JPH0654311B2 - Analytical element for measuring bad breath with nitrite ion - Google Patents

Description

TECHNICAL FIELD The present invention relates to a halitosis measuring method, and more particularly to a simple, rapid and highly accurate halitosis measuring method and an analytical element therefor.

[Background of the Invention]

Volatile sulfur compounds, alcohol compounds, aldehyde compounds, amine compounds, ammonia, etc. have been proved as major odor components in halitosis. It is known that odor components generated from the oral cavity are mainly composed of mercapto compounds such as hydrogen sulfide, methyl mercaptan and dimethyl sulfide. Therefore, a method for detecting a mercapto compound in exhaled breath or saliva has been proposed for the purpose of measuring bad breath.

JP-A-57-135360 discloses a method for measuring a mercapto compound in saliva using 4,4'-bisdimethylaminodiphenylcarbitol, and JP-A-57-148252 uses dithiobisnitrobenzoic acid. JP-A-58-191957 discloses a method of measuring a mercapto compound in exhaled breath by reacting with a mercapto group coloring reagent (NAM) and fluorescence.
-178828 describes a method for measuring mercapto compounds in saliva from the degree of fading using permanganate.

However, these methods have drawbacks such as low measurement sensitivity for sufficiently detecting bad breath, and sufficient measurement sensitivity, but poor reagent stability, and it is not possible to accurately measure bad breath. could not.

Further, in Japanese Patent Application No. 61-137947, it is impossible to detect halitosis with a mercapto compound as an index, so nitrite ion is used as a method for detecting halitosis with nitrite ion present in saliva as an index. A reagent that develops a color by a diazo reaction and a test paper impregnated with the reagent have been proposed. Although this method has improved detection sensitivity, simplicity, etc., the color-forming reagent or the test paper impregnated with the color-forming reagent develops color by the nitrite ion in the air or by the oxygen in the air. Has the drawback that it is oxidized and colored.

Also, this test paper is useful because it is easy to handle and the results can be obtained directly, but due to its constitution, it remains in the area of semi-quantitative or qualitative analysis, and improvement of these problems is desired. It is rare.

[Object of the Invention]

In view of the above points, the present invention is to provide a stable reagent that develops nitrite ion in a diazo reaction, and another object is a simple and rapid method capable of quantitative analysis of nitrite ion and an analytical element. Is to provide.

Still another object is to provide a method for detecting bad breath using this color-forming reagent or analytical element.

[Structure of Invention]

The object of the present invention is to provide a reagent layer containing a compound that develops a color upon diazo reaction with nitrite ions and a polymer acid on a liquid-impermeable support, and further spread a fluid sample uniformly thereon. This is achieved by an analytical element for measuring halitosis by nitrite ion, which is provided with a developing layer which is inactive and permeable to nitrite ion.

The method of the present invention comprises a reagent containing the above compound and a polyamic acid to develop a color by a diazo reaction (hereinafter simply referred to as a color reagent).
Since it is constituted on the basis of the reaction of swiftness / convenience and its stability, there is no limitation on the utilization means as long as the color reagent can be used, but as a means for maximizing the effect of the present invention, This is to construct an analytical element incorporating the coloring reagent.

As a basic mode of the analytical element, a reagent layer containing the color reagent is provided on a liquid-impermeable support, and a reagent layer which is inactive and permeating to a nitrite ion for uniformly spreading a fluid sample thereon is further provided. By providing the developing layer having the property of being permeable, the fluid sample is uniformly and quickly supplied to the reagent layer, and the colorimetric quantitative analysis of nitrite ion is enabled.

Further, various auxiliary members and auxiliary constituent layers can be attached to the analytical element for convenience of analytical operation and complement of performance of the analytical element.

In the present invention, the compound used in the present invention to develop a color by a diazo reaction (hereinafter referred to as a diazo reaction color developing reagent)
Is composed of a diazo component and a coupling component.

The diazo component is a compound that reacts with nitrous acid to form a diazonium salt, and is an amine compound having a primary amino group. Preferred amine compounds include heterocyclic amines and aromatic amine derivatives. For example, aniline, p-nitroaniline, sulfanilic acid, p-cyanoaniline, p-chloroaniline, anisidine, toluidine, 2,4,6-trichloroaniline, 1-naphthylamine, 4-aminopyridine, 4-aminoantipyrine and the like, and These derivatives are mentioned. These amine compounds are preferably used in the form of inorganic salts such as hydrochlorides, sulfates and organic acids.

The coupling component is a compound that reacts with a diazonium salt, and is an aromatic compound or derivative having an amino group and / or a hydroxyl group.

