JP3475228B2 - Capillary device provided with enzyme test paper used for analysis of trace sample, and measurement method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a capillary tool carrying an enzyme test paper, which is used for the analysis of a trace amount of a sample that undergoes a color reaction due to a catalytic action on a specific component in a body fluid of a living body,
And a measuring method for measuring the concentration of a specific component in a body fluid.

[0002]

2. Description of the Related Art Conventionally, for example, a diabetic patient collects blood a necessary number of times a day by an injector-type blood sampler for measuring blood glucose level. Then, the collected blood is injected into the blood glucose level measuring device, and after the blood glucose level is measured, insulin is injected into the diabetic patient in accordance with the measured blood glucose level. You have to endure pain and emotional distress. Therefore, there is a strong demand for a means that does not cause physical pain or mental pain due to blood collection to a subject such as a diabetic patient.

[0003]

As described above, non-invasively collecting a small amount of body fluid other than the subject's blood as a means for preventing the subject from suffering physical pain or mental distress due to blood sampling. A means for measuring the blood glucose level of a subject based on the glucose concentration in the collected body fluid can be considered. There is a capillary tool for non-invasively collecting a minute amount of body fluid other than the subject's blood, such as gingival crevicular fluid or saliva. In this case, when the tip of the capillary tool is inserted into the gingival sulcus or the oral cavity of the subject, gingival crevicular fluid or saliva is inhaled from the gingival sulcus or the oral cavity into the capillary tool. Then, when the gingival crevicular fluid or saliva inhaled into the capillary tool is brought into contact with the color test paper, the color test sheet develops a color corresponding to the concentration of glucose contained in the gingival crevicular fluid or saliva. It is possible to measure the blood glucose level of the subject. However, the above method has the following problems. The concentration of a specific component (for example, glucose) in gingival crevicular fluid or saliva is extremely low compared to that in blood, and it is necessary to improve the sensitivity of the color test strip in order to achieve the required measurement accuracy. Furthermore,
It is also an issue to realize a mechanism for using the collected trace amount sample in the color reaction without waste. In addition, when the subject's gingival crevicular fluid or saliva is inhaled into the capillary tool, the substance of the color test paper is eluted into the gingival crevicular fluid or saliva, and the eluted substance touches the subject via the inhaled gingival crevicular fluid or saliva. there is a possibility. Therefore, the color-developing test paper provided on the capillary tool needs to be considered in terms of safety to the living body.

Therefore, the present invention has sufficient sensitivity for the measurement of low-concentration specific substances, allows a trace amount of sample to be involved in the color reaction for the measurement without waste, and enhances the safety to the living body as the subject. It is an issue to be solved to provide a capillary tool.

[0005]

The above-mentioned problems can be solved by the inventions described in the claims.

According to the capillary tool of claim 1.
For example, if the tip is inserted into the subject's gingival sulcus,
The gingival crevicular fluid inhaled into the tract is built into the capillary tool.
Direct contact with enzyme test strips and absorption
A small amount of gingival crevicular fluid is effectively colored
Contribute to the reaction. The enzyme test paper also contains N, N-die.
Using tyl-p-phenylenediamine and oxidoreductase
Therefore, it is possible to increase the safety for the subject.
In addition, the color reaction sensitivity can be increased.

The capillary tool according to claim 2 is
Adhesive members such as adhesives for assembly by sonic welding
Since it does not use, it can be made thinner.

According to the measuring method of claim 3,
Collected with the capillary tool according to item 1 or 2.
Based on the color of the enzyme test paper that reacted with specific components in the gingival crevicular fluid
Then, the concentration of the specific component in the gingival crevicular fluid can be measured.
Wear. In addition, the measuring method according to claim 4 causes color reaction.
Based on the absorptivity of light of a specific wavelength irradiated on the enzyme test paper
Can measure the concentration of specific components in the gingival crevicular fluid
Therefore, the blood glucose level of the subject can be accurately measured.
It

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Next, embodiments of the present invention will be described. First, an enzyme test paper that causes a color reaction with glucose in the gingival crevicular fluid will be described. This enzyme test paper is made only of highly safe materials without indication of toxicity and carcinogenicity when touching a living body, for example, via gingival crevicular fluid collected by a capillary tool described later. It is safe to contact the gingiva indirectly.

