., 71, 1992) and L
uc Buee et al. (ANALYTICAL BIOCHEMISTRY, 195 , 238
242, 1991), it is possible to quantify the content of glycosaminoglycan in gingival crevicular fluid and synovial fluid by a color reaction with an Alcian blue staining solution using a cellulose acetate membrane or a zeta probe membrane. Reporting.

However, when gingival crevicular fluid is collected using a cellulose acetate membrane or a zeta probe membrane according to the methods of these documents, it is possible to avoid not only glycosaminoglycan but also mucin in saliva from adhering to the membrane. No
Although the color reaction of mucin is also added by Alcian blue staining, there is a risk of making an incorrect judgment, but these documents show that false color reaction due to mucin in saliva that cannot be avoided when collecting gingival crevicular fluid. The existence of is not particularly considered.

[0008]

[Means for Solving the Problems] The present inventors have earnestly conducted research on a tool and a method capable of easily detecting and measuring a diagnostic marker in an extremely small amount of sample sample in which substances of various molecular weights are mixed. As a result, a low molecular weight substance to be measured in the test liquid is permeable, but a substance having a larger molecular weight is substantially impermeable.A water-insoluble and water-permeable porous membrane having a specific pore size. If a sample-collecting device with a water-impermeable membrane releasably bonded to one surface is used, the polymer substance in the test liquid will be applied to the outer surface of the porous membrane (opposite to the side to which the water-impermeable membrane is bonded). The same applies to the non-permeable membrane, since the low-molecular weight substance to be measured can be permeated and transferred to the surface of the porous membrane on the non-permeable membrane side (hereinafter referred to as the bonding surface). After peeling and removing the film, the object to be measured on the joint surface ItaruTsuta a by qualitative or quantitative detection of low molecular substances as readily diagnostic markers without false reaction with polymeric materials, found that it is possible to measure, in the completion of the present invention.

Thus, the present invention provides a sample collecting tool comprising a water-insoluble and water-permeable porous membrane and a water-impermeable membrane releasably bonded to one surface of the membrane, the porous membrane being the subject of inspection. The substance to be measured in the liquid is permeable, but the substance having a larger molecular weight is characterized by having a pore size that is substantially impermeable. Provided is a sample collecting tool for peeling and removing a functional film to detect a substance to be measured.

The present invention also provides that the sample collecting tool of the present invention is brought into contact with a test liquid, the water-impermeable membrane is peeled from the porous film, and the porous film is used as a measurement target in the test liquid. A method for measuring a sample, which comprises qualitatively or quantitatively detecting a substance to be measured in a test liquid by contacting it with a detection reagent capable of reacting with the substance and observing the reaction state of the surface of the porous membrane. Is provided.

Hereinafter, the sample collecting tool of the present invention and the method for measuring a sample using the same will be described in more detail with reference to the drawings.

The sample collecting tool of the present invention comprises a water-insoluble and water-permeable porous membrane (1) and a water-impermeable material that is detachably bonded to one surface thereof, as is apparent from the schematic perspective view shown in FIG. And a flexible film (2).

The material of the porous membrane is not particularly limited as long as it is water-insoluble and water-permeable, and the membrane material is, for example, cellulose acetate,
Nitrocellulose, nylon, polyacrylonitrile,
Examples thereof include polymethylmethacrylate, ethylene vinyl alcohol, polysulfone, polyamide, polyvinyl alcohol, polyethylene, polypropylene, polycarbonate, and hydrophilically treated tetrafluoroethylene. Among them, hydrophilic polymers such as cellulose acetate and polyacrylonitrile are preferable. . Further, the polymer as the film material may have a cationic group or an anionic group.

The porous membrane formed from these materials is
The substance to be measured in the test liquid is permeable, but a substance having a larger molecular weight has a pore size that is substantially impermeable, and the pore size depends on the type of the test liquid and the substance to be measured. Although it needs to be changed depending on the type and the like, it is generally preferable that the thickness is in the range of 0.1 to 3 μm, preferably 0.15 to 1 μm, and more preferably 0.2 to 0.5 μm.

Further, the thickness of the porous membrane is not strictly limited and can be changed according to the type of the test liquid, the site where the liquid is sampled, etc., but is generally 30 to 500 μm.
Preferably 60-350 μm, more preferably 100
It can be within the range of up to 250 μm.

Specific examples of such a porous membrane include cellulose acetate type membranes, nitrocellulose type membranes, and hydrophilic PTFE membranes commercially available from Toyo Roshi Kaisha, Ltd. for particle removal and sterilization filtration; BIO- Examples thereof include Zeta probe nylon membrane ^R manufactured by RAD, and cellulose acetate membrane is particularly preferable.