For example, 1-naphthylamine, N-1-naphthyl-N '
-Diethylethylenediamine, N-1-naphthylethylenediamine, N- (1-naphthylamino) -3-propanesulfonic acid, 1-naphthol, 2-naphthol, phenol, J acid, γ acid, H acid and derivatives thereof I can give you. When the coupling component is an aromatic amine, it may be used as an organic acid salt or an inorganic acid salt.

Further, as another coupling component, 1-phenyl-
A compound having an active methylene group such as 3-methylpyrazolone, 3-hydroxy-1,2,3,4-tetrahydro-7,8
-Benzoquinoline and the like are also used.

These two components are dissolved in water or a water-soluble solvent to give a diazo reaction color developing reagent. In this case, the ratio of the two components and the concentration of the coloring reagent are appropriately adjusted depending on the concentration of nitrite ion to be detected. An acid is used to dissolve the diazo reaction coloring reagent, but with an organic acid, the pH is high and the stability of the coloring reagent deteriorates. The strong inorganic acid has a low pH and thus improves the stability of the color-developing reagent, but it is a problem when the color-developing reagent is contained in the test paper or the analysis element.

Therefore, by using polymer acid, these problems can be solved.

The polymer acid used in the present invention is disclosed in JP-A-59-44659.
No. 59-104552.

Next, specific examples of the polymer acid of the present invention will be shown.

Exemplified polymer acid: (1) Polyacrylic acid (2) Polymethacrylic acid (3) Itaconic acid-styrene copolymer (wt% 50:50) (4) Acrylic acid-butyl acrylate copolymer (wt% 75: 25) (5) Acrylic acid-vinylidene chloride copolymer (wt% 80:20) (6) Ethylene-monobutyl maleate copolymer (wt% 50:50) (7) Styrene-monobutyl maleate copolymer (7) (Wt% 50:50) (8) Monobutyl maleate-methylethylketone copolymer (wt% 50:50) (9) Monobutyl maleate-isobutylene copolymer (wt% 50:50) (10) Methacrylic acid-divinyl Benzene (wt% 98: 2) (11) Acrylic acid-styrene-divinylbenzene (wt% 90: 8: 2) (12) Polystyrene sulfonic acid (13) Polyacrylic amine 2-Methylethanesulfonic acid (14) Amine methacryloyloxypropane sulfonic acid (15) Styrene sulfonic acid-styrene copolymer (wt% 80:20) (16) Styrene sulfonic acid-divinylbenzene copolymer (wt%) 90:10) (17) Acrylamido-2-methylethane sulfonic acid-butyl acrylate-styrene copolymer
(Wt% 15: 80: 5) (18) Methacryloyloxypropane sulfonic acid-2,3-dimethyl-1,3-butadiene-acrylonitrile copolymer (wt% 8:75:17) (19) Styrene sulfonic acid -Vinylidene chloride (wt% 75:25) (20) Methacryloyloxypropane sulfonic acid-methyl methacrylate (wt% 50:50) (21) Poly-2-acryloyloxyethyl phosphate (22) Poly-2-meta Acryloyloxyethyl phosphate (23) 2-Acryloyloxyethyl phosphate-methyl acrylate copolymer (wt% 80:20) (24) 4-methacryloyloxybutyl phosphate-styrene-divinylbenzene copolymer (wt% 50:45) : 5) (25) 2-methacryloyloxyethoxyethyl phosphate- Ethyl acrylate-acrylonitrile copolymer (wt% 15:70:15) (26) 2-Acrylamido-2-methylpropanesulfonic acid-butyl acrylate copolymer (wt% 50:50) (27) 2-acrylamide 2-Methylpropanesulfonic acid-acrylic acid amide copolymer (weight% 75:25) Further, commercially available strong acid and weakly acidic cation ion exchange resins can also be used. For example, Amberlyst 15, Amberlite CG-50, Amberlite IRC-50 (trade name,
Rohm & Hoss). These resins can be used as they are or after being crushed.

The polymer acid of the present invention may be present in such a way that the pH in the layer in which the fluid sample is applied to the coloring reagent solution or the analysis element is about 0.1 to about 2.

The reagent layer of the analysis element according to the present invention can use a hydrophilic polymer substance as a binder. For example, gelatin, gelatin derivatives such as phthalated gelatin, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polymethacrylamide, carboxymethyl cellulose sodium salt, and cellulose derivatives such as hydroxyethyl cellulose are preferable.

Particularly, phthalated gelatin, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone hydroxyethyl cellulose and the like can be usefully used.