FIG. 1 is a production process diagram for producing a "glucose test paper" as an example of the enzyme test paper. This glucose test paper is used as a chromogen for N, N-
Diethyl-p-phenylenediamine sulfate ((C
₂ H ₅ ) ₂ NC ₆ H ₄ NH ₂ · H ₂ SO ₄ ) was used.

The manufacturing process of the glucose test paper will be described below with reference to FIG. (1) Sodium lauryl sulfate 0.0 as a surfactant
A solution A is prepared by adding 5 grams and 0.5 grams of sodium alginate as a stabilizer for the other components to 30 milliliters of distilled water and stirring to dissolve. (2) Glucose oxidase (GOD) and peroxidase (POD) were added to 10 ml of distilled water,
Make a stirred solution B. (3) On the other hand, solution C-1 in which 1.44 g of gelatin was added to 30 ml of distilled water at 40 ° C. to 50 ° C. and 30 ml of neutral phosphate buffer solution (pH 6.86) was added while keeping the temperature. And borate buffer solution (p
H9.18) Solution C-2 was prepared by adding 1.44 g of gelatin to 60 ml of solution C-2.
1. To the solution C-2, N, N-diethyl-p-phenylenediamine sulfate having a different composition (see FIG. 2) is added. (4) The mixed solution obtained by adding the above-mentioned solution A and solution B to solution C
-1, Solution C-2 and Solution D-1 and Solution D-2 are prepared. (5) The filter paper is dipped in the solution D-1 and the solution D-2 for 60 seconds, and then dried with warm air for about 2 hours in a dryer kept at 35 ° C.

The N, N-diethyl-p-phenylenediamine sulphate salt as a chromogen changes its color forming properties depending on the buffer solution. Therefore, as described above, a mixed solution of water as a solvent and a neutral phosphate buffer (solution C-1),
A comparison test was performed on the gradient and the linearity (correlation coefficient) of the color density characteristics of the glucose test paper produced by using two kinds of a mixed solution of a borate buffer solution and (Solution C-2). FIG. 2 shows N, N-diethyl-p-phenylenediamine sulfate (described as DPD sulfate in the figure) in a test for comparing the gradient and linearity (correlation coefficient) of the color density characteristics of the glucose test paper. FIG. 6 is a table showing six kinds of combinations of the used amount of the solvent) and the solvent. FIG. 3 is a characteristic diagram showing changes in the slope a of the calibration curve and the correlation coefficient r depending on the solvent.

As a result of the above test, the following matters were clarified. (1) As shown in FIG. 2, as the amount of DPD sulfate as a chromogen increases, the final pH of the solution decreases and the solution becomes acidic. (2) When the glucose test paper was irradiated with light having a variable wavelength, the reflectance (absorbance) of the glucose test paper was 53 wavelengths.
Since it was found that the change was the largest and the sensitivity was good in the vicinity of 0 nm, the gradient of each solvent at the same wavelength was obtained. As shown in FIG. 3, the smaller the final pH of the solution, the larger the gradient a. found. The wavelength of light used in this test is 532 nm. (3) As shown in FIG. 3, the smaller the final pH of the solution, the larger the correlation coefficient r. In this test, the chromogen amount was 0.375 g / dl, and the solvent was water and a neutral phosphate buffer solution. The best calibration curve was obtained under the condition of mixed solution.

Next, the capillary tool 2 carrying the glucose test paper 1 manufactured as described above will be described with reference to FIGS. 4 and 5. 4 is an exploded perspective view of the capillary tool 2, and FIG. 5 is a plan view showing a state in which the capillary tool 2 is assembled. As shown in FIG. 4, the capillary tool 2 is carried by, for example, an upper plate 3, a middle plate 4, a lower plate 5, and a space between the upper plate 3 and the middle plate 4, which are formed of a plastic film such as a polyester film. It has a disk-shaped glucose test paper 1. Also,
A liquid passage 6 having a role of a capillary is formed in the middle plate 4. The upper plate 3, the middle plate 4, and the lower plate 5 have the same outer shape.