On the other hand, the water-impermeable membrane bonded to one surface of the porous membrane is either non-porous or porous as long as it is substantially impermeable to body fluids such as urine, saliva and filtrate. May be made of such materials, for example, paraffin hydrocarbon film, impregnated paper, coated paper fine paper kraft paper, polyester film, polyethylene film, polypropylene film, cellophane film, oil paper, sticky notes and the like. Among them, paraffin hydrocarbon film and polyethylene film are preferable. More specifically, the American National Can "parafilm"
(PARAFILM ^R ), TOYOBO ENGINEERING CO., LTD.'S TS film (TS-film ^R ), Fuji Photo Film Co., Ltd. Fuji Film Shiron film (FUJI F)
ILM SEALON FILM ^R ), a sticky note "Post-it ^TM " made by Sumitomo 3M Ltd., Asahi Kasei Corporation
Ltd. "Saranratsupu ^R", Kureha Chemical Industry Co., Ltd. can be exemplified "Kureratsupu ^R", etc., in particular commercially available as sticky note "Para film" and office supplies which is commercially available as an experimental auxiliary products "Post-it" is convenient.

The joint between the porous membrane and the water-impermeable membrane does not easily peel off during transportation, storage, or handling, and peels after the sample is collected without substantially damaging the porous membrane. It is possible to do so, and the test liquid is prevented from penetrating into the interface between the porous membrane and the water impermeable membrane. Such joining can be achieved by room temperature or hot pressing, depending on the combination of the two films used, without the use of any adhesive. For example, in the case of a combination of a cellulose acetate membrane and a parafilm, the above-mentioned joining can be achieved only by pressing at room temperature with a press.

In some cases, the above-mentioned joining can be performed using an adhesive (see FIG. 2). It is desirable that the pressure-sensitive adhesive has such an adhesive strength that it is peeled off along with the water-impermeable membrane when the water-impermeable membrane is peeled off from the porous membrane and does not substantially damage the porous membrane. Such a pressure-sensitive adhesive is generally composed of a base resin, a tackifier and an additive, and examples of the base resin include acrylic acid ester, polyisobutylene, styrene-butadiene latex, butyl rubber, chloroprene rubber, and chloride. Vinyl-vinyl acetate copolymer, chlorinated rubber, polyvinyl butyral, etc. are used, and as the tackifier, for example, rosin-based, terpene-based, synthetic petroleum-based, phenol-based, xylene-based, etc. resins are used, and additives Examples thereof include softening agents, fillers, anti-aging agents, cross-linking agents and the like.

As a specific example of joining when such an adhesive is used, a combination of a cellulose acetate membrane and a post-it to which the adhesive has already been applied can be exemplified.

Some commercially available porous membranes have front and back markings. In this case, either surface may be joined to the water impermeable membrane, but in general, It is preferable that the front side of the porous membrane is bonded to the water-impermeable membrane.

The bonding of the porous membrane to the water impermeable membrane is
Usually, as shown in the figure, it is convenient to carry out so that the end (4) of the water-impermeable membrane remains, which facilitates the operation when peeling the porous membrane from the water-impermeable membrane. Become.

The sample collecting tool of the present invention manufactured as described above can take various forms depending on the type of the test liquid, the site where the test liquid is collected, etc. 05-
It is suitable to make a short fence with a length of 0.2 mm and a length of 10 to 50 mm.

Using the sample collecting tool of the present invention, various test liquids such as blood, serum, plasma, urine, saliva, gingival crevicular fluid, tear fluid, synovial fluid, sweat, semen, vaginal secretions and pus are used. A substance to be measured, which can be a diagnostic marker contained in sputum, feces, etc., can be easily separated by molecular weight and collected to be qualitatively or quantitatively detected.

For example, the sample collecting tool of the present invention is brought into contact with the test liquid as described above, washed and / or dried appropriately, and then the impermeable membrane is peeled from the porous membrane, and the porous membrane is removed. Qualitatively or quantitatively detecting the measurement target substance in the test liquid by contacting the detection reagent capable of reacting with the measurement target substance in the test liquid and observing the reaction state on the surface of the porous membrane. You can

When the sample collecting tool of the present invention is brought into contact with the test solution, the test solution permeates from the outer surface of the porous membrane of the collecting tool toward the impermeable membrane side. At this time, by appropriately selecting the pore size of the porous membrane, the high-molecular weight detection interfering substance in the test liquid remains on the outer surface of the porous body, and the target substance having a relatively low molecular weight to be the target is selectively selected. It can be transparent.