Further, the polymer acid according to the present invention can be used as it is as a binder.

The reagent layer composed of the binder may contain the diazo component, the coupling component and the polymer acid according to the present invention separately in the same layer or separate monolayers. Alternatively, a layer containing a diazo component, a coupling component, and a polymer acid may be sequentially laminated to form a laminate. In another embodiment, a layer containing a diazo component, a coupling component and a polymer acid layer (however, the binder may or may not be present) can be sequentially laminated on the support.

Further, it is possible to separately contain the above-mentioned three compounds in three adjacent layers.

As a matter of course, it is also possible to add an additional substance such as a surfactant or a preservative to the above-mentioned reagent layer in carrying out the analysis reaction.

The reagent layer of the present invention, including other layers, may be formed on the support of the present invention by various methods such as dip coating method, air knife method, curtain coating method or extrusion coating method using a hopper as described in US Pat. It is possible to apply by the coating method of US Pat.
It is also possible to apply simultaneously by the method described in 2,761,791 and British Patent 837,095.

Furthermore, the drying temperature of the reagent layer of the analytical element according to the present invention can be arbitrarily selected. For example, it is used in a wide temperature range from room temperature to 100 ° C.

The support of the analytical element according to the present invention may be of any type as long as it is liquid-impermeable, and various polymer materials such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene are used for this purpose. Suitable. The thickness of the support in this case is arbitrary, but is preferably about 50 to 250 microns.
Further, one side surface of the support according to the present invention on the observation side can be optionally processed according to its purpose. Furthermore, an adhesive layer between the reagent layer and the support can be improved by optionally using an undercoat layer on the surface of the support on which the reagent layer is laminated. A transparent support is preferable when the constituent layers of the analytical element are not separated.

The spreading layer according to the present invention has the performance described in JP-B-53-21677, that is, (1) a constant volume of a fluid sample is uniformly spread per unit area of the reagent layer, and (2) an analytical reaction in the fluid sample. If a substance or a factor that inhibits (3) has a function of performing a background action of (3) reflecting the measurement light transmitted through the support when performing the spectrophotometric analysis,
It can be arbitrarily selected. Therefore, the development layer according to the present invention can perform all of the above three functions, but it is also possible to appropriately separate the three functions and use a different layer for each function. Further, it is also possible to combine and use a layer having two functions out of the three functions and a layer having the remaining other functions. For example, a spreading layer of a non-fibrous porous medium called a brush polymer composed of titanium dioxide and cellulose diacetate, JP-A-56-24576 and JP-A-57-12584.
7 and JP-A-57-19746 and the like, the fiber structure developing layer can be mentioned.

The analysis element according to the present invention can take any one of various different arrangements. Further various functional layers of the present invention, various functional layers, reagent-containing layers and members, for example, reagent layer described in U.S. Pat.No. 3,992,158, reflective layer, undercoat layer, 4,0
Radiation blocking layer described in 42,335, barrier layer described in 4,066,403, registration layer described in 4,144,306, migration prevention layer described in 4,166,093, scintillation layer described in 4,127,499, JP-A-5
The cleaning layer described in 5-90859 and the destructible pod-shaped member described in U.S. Pat. No. 4,110,079 and the like can be arbitrarily combined to form an analytical element for the purpose of the present invention.

The method for producing the layer and the method for incorporating the layer into the analytical element according to the present invention are the same as or similar to the method described in the above-mentioned disclosed technique. The disclosed technology also discloses useful materials that can be used for producing such layers.

The analysis element according to the present invention can be visually determined from the side of the support or the side opposite to the support. In addition, colorimetric determination is possible by reflection spectrophotometry. The measurement wavelength is
40 depending on the combination of diazo component and coupling component
A wide range of 0 to 700 nm can be selected.

The analytical element configured as described above, after supplying the fluid sample from the developing layer side, observes the analytical reaction of the reagent layer from the support side or the side opposite to the support, measures the reflection density, and obtains the obtained measurement. The amount of the unknown test substance can be determined by applying the value to the calibration curve prepared in advance.

The amount of the fluid sample applied to the analytical element can be arbitrarily determined, but is preferably about 50μ to about 5μ, and more preferably about 20μ to about 5μ. Normal
It is preferred to apply a 10μ fluid sample.

By using the analysis element of the present invention, it is possible to qualitatively or quantitatively analyze nitrite ions present in various fluid samples, and particularly it is used for analysis of nitrite ions in urine and saliva.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples, but the embodiments of the present invention are not limited thereto.