The upper plate 3, the glucose test paper 1, the middle plate 4 described above
And the lower plate 5 are arranged in the order shown in FIG. 4, and the upper plate 3 and the middle plate 4, using an ultrasonic welding device (not shown),
By stacking and welding the lower plate 5 and the middle plate 4, the capillary tool 2 as shown in FIG. 5 is obtained. When the upper plate 3, the middle plate 4, and the lower plate 5 are laminated and welded without any interposition using the ultrasonic welding apparatus as described above, the capillary tool 2 having a small thickness can be obtained. When manufacturing the capillary tool 2, in addition to laminating and welding the upper plate 3, the middle plate 4, and the lower plate 5 using the ultrasonic welding device as described above, a double-sided tape or an adhesive material is used to form the upper plate 3. The middle plate 4 and the lower plate 5 may be bonded together. However, in this case, the thickness of the capillary tool 2 increases.

Since the upper plate 3, the middle plate 4 and the lower plate 5 are made of a plastic film, they have water repellency. Therefore, when the gingival crevicular fluid is collected from the gingival crevice by the capillary tool 2 as described later, the gingival crevicular fluid is collected in the intermediate plate
Is difficult to be sucked into the liquid passage 6. Therefore, in order to smoothly suck the gingival sulcus liquid of the gingival sulcus into the liquid passage 6 of the middle plate 4 by the capillary phenomenon, ethanol is applied to the inner surface of the upper plate 3 and the inner surface of the lower plate 5 before assembling the capillary tool 2. 5%
By applying a diluted surfactant (DK ester, sucrose fatty acid ester) 7 and drying it, the area of the liquid passage 6 of the assembled capillary tool 2 is made hydrophilic. The surfactant 7 has been sufficiently confirmed to be biosafe and environmentally safe.

As shown in FIG. 4, the measurement hole 8 formed in the upper plate 3 of the capillary tool 2 exposes the glucose test paper 1. That is, since the glucose test paper 1 develops a color corresponding to the glucose concentration in the gingival crevicular fluid collected from the gingival sulcus by the capillary tool 2, the glucose test paper 1 is irradiated with light of a predetermined wavelength as described later, The glucose concentration in the gingival crevicular fluid is measured. The method for optically measuring the glucose concentration in the gingival crevicular fluid will be described later.
In addition, the two holes 9 and 10 of the capillary tool 2 shown in FIGS. 4 and 5 are provided on the optical measuring device 21 (see FIG. 6) for optically measuring the glucose concentration in the gingival crevicular fluid. It serves as a positioning hole for setting the tool 2.

The capillary tool 2 manufactured as described above is inserted into the gingival sulcus of the subject at the tip end where the liquid passage 6 is opened, and a small amount of gingival crevicular fluid (a small amount of sample) secreted in the gingival sulcus is drawn into a capillary tube. Due to the phenomenon, the gingival crevicular fluid is collected by sucking into the liquid passage 6. When the gingival crevicular fluid thus collected in the capillary tool 2 flows through the liquid passage 6 and comes into contact with the glucose test strip 1, the glucose test strip 1 is colored corresponding to the glucose concentration in the gingival crevicular fluid.

Here, the principle that the glucose test paper 1 carried by the capillary tool 2 is colored according to the glucose concentration in the gingival crevicular fluid will be described. When the glucose in the gingival crevicular fluid adheres to the glucose test paper 1, it is combined with oxygen by the catalytic action of glucose oxidase to produce gluconolactone and hydrogen peroxide. C ₆ H ₁₂ O ₆ (glucose) + O ₂ → C ₆ H ₁₀ O ₆ (gluconolactone) + H ₂ O ₂ Then, the hydrogen peroxide oxidizes chromogen by the catalytic action of peroxidase to give a reddish purple color. H ₂ O ₂ + Chromogen → Coloring (white → magenta)

The above-mentioned capillary tool 2 has the following features. (1) Since the carried glucose test paper 1 is neither toxic to a living body nor carcinogenic, it is possible to safely collect a small amount of gingival crevicular fluid. (2) Generally, the gingival crevicular fluid (sample) of a subject whose secretion amount is only about 1 microliter can be automatically collected, and the collected sample is guided to the sealed liquid passage 6 except for the tip. Therefore, the concentration fluctuation due to water evaporation of the sample does not occur within the time when the coloring is completed corresponding to the glucose concentration in the gingival crevicular fluid. (3) Since the tip has a small thickness and width, it can be easily inserted into the gingival sulcus with a groove width of about 0.1 mm without causing pain to the subject. (4) The amount of sample collected is constant. (5) Since it is inserted into the oral cavity, it needs to be disposable, but it is suitable for disposable because it is inexpensive.