Next, if necessary, the sampling tool is washed to remove excess test liquid adhering to the surface of the tool and dried to transfer the permeated substance to be measured to the surface of the porous membrane to be joined. It can be adsorbed and immobilized. After that, the water-impermeable membrane bonded to the porous membrane is peeled off, and at least the bonding surface of the porous membrane is reacted with the detection reagent capable of reacting with the substance to be measured, and the reaction state is observed. The detection reagents that can be used here differ depending on the type of substance to be measured,
Usually, it is convenient to use a reagent that exhibits a color reaction.

Thus, by observing the coloration state of the surface of the peeled porous film, it is possible to qualitatively or quantitatively detect the substance to be measured that has permeated and transferred to the joint surface of the porous film.

The case where the method for measuring a sample using the sample collecting tool of the present invention is used for the diagnosis of periodontal disease will be described in more detail below.

First, the gingival crevicular fluid is collected by inserting the short fence-shaped sample collecting tool of the present invention into the periodontal pocket of the subject. The gingival crevicular fluid adhering to the outer surface of the porous membrane of the sample collecting tool penetrates into the porous membrane. In the gingival crevicular fluid, the mucin originally contained in saliva (molecular weight of about 1,000,000-
4 million) and various glycoproteins,
It contains glycosaminoglycan (molecular weight of about 55,000 to 100,000) released from the periodontal inflammation site of patients with periodontal disease.
When glycosaminoglycan is detected by the staining method described below, mucin is also stained at the same time and shows a pseudo reaction, so that as the porous membrane, mucin and other polymer substances remain on the outer surface of the porous membrane, and glycosamino It is desirable to use a low molecular weight substance such as glycan having a pore size that allows it to pass therethrough. As such a porous membrane, the pore size is 0.2 to 0.2.
Within the range of 45 μm and the film thickness is 100 to 250 μm
Those in the range can be exemplified.

By using such a porous membrane, glycosaminoglycans that may be present in the gingival crevicular fluid can be separated from a macromolecular substance such as mucin and permeated to the joint surface side of the porous membrane. it can.

As described above, the tool from which the sample has been collected is washed, if necessary, after removing excess gingival crevicular fluid adhering to the tool and / or after drying. The water-impermeable film is peeled off from the porous film.

Next, glycosaminoglycan that may be present on the surface (bonding surface) of the porous membrane from which the water-impermeable membrane has been peeled off is detected by, for example, a critical electrolyte concentration method.
ration-J. E. Scott and J. Dorling, Histochemie,
5 , 221, 1965) according to the staining method
When it is brought into contact with an Alcian blue dyeing solution having a predetermined cationic electrolyte (eg, magnesium chloride) concentration, the dyeing concentration changes depending on the concentration of glycosaminoglycan present. As the staining solution, other staining solutions such as cupronic blue and safranin-O can be used.

Therefore, the porous membrane is washed with a washing liquid as necessary to remove excess reagents, and then dried if necessary, and the coloration state or the degree of coloration reaction is visually observed or measured with a densitometer or the like. By comparing the measurement results with a standard curve prepared in advance, if necessary, glycosaminoglycan in the test solution can be qualitatively or quantitatively determined, and thereby the patient's symptoms can be activated. It is possible to diagnose whether the period is stationary or stationary.

In practical use, the sample collection tool of the present invention can be made into a kit by combining it with a liquid containing a detection reagent capable of reacting with the substance to be measured in the test liquid. For example, in the case of diagnosis of periodontal disease described above,
The sample collecting tool of the present invention (for example, width 0.05 to 0.2 m
m, a short fence with a length of 10 to 50 mm) and an Alcian blue dyeing solution (Alcian blue 0.05 to 0.2%,
Sodium chloride 0.03 to 0.07 M, magnesium chloride 0.0 to 1.0 M, pH 2.5 to 6.0) and washing solution (eg phosphate buffered saline, Tween 205%, pH 7.2).
Can be combined into a kit.

[0036]

[Examples] Hereinafter, a sample collecting tool of the present invention and a method for measuring a test substance in a sample using the sample collecting tool will be described with reference to the case of detecting glycosaminoglycan which is an inflammatory substance in gingival crevicular fluid. Further, the present invention will be described in more detail with reference to Test Examples, but the present invention is not limited thereto.