Comparative Example (1) Preparation of test paper Dimethyl-α-naphthylamine 0.5 g Sulfanilic acid 0.2 g Polymeric acid (Ex. No. 4) 2.5 g Dissolved in 100 m of water to prepare a coloring reagent. Glass fiber paper (GC-50, Toyo Paper Co., Ltd.) was immersed in this solution.
0.5c after removing excess solution and drying in freeze dryer for 2 hours
It was cut into a size of m × 0.5 cm and attached to a plastic stick of 0.6 cm × 4 cm with a double-sided tape to give a test paper A.

In the above color-developing reagent, except for polyamic acid, instead of water
A test paper B was obtained by performing the same operation as the test paper A except that 100 m of 10% acetic acid was used, and used as a comparative example.

(2) Detection of nitrite ion with test paper Standard solution of sodium nitrite (0.5 μg / m, 10 μg /
m, 100 μg / m) on each test strip,
The degree of color development was visually determined. The results are shown in Table 1.

When the test papers A and B were left to stand at room temperature in the air, the test paper B was colored pink after several hours, and the coloration proceeded with the passage of time.

However, with respect to the test paper A, no coloring was observed and the white state was retained.

Similar sensitivity can be obtained by using polymer acid,
Moreover, it can be seen that the storability of the test paper is improved.

Example-1 (1) Preparation of Analytical Element A reagent layer having the following composition was coated on a transparent polyethylene terephthalate support having a thickness of about 180 microns.

N-1- naphthyl -N'- diethyl ethylene amine oxalate 0.51 g / m ² sulfanilic acid 0.23 g / m ² polymeric acid as a reagent layer (illustrated No.26) 2.52g / m ² octylphenoxypolyethoxyethanol ( trade name Triton X-100; Rohm &, the porous spreading layer made of lower composition onto Hass Co.) 0.80 g / m ² consisting of polyacrylamide 12.51 g / m ² dry film thickness of about 20μm layer further reagent layer Laminated.

As a fiber spreading layer Powder paper (D) 101.7 g / m ² (Toyo Paper Co., Ltd.) Styrene-glycidyl methacrylate copolymer (polymerization ratio 9: 1) 25.56 g / m ² Octylphenoxypolyethoxyethanol (Triton X- 100) 14.00g / m ² fiber layer with a dry film thickness of about 300μm.

(2) Measurement of nitrite ion with an analytical element Sodium nitrite was added to saliva collected in a clean state to adjust saliva containing sodium nitrite at various concentrations, and 10 μ of saliva was spread on the developed layer of the analytical element. Dropped.

30 seconds after the dropping, the reflection density (Dr) was measured at 546 nm. First
As shown in the figure, there is a good correlation between the reflection density and the sodium nitrite concentration level, and the quantification is possible.

Example-2 By using a combination as shown in Table 2 instead of sulfanilic acid and polymer acid (Ex. No. 26), an analytical element was prepared and detected in the same manner as in Example-1.

Diazo component Polymeric acid 4-Aminoantipyrine HCl Exemplified No. 4 4-Nitroaniline Exemplified No. 13 4-Cyanoaniline Exemplified No. 23 Similar to Example-1, good between the concentration level of sodium nitrite and the reflection concentration. There was a strong relationship.

Further, no coloring was observed even when the analytical elements prepared in Examples-1 and 2 were left at room temperature in the atmosphere.

Example-3 The concentration of nitrite ion in saliva of the analytical element prepared in Example 1 was measured using a calibration curve, and a sensory test was also conducted at the same time.

Evaluation criteria for sensory evaluation None to low odor No unpleasant odor 0 Low to medium low odor Unpleasant odor 1 Medium low to strong bad odor Unpleasant odor 2 Strong bad odor Strong unpleasant odor 3 Good correlation as shown in Fig. 2 Indicated.

〔The invention's effect〕

According to the present invention, the nitrite ion, which is an index of halitosis, can be easily and accurately measured in such a short time, and the degree of halitosis can be known by measuring the nitrite ion in saliva. .

[Brief description of drawings]

FIG. 1 shows the correlation between the nitrite ion concentration and the reflection concentration (Dr), and FIG. 2 shows the correlation between the nitrite ion concentration and the halitosis sensory test.

Claims (1)

[Claims] 1. A liquid impermeable support, on which a reagent layer containing a compound that develops a color by a diazo reaction with nitrite ions and a polymer acid is provided, and further nitrous acid on which a fluid sample is uniformly spread. An analytical element for measuring bad breath by nitrite ion, which is provided with a developing layer which is inactive and permeable to ions.