Next, when the gingival crevicular fluid is collected by the capillary tool 2 and the carried glucose test paper 1 is colored based on the above-mentioned coloring principle, the glucose concentration in the gingival crevicular fluid is measured. The measuring method will be described. FIG. 6 shows that the capillary tool 2 in a state where the carried glucose test paper 1 is colored is set at a predetermined position, and
By irradiating the glucose test paper 1 with a laser beam,
It is a structure explanatory view which showed roughly the structure of the optical measuring device 21 for optically measuring the glucose concentration in the collected gingival crevicular fluid. Light emitted from the light emitting diode (LED) may be used as the light with which the glucose test paper 1 is irradiated.

The optical measuring device 21 comprises a semiconductor laser 22 for irradiating the glucose test paper 1 of the capillary tool 2 set at a predetermined position with a beam-shaped laser beam at an oblique angle of 45 degrees, and the glucose test paper 1 A lens 23 that collects the laser beam reflected by the lens 23, a photodiode 24 that receives the laser beam collected by the lens 23, and outputs an electrical signal corresponding to the amount of received light;
4 has a concentration measuring unit 25 for measuring the glucose concentration in the collected gingival crevicular fluid on the basis of the electric signal output from No. 4 and displaying the concentration.

In the optical measuring device 21, the laser beam emitted from the semiconductor laser 22 to the glucose test strip 1 has the highest sensitivity in the vicinity of the wavelength of 530 nm where the reflectance (absorbance) of the glucose test strip 1 is as described above. The wavelength is set to 532 nm as it has been found to be good.

The color density characteristics of the glucose test paper 1
That is, the coloring concentration with respect to the glucose concentration in the gingival crevicular fluid is
As a result of the experiment, since the density measuring unit 25 has a very good linearity, the density measuring unit 25 outputs an electric signal output from the photodiode 24 (the electric signal is in a proportional relationship with the color density). And a correspondence table of the glucose concentration corresponding to the electric signal is stored in the storage unit. Therefore,
When the electric signal output from the photodiode 24 is input, the concentration measuring unit 25 searches the above correspondence table, determines the glucose concentration corresponding to this electric signal, and displays the value.

In the embodiments of the invention described above, (1) an enzyme test paper for measuring the glucose concentration by analyzing gingival crevicular fluid as a trace amount sample, that is, glucose test paper 1, and (2) collected Capillary tool 2 that carries glucose test paper 1 that changes color depending on the glucose concentration in the gingival crevicular fluid as an enzyme sensor, and (3) Glucose test that changes color depending on the glucose concentration in the collected gingival crevicular fluid. Capillary tool 2 carrying paper 1
Was set in the optical measuring device 21 to optically measure the glucose concentration in the gingival crevicular fluid.

The optical measuring device 2 measures the glucose concentration in the gingival crevicular fluid collected by the capillary tool 2 as described above.
When measured by 1, the glucose concentration measurement data is input to a diagnostic computer (not shown).
This diagnostic computer diagnoses a subject's diabetes based on the input glucose concentration measurement data and outputs the diagnostic data.

The capillary tool 2 described in the above embodiment of the invention is not limited to the one having the structure shown in FIGS. 4 and 5, but the features (1) of the above-described capillary tool 2
What is necessary is that it is configured so as to satisfy (5).

In addition to measuring the glucose concentration in the gingival crevicular fluid collected by the capillary tool 2 with the optical measuring device 21, the above-mentioned chemical formula C ₆ H ₁₂ O ₆ (glucose) + O ₂ → C ₆ H ₁₀ Hydrogen peroxide concentration in O ₆ (gluconolactone) + H ₂ O ₂ (proportional to glucose concentration)
The glucose concentration can also be measured by a known measuring means using an electrode reaction for quantifying. Alternatively, the glucose concentration in the gingival crevicular fluid collected by the capillary tool 2 can be semi-quantitatively determined by visually observing the color density of the glucose test paper 1 of the capillary tool 2.