[0037] Example 1 (Preparation of Sample Collection Equipment) commercially available para film "PARAFILM ^R, (American
National Can Co., Ltd.) and cellulose acetate type membrane “Pore size (thickness): 0.20 (125), 0.4
5 (125), 0.80 (125), 3.00 (135)
μm ”or nitrocellulose type membrane“ pore size (thickness): 0.10 (110), 0.30 (140),
0.45 (145), 1.00 (150), 5.00 (1
60) μm ”was pressed by a press and cut into strips to prepare a sample collecting tool as shown in FIG.

Example 2 (Preparation of Sample Collecting Tool) Post-made by Sumitomo 3M Co., Ltd., which is a commercially available sticky note.
it ^TM of special pressure-sensitive adhesive with a portion of the cellulose acetate type membrane "pore size (thickness): 0.20 (12
5), 0.45 (125), 0.80 (125), 3.0
0 (135) μm "or nitrocellulose type membrane" pore size (thickness): 0.10 (110), 0.30 (1
40), 0.45 (145), 1.00 (150), 5.
00 (160) μm ”was affixed and cut into strips to prepare a sample collecting tool as shown in FIG.

Experimental Example 1 (Color reaction of various samples with magnesium chloride-free Alcian blue dyeing solution) The sample collecting tool prepared in Example 1 was used for human and saliva of a healthy person, hyaluronic acid (12.5γ / ml). ), Heparan sulfate (12.5γ / ml) or chondroitin-4-sulfate (12.5γ / ml) solution for 1 minute and then dried.
AFILM ^R ) "was peeled off. The membrane was dyed with Alcian Blue dyeing solution [Alcian Blue 0.2% (W /
V), sodium chloride 0.05 M, pH 2.7] and stained for 15 minutes.

Next, a washing solution (phosphate buffer solution + Tween 2)
The membrane was washed with 0.5%, pH 7.2) and dried, and then the coloration state of the bonded surface of the membrane (the surface to which the water-impermeable membrane was attached) was evaluated. The evaluation criteria are as follows:-: No coloring ±: Slightly coloring +: Coloring ++: Strongly coloring The results are shown in Table-1.

[0041]

[Table 1]

According to the results shown in Table 1 above, glycosaminoglycans such as hyaluronic acid, heparan sulfate and chondroitin sulfate, which may be contained as inflammatory products in the gingival crevicular fluid of patients with periodontitis and gingivitis, have a pore size of 0. It can be seen that through the porous film of 0.1 to 0.45 μm, a color reaction is exhibited at the joint surface.
On the other hand, since glycosaminoglycan is not present in humans and saliva of healthy persons, a porous membrane having a pore size of 0.1 to 0.45 μm does not show a color reaction at the joint surface, but has a pore size of 0.1.
In the porous membrane having a thickness of 8 μm or more, a high-molecular weight glycoprotein such as mucin existing in saliva begins to permeate, and a color reaction appears on the joint surface.

From the above results, by using the sample collecting tool of the present invention, by appropriately selecting the pore size of the porous membrane, glycosaminoglycan is selectively separated from glycoprotein such as mucin in saliva by selective selection. It turns out that it can be detected.

Experimental Example 2 (Color Reaction of Various Samples with Alcian Blue Staining Solution of Various Magnesium Chloride Concentration) The following experiment was conducted in order to examine the influence of the magnesium chloride concentration in the Alcian blue staining solution on various samples. It was

As samples, human and saliva of healthy people, 1
2.5γ / ml hyaluronic acid aqueous solution, 12.5γ / ml
Heparan sulfate aqueous solution and 12.5γ / ml chondroitin sulfate aqueous solution were prepared, and a cellulose acetate type membrane (pore size 0.20 μm, thickness 125
μm) for 1 minute and dried, and then the membrane is stained with Alcian blue dyeing solution of various magnesium chloride concentrations [Alcian blue 0.2% (W / V), sodium chloride 0.2%.
Magnesium chloride was added to each of the 05M solutions at 0.0,
0.1, 0.15, 0.4M was added and the solution was adjusted to pH 2.7] and stained for 15 minutes.

Next, a washing solution (phosphate buffer solution + Tween 2)
It was washed with 0.5%, pH 7.2) and dried to evaluate the coloration state. The results are shown in Table-2.

[0047]

[Table 2]

From the results shown in Table 2 above, in the case of human and saliva of healthy persons, no color reaction was observed by the Alcian blue dyeing solution regardless of the magnesium chloride concentration, but hyaluronic acid showed magnesium chloride. Colored with Alcian Blue dyeing solution that does not contain MgCl.
No color reaction was observed with Alcian Blue dyeing solution containing 1M or more concentration, and heparan sulfate and chondroitin sulfate were detected with Alcian Blue dyeing solution with magnesium chloride concentrations of 0.1M and 0.15M or less, respectively. However, it is found that the Alcian blue dyeing solution containing magnesium chloride at a higher concentration does not show a color reaction.