In the above-described embodiment of the invention, the glucose test paper 1 produced as an enzyme test paper that causes a color reaction by a catalytic action on a specific component in a body fluid of a living body is a gingival crevicular fluid.
Explained that it has sufficient sensitivity to measure low-concentration specific substances in saliva, and that it does not indicate toxicity or carcinogenicity, and that the concentration of specific substances can be measured accurately and safely. . As described above, if an enzyme test paper that produces a color reaction by a catalytic action on a specific component in a body fluid of a living body is produced using a substance that is highly safe for the living body, other than measuring the glucose concentration in the gingival crevicular fluid For example, the concentration of a specific component contained in saliva can be measured.

It is considered that saliva contains substances as shown in the right column of FIG. Therefore, the concentration of each substance in the collected saliva can be measured by producing an enzyme test paper that causes a color reaction by a catalytic action on each of these substances and supporting it on a capillary tool or the like. Then, each disease as shown in the left column of FIG. 7 can be diagnosed based on the measured concentration of each substance in saliva.

[0031]

As described above in detail, the use of the capillary tool containing the enzyme test paper used for the analysis of the trace amount sample of the present invention makes it possible to accurately, safely and easily determine the specific component in the gingival crevicular fluid.
Can be measured.

[Brief description of drawings]

FIG. 1 is a production process diagram for producing a glucose test paper.

FIG. 2 is a table showing color development characteristics in a process of manufacturing a glucose test paper.

FIG. 3 is a characteristic diagram of a calibration curve gradient and a correlation coefficient depending on a solvent of glucose test paper.

FIG. 4 is an exploded perspective view of a capillary tool.

FIG. 5 is a plan view showing a state in which the capillary tool is assembled.

FIG. 6 is a structural explanatory view of an optical measuring device for optically measuring a glucose concentration.

FIG. 7 is a table showing the relationship between substances in saliva and diseases.

[Explanation of symbols]

1 glucose test paper 2 Capillary tools 3 Upper plate 4 Middle plate 5 Lower plate 6 liquid passages 7 Surfactant 8 exposed holes 9,10 holes 21 Optical measuring device 22 Semiconductor laser 23 lenses 24 photodiode 25 Concentration measurement unit

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeru Wada 8 Higashi-Katabata-cho, Higashi-ku, Nagoya-shi, Aichi Incorporated company Suzuken (72) Inventor Tomonari Kamei 8-8 Higashi-Katabata-cho, Higashi-ku, Nagoya, Aichi Prefecture (72) Inventor Takeshi Miyabayashi 15-1 Naedai-cho, Mizuho-ku, Nagoya-shi, Aichi Brother Industry Co., Ltd. (72) Inventor Yasuhiro Watanabe 15-1 Naeyo-cho, Mizuho-ku, Nagoya-shi, Aichi Brother Industry Co., Ltd. ( 72) Inventor Shoji Yamada 15-1 Naeshiro-cho, Mizuho-ku, Nagoya-shi, Aichi, Brother Industries, Ltd. (56) References JP-A-11-326321 (JP, A) JP-A-10-197526 (JP, A) JP-A-1-109261 (JP, A) JP-A-9-72900 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 33/52 C12M 1/40 C12Q 1/00 C12Q 1/26 G01N 21/78

Claims (4)

(57) [Claims] 1. A fine tip whose tip can be inserted into the gingival sulcus of a subject.
Middle plate made of thin film formed in width shape and 2
A state in which the outer plates and the middle plate are joined together so as to sandwich the middle plate.
When the tip portion is inserted into the gingival sulcus in the state,
In front of the liquid passage that sucks the gingival crevice fluid from the gingival crevice by capillarity
A specific component in the gingival crevicular fluid, which is formed on the plate
, N-diethyl that undergoes a color reaction by catalysis of
-Fermentation having p-phenylenediamine and oxidoreductase
Contact the elementary test paper with the gingival crevicular fluid sucked into the liquid passage.
For capillaries characterized by being built in at the position to be touched
Ingredient 2. The capillary tool according to claim 1.
In the assembly process, the ultrasonic welding means
A step of joining the outer plate to the middle plate.
Capillary equipment. 3. The capillary according to claim 1 or 2.
The gingival sulcus based on the coloration of the enzyme test paper placed in the tool.
Measurement characterized by measuring the concentration of a specific component in the liquid
Method. 4. The measuring method according to claim 3, wherein
Specific Waves Irradiated on Enzyme Test Paper Used for Analysis of Gingival Crevicular Fluid
Of specific components in the gingival crevicular fluid based on the absorption rate of long light
A measuring method characterized by measuring the concentration.