Therefore, the type of glycosaminoglycan to be detected can be distinguished by adjusting the magnesium chloride concentration in the Alcian blue staining solution.

Test Example 1 (Color reaction of periodontal disease patient's gingival crevicular fluid with Alcian blue staining solution of various magnesium chloride concentration) Periodontitis patient, gingivitis patient and healthy person, gingival crevicular fluid of 6 persons each The sample collecting tool prepared in Example 1 [porous membrane: cellulose acetate type membrane, pore size (thickness),
0.45 (125) were taken at [mu] m], dried, and peeled off the water-impermeable film is adhered to the membrane "para film (PARAFILM ^R)". Alcian blue dyeing solution of various magnesium chloride concentration [Alcian blue 0.2% (W / V), sodium chloride 0.05M solution, magnesium chloride 0.05% respectively.
0, 0.1, 0.15, 0.4M was added and the solution was adjusted to pH 2.7] and stained for 15 minutes.

Next, a washing solution (phosphate buffer solution + Tween 2)
After being washed with 0.5% and pH 7.2) and dried, the coloration state of the bonded surface of the membrane was evaluated. The results are shown in Table-3.

[0052]

[Table 3]

According to the results shown in Table 3 above, chondroitin sulfate was detected in the gingival crevicular fluid of patients with periodontitis except for Voluntia No. 1, but hyaluronic acid was found in the gingival crevicular fluid of patients with gingivitis. Alternatively, it can be seen that heparan sulfate is detected, but chondroitin sulfate is not detected, and glycosaminoglycan is not detected in the gingival crevicular fluid of healthy subjects.

[Brief description of drawings]

FIG. 1 is a schematic perspective view of an example of a sample collecting tool of the present invention.

FIG. 2 is a schematic perspective view of another example of the sample collecting tool of the present invention.

[Explanation of symbols]

 1 porous film 2 water impermeable film 3 adhesive layer 4 edge of water impermeable film

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kyoji Kito 701 Bentogaoka, Moriyama-ku, Nagoya-shi Omoridai Housing 2 404

Claims (8)

[Claims] 1. A sample collecting tool comprising a water-insoluble and water-permeable porous membrane and a water-impermeable membrane releasably bonded to one surface of the membrane, the porous membrane being used for measurement in a test liquid. A target substance is permeable, but a substance having a higher molecular weight is characterized by having a pore size that is substantially impermeable. Peeling the non-permeable membrane from the porous membrane after collecting a sample. A sample collection tool for removing and detecting the substance to be measured. 2. The sample collecting tool according to claim 1, wherein the porous membrane has a pore size in the range of 0.1 to 10 μm and a thickness in the range of 30 to 500 μm. 3. The sample collecting tool according to claim 1, wherein the porous membrane is made of a material selected from the group consisting of cellulose acetate and polyacrylonitrile. 4. The sample collecting tool according to claim 1, wherein the water-impermeable membrane is releasably bonded to one surface of the porous membrane by room temperature or thermocompression bonding or an adhesive. 5. The sample collecting tool according to claim 1, wherein the water-impermeable membrane is made of a material selected from the group consisting of paraffin hydrocarbons and polyethylene. 6. The test sample collecting tool according to claim 1 is brought into contact with a test liquid, the water-impermeable membrane is peeled from the porous membrane, and the porous film is used as the test liquid. It is characterized in that the substance to be measured in the test liquid is detected qualitatively or quantitatively by bringing it into contact with a detection reagent capable of reacting with the substance to be measured and observing the reaction state on the surface of the porous membrane. How to measure a sample. 7. The sample collecting tool according to claim 1, wherein the porous membrane has a pore size in the range of 0.2 to 0.45 μm and a thickness in the range of 100 to 250 μm. After contacting with the liquid, peel the non-permeable membrane from the porous membrane,
By contacting the porous membrane with Alcian blue and observing or measuring the coloration state or the degree of coloration on the surface of the porous membrane, the glycosaminoglycan in the gingival crevicular fluid is qualitatively or quantitatively A method for diagnosing periodontal disease, which comprises detecting. 8. A sample comprising the sample collecting tool according to any one of claims 1 to 5 and a liquid containing a detection reagent capable of reacting with a substance to be measured in a test liquid. Measuring kit.